Food Additives and Contaminants: Part B Vol. 1, No. 1, July 2008, 51–57
Survey on ochratoxin A in Indian green coffee destined for export T.N. Gopinandhana*, G.S. Kannanb, P. Panneerselvamc, K. Velmourouganed, Y. Raghuramulue and Jayaramae a
Analytical Laboratory, Coffee Board, No. 1 Dr. B.R. Ambedkar Veedhi, Bangalore-560 001, Karnataka, India; bFaculty of Agriculture and Animal Husbandry, Gandhigram Rural University, Gandhigram-624 302, Dindigul, Tamilnadu, India; cDivision of Soil Science and Agricultural Chemistry, Indian Institute of Horticultural Research, Bangalore, India; dCrop Production Division, Central Institute for Cotton Research (CICR), Indian Council of Agricultural Research, Post Box No. 2, Shankar Nagar, Nagpur, India; eCentral Coffee Research Institute, Coffee Research Station, Chikmagalur-577 117, Karnataka, India (Received 7 October 2007; final version received 29 January 2008) Ochratoxin A (OTA) is a toxic metabolite, produced by Aspergillus spp. and Penicillium verrucosum, that is nephrotoxic and possibly carcinogenic to humans. The aim of this study was to evaluate OTA contamination in batches of green coffee destined for export. Analysis of 80 green coffee samples indicated that, although a high incidence (74%) of OTA contamination (0.2–13.5 ng g1) was recorded, the overall mean OTA level (2.17 2.45 ng g1) was low. The highest recorded OTA concentration was 13.5 ng g1 in a robusta cherry sample and only five samples had OTA above 5 ng g1 level. The mean OTA level was higher in cherry (range: 1.63 0.97–4.8 3.90) than parchment (0.56 0.35–1.10 0.28), indicating a correlation between processing method and OTA contamination. Keywords: coffee; ochratoxin A; Aspergillus; high performance liquid chromatography
Introduction Ochratoxin A (OTA) is an extra-cellular mycotoxin (Filternborg et al. 1983) produced by several toxigenic species of two common fungal genera, Aspergillus and Penicillium. OTA has been shown to be produced by Aspergillus ochraceus and related species belonging to section circumdati (Hesseltine et al. 1972; Varga et al. 1996), A. carbonarius, A. niger in section nigri (Abarca et al. 1994; Varga et al. 1996; Heenan et al. 1998) and Penicillium verrucosum (Pitt 1987). OTA has been extensively documented as a global contaminant of a wide variety of commodities, including cereals (Chelkowski et al. 1983), figs (Steiner et al. 1993), beer (Guldborg 1997), dried vine fruit (MacDonald et al. 1999), quinces (Yashpal and Geeta 1999), cocoa (Matissek and Raters 2000), spices (Thirumala et al. 2000), liquorice (Majerus et al. 2000), grape juice (Majerus et al. 2001), wine (Otteneder and Majerus 2000) and coriander (Celeste et al. 2006). The first report on the occurrence of OTA in coffee appeared in 1974 (Levi et al. 1974). Since then, several reports have confirmed the presence of OTA in green coffee beans (Tsubouchi et al. 1987; Nakajima et al. 1997; Romani et al. 2000; de Moraes and Luchese 2003), roasted coffee (Studer-Rohr et al. 1995; Leoni et al. 2000) and instant coffee (Patel et al. 1997; Lombaert et al. 2002). OTA has also been detected in *Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2008 Taylor & Francis DOI: 10.1080/19393210802236984 http://www.informaworld.com
the final coffee brew prepared under normal methods (van der Stegen et al. 1997; Perez de Obamos et al. 2005). OTA is the main mycotoxin reported in coffee (Le-Bars and Le Bars 2000) with the contamination level ranging from 0.1 to 80 ng g1 in green coffee (Nakajima et al. 1997). As far as coffee is concerned, three Aspergilli, Aspergillus ochraceus, A. carbonarius and A. niger, have been reported to be responsible for OTA accumulation (Taniwaki et al. 1999; Bayman and Baker 2006). However, A. ochraceus is believed to be the major source of OTA in green coffee (Frank 1999). Several studies have shown a high occurrence of A. ochraceus isolates in green coffee (Taniwaki et al. 2003) and a high percentage (75–90%) of isolates with a capacity to produce OTA has been detected (Taniwaki et al. 2003). Extensive sampling of green coffee from all origins and both types of coffees (arabica and robusta) has shown that OTA contamination may be more frequent in some areas but no producing country is entirely free from contamination (Taniwaki 2006). In India, coffee is grown mainly in the southern states of Karnataka, Kerala and Tamilnadu. Karnataka accounts for 59% of the planted area (29% arabica and 30% robusta) and 72% of Indian production. Kerala with 22% (1% arabica and 21% robusta) and Tamilnadu with 8% (6.5% arabica and
52
T.N. Gopinandhan et al.
