Occurrence , harmful effects and analytical determination of Ochratoxin A in coffee

Article history: Received on: 05/11/2014 Revised on: 22/11/2014 Accepted on: 04/12/2014 Available online: 30/01/2015 Coffee is an important stimulant and a good source of bioactive constituents with beneficial health effects. Coffee beans are readily attacked by the growth and proliferation of several fungal species from the genera Aspergillus and Penicillium. Ochratoxin A, an important toxin produced by these fungal entities, is commonly found in coffee products, from farm to cup-thus challenging the popularity of this world-acclaimed beverage. The toxin has been shown to be nephrotoxic and hepatotoxic, among other deleterious effects. This has prompted the European Commission to set limits for ochratoxin A in coffee products. In order to abide by these regulatory codes, various analytical methods have been developed and deployed in the analysis of the toxin in green coffee beans, roasted coffee and instant coffee. This review will discuss the occurrence of ochratoxin A, its harmful effects and various analytical methods that have been applied in its detection and quantification in coffee products.


INTRODUCTION
Since its introduction into Arabia across the Bahr al-Yaman, "Sea of Yemen" (Red Sea), many centuries ago (Miran, 2012), popularity of coffee as an important beverage has continued to grow.Apart from its organoleptic properties, coffee is a functional beverage with many beneficial effects attributed to its consumption due to the presence of some major and minor constituents with bioactive potentials (Esquivel and Jiménez, 2012;Nuhu et al., 2014).These chemical compounds include caffeine (Prediger, 2010), trigonelline (Dijk et al., 2009;Yoshinari and Igarashi, 2010), chlorogenic acid (Moon et al., 2009) and diterpenes (Sridevi et al., 2011).Coffee is produced from green and roasted coffee beans of different varieties; but two main species, Coffea arabica and Coffea canephora have remained the most economically viable ones (Ky et al., 2001).This cash crop is now significantly produced as an export commodity in three geographical regions of the world: America, Asia and Africa (Serra et al., 2005).Challenges to this important business include market shocks, extreme weather, pests, parasites .and import regulations (Martins et al., 2003;Tucker et al., 2010;Burbano et al., 2012;Nugroho, 2014).The quality of coffee bean and its economic value are partly determined by its physical characteristics (Kathurima et al., 2009;Iamanaka et al., 2014).A daunting challenge faced by producers is how to prevent coffee products from the growth and proliferation of mycotoxin producing molds.From farm to cup, mycotoxins, especially ochratoxin A (OTA), one of the most widespread, can now be found in coffee beans and its beverage (Bucheli and Taniwaki, 2002;Duarte et al., 2009;Velmourougane et al, 2010).
This mycotoxin is produced by mainly two genera of microorganisms, Penicillium and Aspergillus (Pardo et al., 2004;Perrone et al., 2007;Cabanes et al., 2010;Fernández-Baldo et al., 2011).Because of the risk factors associated with its presence in biological systems (Clark and Snedeker, 2006), there is a great need for sound analytical methods for its characterization and quantitative analysis.Some general reviews have been written on methods for the determination of mycotoxins in foods and drinks (Turner et al., 2009;Koppen et al., 2010;Kaushik et al., 2013).This work is particularly dedicated to methods that have been applied in the analytical determination of OTA in coffee due to its social and economic importance.
Importantly, it has been detected in coffee beans (Studer-Rohr et al., 1995;Palacios-Cabrera et al., 2004) and in ready-todrink coffee (Noba et al., 2009).Analysis of green coffee beans by Micco et al. (1989) showed up to 58% contamination level.Interestingly, Nehad et al. (2005) found that decaffeination of coffee may even encourage the growth of OTA-producing species.
Although roasting causes degradation of OTA (Romani et al., 2003;Castellanos-Onorio et al., 2011), some amount may still be found in commercial roasted coffee (Tsubouchi et al., 1988).Mounjouenpou et al. (2013) found that, compared to pure Arabica and mixed coffee brands, pure Robusta coffee was the most contaminated in the range of 0.6-18 µgKg -1 .This result may be worrisome considering that the maximum contaminant level set by the European Commission is 5 µgKg -1 for roasted coffee beans and 10 µgKg -1 for instant coffee (The Commission of the European Communities, EC No. 1881/2006).