1.5% robusta) of planted area accounts for 21 and 7% of Indian coffee production, respectively. There are 60 licensed curing factories located in different parts of India; 40 of them are in Karnataka, 13 in Tamilnadu and six are in Kerala (Anonymous 2006). In India, 75% of arabica and 15% of robusta are processed by the wet method. While dry processing is predominately followed in processing India’s robusta and a proportion of arabica coffee. With the wet method, only ripe fruits are passed through a pulping machine, which removes the fruit skin. Later, the mucilage surrounding the bean is removed either by natural fermentation or by aquawashers. After removal of the mucilage, the coffee beans are subjected to sun-drying for 6–7 days by spreading them evenly on drying yards. The coffee thus prepared is called parchment coffee. In contrast, the dry method is relatively simple. The harvested fruits are subjected to sun-drying by spreading them on drying yards for 12–15 days. Coffee dried by this method is called cherry coffee (Anonymous 2003). Although surveys of OTA in green coffee from several origins have been published, only a few studies have reported its occurrence in green coffee samples of Asian origin with a limited number of samples from India (Nakajima et al. 1997; Romani et al. 2000; Yani 2004; Pardo et al. 2004; Perez de Obanos et al. 2006; MAFF 2006). Furthermore, data on the presence of OTA in Indian coffees are limited and, to our knowledge, only two studies (Ramesh and Vasanthi 2005; Gopinandhan et al. 2007) have recorded OTA contamination in coffee samples of Indian origin. Ramesh and Vasanthi (2005) examined 158 green coffee samples collected from different coffee-growing
regions in Karnataka state and reported that OTA was present in 60% of the samples analyzed, but the majority of the samples (88%) contained OTA below 5 ng g1. In our previous study (Gopinandhan et al. 2007), a total of 129 green coffee samples, randomly collected from curers, traders and local auction platforms, were tested for OTA. The results indicated that 81% of samples had OTA level below 5 ng g1. Only three samples had OTA above 5 ng g1and the highest level of OTA was 11.7 ng g1 in a robusta cherry sample. To date, there are no reports on OTA levels on Indian green coffee exclusively destined for export. With this objective in mind, the present survey was designed to assess OTA level in green coffee samples intended for export and collected from various curing factories located in different parts of the country.
Material and methods For the present study, 48 green coffee samples were collected from four curing works in each of the Chikmagalur, Hassan and Kodagu districts in Karnataka state. Four green coffees (one arabica parchment, one arabica cherry, one robusta parchment and one robusta cherry) were taken from each curing works, making a total of 48 samples (i.e. 4 samples 12 curing works). Seventeen green coffees (two robusta parchment and 15 robusta cherry) were collected from two curing works in two districts of Kerala state and 15 coffees (10 arabica parchment and five arabica cherry) from two curing works in two districts of Tamilnadu state. Details of sampling locations and sample types collected are given in Table 1.
Table 1. Composition of samples according to location and coffee type. Location
No. of curing works
No. of sample
Type
Karnataka State Chikmagalur
4*
16
Hassan Coorg
4* 4*
16 16
Arabica parchment (4 samples) Arabica Cherry (4 samples) Robusta parchment (4 samples) Robusta cherry (4 samples) As above As above
Kerala State Wayand
1
7
Palkad
1
10
Tamilnadu State Dindigal
1
8
Salem
1
7
Total
16
80
Robusta parchment (2 samples) Robusta cherry (5 samples) Robusta cherry Arabica Arabica Arabica Arabica
parchment (5) cherry (3) parchment (4) cherry (3)
*From each curing works, four samples (one arabica parchment, one arabica cherry, one robusta parchment and one robusta cherry) were collected, making a total of 16 samples.
Food Additives and Contaminants: Part B
53
Sampling the green coffee
Extraction and clean-up
To study the natural distribution of OTA in batches of export coffee, a total of 50 bags of 60 kg each were randomly selected from the identified lot. From each bag, a composite sample of 100 g of beans was taken, pooled and 1 kg of green coffee taken from this pool. Samples were drawn from all sides (center, top, bottom, right side and left side) of the bags using a sleeve-type seed trier. In Karnataka state, one arabica parchment, one arabica cherry, one robusta parchment and one robusta cherry (1 1 kg each) were collected from each curing works. In Kerala state, two robusta parchment (2 1 kg) and five robusta cherry (5 1 kg) samples from a curing works in the Wynad district and 10 robusta cherry (10 1 kg) samples from a curing works in the Palkad district were collected. In Tamilnadu state, five arabica parchment (5 1 kg) and three arabica cherry (3 1 kg) from a curing works in the Dindigul district and four arabica parchment (4 1 kg) and three arabica cherry (3 1 kg) from a curing works in the Salem district were collected. Samples were collected during the 2006–2007 harvest seasons. Arabica and robusta samples were collected during January and April 2007, respectively.
OTA was determined using the immunoaffinity column clean-up procedure of Pittet et al. (1996). In brief, OTA was extracted from 25 g of finely milled green coffee with 500 ml of a methanol/3% aqueous sodium hydrogen carbonate (1:1, v/v) solution. Extraction was carried out for 3 min in a 500-ml glass jar using a high-speed blender. A 50-ml aliquot of sample extract first centrifuged at 5000 rpm for 10 min and the supernatant filtered through a GF/B microfiber filter under vacuum. Then, 4 ml of filtrate was transferred to an amber-colored, graduated cylinder and diluted to 100 ml with phosphate-buffered saline. All the diluted extract was applied to an immunoaffinity column at a flow rate of 2–3 ml min1. After washing the column with 10 ml of Milli-Q water, OTA was eluted with 4 ml of HPLC-grade methanol. To ensure the complete removal of bound toxin, the methanol was left in column for at least 3 min by reversing the flow of methanol (back flushing) two or three times. The eluate was then evaporated to dryness under a stream of nitrogen at 40 C and the residue was redissolved in 500 ml of HPLC mobile-phase.
Sample preparation The whole green coffee sample (1 kg) was ground in a Romer analytical sample mill. To overcome the problem of the non-homogeneity of OTA distribution, the ground green coffee was mixed thoroughly and two 25-g aliquot taken from the mixed, homogenized sample for OTA analysis.
Chromatographic conditions for OTA analysis OTA in green coffee was quantitated by reverse-phase HPLC with fluorescence detection (Shimadzu–LC 10A Series). The HPLC analysis was performed using 45% acetonitrile–55% 4 mM sodium acetate/acetic acid (19:1 v/v) on a Spherisorb ODS II of 5 mm, 250 4.6 mm I.D., connected to a ODS Hypersil precolumn of 5 mm, 25 4.6 mm I.D. Separation was performed at ambient temperature and a flow rate of 1 ml min1; excitation maximum was 330 nm and emission 470 nm.