Harmful effects
As a food contaminant, OTA does not only affect the physical characteristics of coffee, its presence also contributes to human exposure (Studer-Rohr et al., 1995;Duarte et al., 2011;Hennemeier et al., 2012).In Saudi Arabia where Qahwah (coffee) is a very popular beverage, high degree of OTA contamination was found (Bokhari, 2007).OTA has been detected in human milk and cord serum, and values corresponded with maternal dietary habits (Biasucci et al., 2010).
Its transformation into OT-α, OTA-glucosides and other metabolites may elicit some negative responses in different biological systems (Zepnik et al., 2003).OTA is hepatotoxic, and this effect on human hepatoma cells HepG2 is strongly dependent on concentration of proteins in the culture medium (Bösch-Saadatmandi et al., 2006).
Probably due to its phenolic component, OTA has a high affinity for human serum albumin at the same binding site of natural flavonoid aglycones (Poor et al., 2012).Cremer et al.(2012) have found that OTA has a strong pro-inflammatory effect on nasal epithelial cell cultures, significantly stimulating the increased release of interleukins, IL-6 and IL-8.OTA fed to gestating rabbits resulted in skeletal and soft tissues anomalies, a strong indication of its teratogenic effect (Wangikar et al., 2004).Two doses of this mycotoxin (1000 and 2000 µg/kg of feed) fed to white Leghorn cockerels for seven weeks resulted in significant decrease in the counts of important hematological and serum biochemical parameters, including erythrocytes, leukocytes, hemoglobin and pack cell volume (Ahmad et al., 2012).In a different study, endocrine disrupting activity was displayed through steroidogenic effect after exposing H295R cells to OTA at concentration range of 0.1-1000 ng/ml, resulting in increased production of the hormone estradiol, three times that of solvent control (Frizzell et al., 2013).This effect was further investigated in human placental cells JEG-3 in vitro.After 72-96 h exposure at both cytotoxic and non-cytotoxic levels, 3βhydroxysteroiddehydrogenase/isomerase mRNA expression was increased by 281-378% (Woo et al., 2013).
OTA may hinder the normal functioning of kidney by decreasing the production of vascular endothelial growth factor (Stachurska et al., 2011).Elsewhere, Abdu et al. (2011) have observed several histopathological changes in the renal corpuscles of rats exposed to OTA which might ultimately lead to renal failure.Previously, Stoev (2010) has examined the carcinogenic effects of OTA in various internal organs of Plymouth Rock chicks.In this study, only liver and kidney were the target organs for OTA carcinogenicity, even though many degenerative effects were observed in brain, kidney, liver and lymphoid organs.A sitespecific response was observed when gpt-δ rats were exposed to carcinogenic doses of OTA for 13 weeks, leading to induction of karyomegaly and apoptosis at the outer stripe of the outer kidney medulla (Hibi et al., 2011).OTA forms covalent adduct with DNA (Mantle et al., 2010) and causes direct or oxygenic DNA damage (Ali et al., 2011).The key to these cytotoxic and carcinogenic effects of OTA might be its perturbation of mitotic processes through inhibition of histone acetyltransferases (Czakai et al., 2011).

Extraction and Clean-Up Methods for ota in Coffee
Detection of OTA in coffee can be done without going through sample preparation steps of extraction and sample cleanup.In many instances, however, these steps are necessary in order to remove interferences and improve analytical sensitivity.To this end, liquid-liquid extraction (LLE) and solid-liquid extraction (SLE) of both soluble and solid coffee using suitable solvents can be encountered (Mun et al., 2013;Ventura et al., 2005).In order to expedite desorption of analytes from their parent matrixes, these processes are sometimes driven or facilitated by ultrasonic agitation (Fujii et al., 2007)or microwave heating (Graziani et al., 2012).A mixture of methanol and sodium bicarbonate is commonly applied as extraction solvent for OTA in different matrixes (Bandeira et al., 2012a;Santos and Vargas, 2002;Vargas et al., 2005).Arguably, the most commonly applied clean-up techniques are liquid-liquid (LL),solid-phase (SP), and immunoaffinity column (IAC) procedures.I n some cases, both extraction and clean-up procedures are carried out simultaneously (Sibanda et al., 2002).

Immunoaffinity column clean-up methods
In the sample preparation of OTA for analysis, IAC is used for the clean-up of extracts from green coffee beans, roasted coffee beans and soluble or instant coffee (Pittet et al., 1996;Diaz et al., 2004).IAC, as a clean-up system for OTA (at concentration > 1.2 ng/g), is officially recommended by the Association of Official Analytical Chemists (Entwisle et al., 2001;Ahmed et al., 2007).After washing the column with water, the prepared extract is usually diluted in phosphate buffered saline (PBS) and then run through the column at an appropriate flow rate; the column is then rinsed with the PBS, dried under vacuum and the OTA content eluted with a suitable solvent such as methanol (Mounjouenpon et al., 2013).

Solid-phase clean-up methods
For some time now, various solid phase media have been employed in the extraction and sample clean-up of many different organic compounds, including OTA in coffee (Lindenmeier et al., 2011;Kathurima et al., 2012;Caprioli et al., 2013;Fan et al., 2013).The type of sorbent material used partly determines the performance of solid-phase extraction (SPE) and clean-up methods.In most cases, a sorbent with high affinity for the analyte in mobile phase is used to effect separation and clean-up.However, Lobeau et al.(2005) have developed a tandem assay column having a layer for adsorbing interfering substances in the sample.To prevent false positives in both high performance liquid chromatography (HPLC) and flow-through enzyme immunoassay (FTEIA), aminopropyl clean-up was used to restrict cross-reacting compounds while allowing the chromatographic elution of OTA (Sibanda et al., 2002).
In a different method, a polymeric multifunctional column (MFC) which possessed reversed-phase and anion exchange functionalities was used for clean-up of OTA following its extraction with 1% sodium bicarbonate (Ventura et al., 2003).High specificity of molecularly imprinted polymer (MIP) and simplicity of micro-solid phase extraction (µ-SPE) were combined in the method explained by Lee et al.(2012a).In this method, a porous membrane-protected µ-SPE column, loaded with MIP, was employed in the extraction of OTA; desorption was subsequently performed under the influence of ultrasonication.Selection of the type and amount of mobile phase or elution solvent also significantly affect the performance of this approach.Sibanda et al. (2002) have found that the use of a mixture of methanol and 5% aqueous sodium bicarbonate, both as extraction solvent and as mobile phase, achieved a high partition coefficient for the extraction and clean-up of OTA.Although the introduction of SPE has resulted in a relative decrease in solvent consumption compared to LLE procedures, there is still a need for a greener approach.This is provided by the µ-SPE method explained by Lee et al.(2012b)in which only 400 µL of methanol was used to desorb OTA from Zeolite Linde Type L sorbentunder 5 min ultrasonication,.

Detection and Quantification of ota in Coffee
A study conducted by Vecchio et al. (2012) has revealed a high frequency of OTA contamination in instant coffee samples.Out of 50 commercial coffee products tested for different mycotoxins, 36% were positive for OTA (Vaclavik et al., 2013).The daily consumption of OTA from coffee in Europe is 2-3 ng/kg bw (Mounjouenpou et al., 2013), andPittet et al.(1996) have found higher than normal figures of OTA in green coffee beans purchased in East European countries.Using a certified material and spiked samples, 69% of coffee beans, ground roasted coffee and soluble coffee products imported by Argentina were found contaminated by OTA (Vanesa and Ana, 2013).About 0.46 ng/g of this toxin was also found in sound Brazilian green coffee beans destined for exports (Gollucke et al., 2004).Considering the importance of these findings vis-à-vis health implications of OTA, and to assist in complying with stipulated maximum limits, it becomes extremely necessary to continue to deploy sensitive, fast and accurate analytical methods for its detection in coffee.
While polymerase chain reaction (PCR) has been deployed as an important tool for the identification of OTA producing fungal species (Schmidt et al., 2004;Morello et al., 2007), methods are most importantly directed toward detection of the toxin in different food items.The bulk of these methods that have been applied for the quantification of OTA in coffee are by liquid chromatography (LC) in combination with several detection systems, such as fluorescence detection (FLD) or mass spectrometry (MS).These methods can be very selective and often have low limits of detection (LOD) that are sufficient for the sensitive detection of OTA in samples.Other applications in this regard include enzyme-linked immunosorbent assay (ELISA), and deoxyribonucleic acid (DNA) and peptide-based biochips.These applications are summarized in Table 1.