Chemical and reagents The OTA and phosphate-buffered saline tablet were obtained from Sigma–Aldrich (Bangalore, India). Analytical grade methanol, sodium acetate trihydrate, sodium hydrogen carbonate and HPLCgrade methanol, acetonitrile, glacial acetic acid and toluene were purchased from S.D. Fine Chemicals (Bangalore, India). The Ochratest immunoaffinity column (Vicam) was purchased from M/s. Special Instrument Consortium (Chennai, India). Water was obtained from a Milli-Q water purification unit. All solvents were filtered through a 0.45 mm nylon filter and degassed by sonication in a water bath. A stock solution of OTA was prepared by dissolving the crystalline material in HPLC-grade toluene/acetic acid (99:1, v/v) and the concentration was verified spectrophotometrically (Bacha et al. 1988).
Calibration curve The calibration curve was obtained using the linearsquares regression procedure of the peak area versus concentration. Linearity for OTA in the working standard solutions at three determinations of six concentrations levels (0.1, 1, 5, 10, 20 and 30 ng g1) was good, as shown by the correlation coefficient (r2) of 0.9913.
Recovery test Recovery testing were performed in triplicates by spiking standard solution at 5 and 10 ng g1 into OTA-free samples and analysis as described for samples. The average recovery was 89 and 82%, respectively, for the two spiking levels. The lower
54
T.N. Gopinandhan et al.
Table 2. OTA contamination in green coffee samples from Karnataka State. OTA (ng g1) Location Chikmagalur
Hassan
Coorg
Type
No. of samples
Frequency
Range
Mean (S.D.)
Arabica parchment Arabica cherry Robusta parchment Robusta cherry Arabica parchment Arabica cherry Robusta parchment Robusta cherry Arabica parchment Arabica cherry Robusta parchment Robusta cherry
4 4 4 4 4 4 4 4 4 4 4 4
2 4 3 4 0 2 2 4 1 3 2 4
0.2 – 0.9 0.5 – 2.3 0.5 – 1.1 0.9 – 2.3 0 1.1 – 1.8 0.5 – 0.8 0.7 – 4.5 0.6 0.9 – 3.6 0.8 – 1.4 0.5 – 2.9
0.55 1.25 0.83 1.47 0 1.45 0.65 2.07 0.6 2.26 1.10 1.50
( 0.49) ( 0.80) ( 0.30) ( 0.60) ( 0.49) ( 0.21) ( 1.67) ( 1.35) ( 0.42) ( 1.05)
Table 3. OTA contamination in green coffee sample from Kerala and Tamilnadu States. OTA (ng g1) Location Kerala Wayand Palkad Tamilnadu Dindigul Salem
Type
No. of samples
Frequency
Range
Mean (S.D.)
Robusta parchment Robusta cherry Robusta cherry
2 5 10
2 5 8
0.9 – 1.3 0.7 – 7.8 0.5 – 13.5
1.10 ( 0.28) 3.46 ( 2.82) 5.63 ( 4.43)
Arabica parchment Arabica cherry Arabica parchment Arabica cherry
5 3 4 3
3 3 4 3
0.4 – 1.3 1.2 – 4.3 0.4 – 1.4 0.5 – 3.7
0.86 3.03 0.97 1.93
Table 4. Mean OTA levels in green coffees of different origins.
Type
Mean OTA (ng g1, S.D.)
Arabica parchment Arabica cherry Robusta parchment Robusta cherry Robusta parchment Robusta cherry Arabica parchment Arabica cherry
0.56 ( 0.35) 1.63 ( 0.97) 0.85 ( 0.32) 1.66 ( 1.11) 1.10 ( 0.28) 4.80 ( 3.90) 0.92( 0.40) 2.46 ( 1.57)
Origin Karnataka
Kerala Tamilnadu
limit of detection (LOD), based on a signal-to-noise ratio of 3, was 0.1 ng g1.