Methods based on liquid chromatography/High performance liquid chromatography
Techniques of LC are applied for the separation of components of a matrix between a stationary phase and a liquid mobile phase.This liquid phase varies with the type of application.The technique is sub-divided into two main forms, column and planar, based on the form and design of the supporting material for the liquid or solid stationary phase.The liquid mobile phase is commonly driven by gravity or capillarity.In some instances, a low to moderate pressure is applied by mechanical means.Where high pressure is applied, the technique is referred to as HPLC.LC is also divided into normal and reversed-phase modes based on the polarity of the development solvent or mobile phase.The technique is commonly used for the analytical separation of mixture of organic compounds for both preparative and quantitative applications.
Methods based on the column type, in combination with various types of detection systems, were applied in the determination of OTA in coffee (Vargas et al., 2004;Sugita-Konishi et al., 2006;Bandeira et al., 2008).Ventura et al.(2003) have developed a sensitive method (LOD, 0.1 ng/g) for the determination of OTA in 20 different coffee samples from different sources using a narrow-bore reversed-phase HPLC analysis with fluorescence detector and acetonitrile/water (0.1% formic acid) (40:60) mobile phase; results were confirmed by single quadruple-MS with ESI source.The official AOAC method for detection of OTA is IA-HPLC with FLD (Ahmed et al., 2007), and this was applied in the determination of OTA in coffee at excitation wavelength (λ) of 332 nm and emission λ of 476 nm (Vargas et al., 2005).A similar method was developed and validated by Diaz et al.(2004) for the analysis of OTA in soluble coffee and green coffee samples with respective limit of quantitation (LOQ) of0.6 µg/kg and 0.3 µg/kg.Previously, a value comparable to that of soluble coffee was obtained for roasted coffee beans following a clean-up procedure with sequential phenylsilane and IAC (Entwisle et al., 2001).During roasting of coffee beans, about 90% of OTA may be degraded, and two of its degradation products, one as a result of isomerization (14-(R)-OTA) and the other as result of decarboxylation (14-decarboxy-OTA), were quantified by HPLC-MS/MS (Cramer et al., 2008).
The dearth of certified reference material (CRM) has been a major setback in the analysis of OTA.To overcome this challenge, an LC method with tandem-MS detection (LC/MS/MS) was proposed for the production of CRM that would be suitable for application in the determination of OTA in food samples (Bandeira et al., 2008).This was achieved by the development of the European Reference Material for the determination of OTA in roasted coffee (ERM-BD475) (Koch et al., 2011).Another LC/MS/MS was described for OTA determination in coffee using a SynergiHydro C 18 column, a mixture of 0.05% of trifluoroacetic acid and methanol (20:80, v/v)as mobile phase, and Selective Reaction Mode with positive electrospray ionization (SRM-ESI+) for detection (Bandeira et al., 2012a); for the quantification of OTA, [M+H] + m/z 239 and m/z 358, fragmentation productsof a pseudo-molecular precursor ion, [M+H] + m/z 404, were used.Argon and high purity nitrogen were employed as collision gas and nebulizer gas, respectively, and matrix-matched calibration curve was linear between OTA concentrations of 3.0 and 23.0 ng/g (Bandeira et al., 2012b).

Thin layer chromatography
Thin layer chromatography (TLC) is an example of the planar LC in which the stationary phase material is thinly spread on a planar solid support such as glass.Development can be onedimensional or two-dimensional.Detection of components of a sample is commonly through visual inspection.Applications of TLC are routinely found in analytical determinations of organic compounds.
One-dimensional TLC method, in both normal and reversed-phase modes, was developed for OTA determination in green coffee using visual and densitometric analyses, and results were positively correlated (r > 0.99) with those obtained from HPLC analysis (Santos and Vargas, 2002).No false positive or false negative was observed in the TLC screening method described by Pittet and Royer (2002) for the detection of OTA in green coffee at control level of 10 µg/kg.Ventura et al.(2005) have developed a cheap, rapid and easy method, based on normal two-dimensional TLC, for the determination of OTA in different coffee samples from different origins; toluene-methanol-formic acid (8:2:0.03,v/v/v) and petroleum ether-ethylacetate-formic acid (8:10:1, v/v/v) were used as the first and second dimension development solvents, respectively, and OTA content was estimated by visual inspection under UV lamp at 365 nm.

Other methods
Researchers are deploying a lot of efforts and resources in the development of new methods for the detection of OTA in coffee.Experiments with ELISA and biochips are showing promising results.A method based on indirect competitive ELISA that uses a monoclonal antibody (mAb) for the detection OTA in freshly harvested coffee was described (Fujii et al., 2006).Result obtained by this method showed positive correlation to that obtained by HPLC analysis (r= 0.90), and no cross-reaction was observed between the antibodies and two analogs of OTA (OTB and OTα).Due to possibly the presence of matrix effect in ELISA, higher values of OTA were recorded compared to HPLC results, with the highest calculated ELISA/HPLC values for green coffee, roasted coffee and instant coffee as 1.46, 1.11, and 1.82, respectively.In order to match results of ELISA with findings from stable isotope dilution assay (SIDA), therefore, IA chromatography was performed as an extract clean-up before ELISA (Lindenmeier et al., 2011). Sibanda et al.(2001) had earlier developed an FTEIA-based method which did not display any false positives or negatives during screening trials performed on green coffee bean samples.
Combining a flow-through reagent, a stand-alone automated Munich Chip Reader 3 (MCR3) platform, and chemiluminescence detection, a fast method of indirect competitive immunoassay was developed by Sauceda-Friebe et al. (2011)for the screening of OTA in green coffee extracts.The biochip was constructed by contact spotting immobilization of a peptide-functionalized OTA analog on a glass plate.Recently, Mun et al. (2013) have described a method with the shortest reported total assay time of 10 min for the detection of OTA in coffee.In this method called chemiluminescence resonance energy transfer (CRET), a biosensor was constructed using a singlestranded DNA, and this can detect as low as 0.5 parts per billion (ppb) concentration of OTA in coffee without cross reaction to other mycotoxins.A highly stable colorimetric aptamer sensorbased method was described for the detection of OTA at a very low concentration of about 1nM (Lee et al., 2014).This detection limit could be further lowered to 0.75 pM by using mythelene blue as a redox indicator for a two-level cascaded signal amplification strategy (Yang et al., 2014).Apart from very low detection limits, another advantage of using such specific aptamers is their high selectivity for OTA to the detriment of its analogs such as Nacetyl-l-phenylalanine and zearalenone (Lv et al., 2014).

CONCLUSIONS
OTA is an important mycotoxin produced, as a secondary metabolite, by primarily two genera of fungus, Aspergillus and Penicillium.Research findings have indicated its nephrotoxicity and hepatotoxicity among other deleterious effects.Occurrence of this toxin in coffee, a world-acclaimed beverage and a source of important chemicals, is a serious source of concern to both producers of coffee beans and to consumers of coffee products alike.This has prompted the European Commission to stipulate limits for the level of OTA in roasted coffee beans and in instant coffee.To assist in abiding by these regulatory codes, different analytical methods, including chromatographic applications, and peptide and DNA biochips, have been developed and applied to the determination of OTA in coffee products.There is, however, a continuous need for improvement in these methods in order to ultimately cut down the total analysis time and to improve sensitivity of determination.This measure will aid in safeguarding consumer health and ensuring quality of coffee as an important stimulant and a good source of bioactive compounds with many important health benefits.

Table 1 :
Summary of analytical methods for the determination of ochratoxin A in coffee.