Results and discussion OTA levels in green coffee samples collected from different export batches are summarized in Tables 2 and 3. Of the 80 green coffee samples, 74% contained detectable amount of OTA with concentrations
( 0.45) ( 1.62) ( 0.43) ( 1.62)
ranging from 0.2 to 13.5 ng g1. The highest concentrations of OTA were 13.5 ng g1 in a robusta cherry sample, followed by 4.3 and 1.4 ng g1 in arabica cherry and parchment of both arabica and robusta samples, respectively. The frequency of OTA in different types of coffee were 10 of 21 (48%) in arabica parchment, 15 of 18 (83%) in arabica cherry, 9 of 14 (64%) in robusta parchment and 25 of 27 (93%) in robusta cherry samples. Similarly, the frequency of OTA in coffee from different states was 65% in Karnataka, with a very low average of 1.37 ng g1. In contrast, the frequency of OTA was high in green coffees from Kerala (88%) and Tamilnadu (87%), with mean values of 4.3 and 1.64 ng g1, respectively. The mean OTA level was higher in cherry (range: 1.63 0.97–4.8 3.90) than parchment (0.56 0.35– 1.10 0.28) samples (Table 4). The frequency distribution of OTA contamination for green coffee samples is shown in Figure 1. Of the 80 samples examined, 21 were below the detection limit of 0.1 ng g1 and over 90% of samples were in the range 0.2–5 ng g1. Only five samples had OTA level above 5 ng g1 and all were robusta cherry samples from Kerala state.
Food Additives and Contaminants: Part B 25
23 21
Sample Number
20
18
15
10 5
5
0
< 0.1
0.1-1.0
1.1-2.0
4
4
5
2.1-3.0 3.1-4.0 4.1-5.0 5.0-15.0
OTA content range (ng g−1)
Figure 1. Frequency distribution of OTA in green coffee samples. Numbers on the histogram indicate the total number of samples in each range.
Table 5. Statistical analysis of influence of processing on the level of OTA contamination in green coffee beans. Type of coffees Arabica parchment Robusta parchment Arabica cherry Robusta cherry S.E. CD (p ¼ 0.05)
Overall mean OTA (ng g1) 0.82a 0.91a 1.97b 3.30c 0.105 0.230
Data marked with same superscript are not significantly different at a p value of 0.05 level.
Post-harvest practices, particularly drying, affect OTA contamination (de Moraes and Luchese 2003). The high occurrence of OTA in coffee from Kerala state could be linked to the post-harvest practices followed in that region. The majority of coffee-growers in Kerala, dry their coffee on low-quality drying surfaces, such as bare or cow dung-plastered soil (Reddy 2000), thereby, affecting the overall quality. This is in accordance with the findings of de Moraes and Luchese (2003) who reported that coffee dried on a compacted soil floor had a higher level of OTA contamination (563 ng g1) compared to samples dried on a cement floor (10 ng g1). Statistical analyses to compare OTA contamination between sample types (parchment versus cherry), show that mean OTA concentration in cherry samples (1.97–3.30 ng g1) was significantly higher (p ¼ 0.05) than parchment (0.82–0.91 ng g1) samples (Table 5), indicating processing had a significant influence on accumulation of OTA in green coffee beans. This result is in good agreement with the findings of Ramesh and Vasanthi (2005) who reported that cherry samples had higher levels of OTA contamination than parchment coffee. Frank (1999)
55
reported that parchment coffee is less susceptible to infection by Aspergillus species and OTA contamination. Since, the fruit pulp is an excellent substrate for the growth of OTA-producing strains (Joosten et al. 2001), its removal during wet processing eliminates a suitable substrate for proliferation of moulds. In the present study, the level of OTA contamination was in the range 0.2–13.5 ng g1 with an overall mean OTA level of 2.17 2.45 ng g1. In comparison, Pardo et al. (2004) analyzed 14 green coffee samples of Asian origin and reported OTA concentrations ranging from 1.6 to 31.5 ng g1 with an overall mean of 6.0 7.9 ng g1. Similarly, Romani et al. (2000) showed that 11 of 18 Asian green coffee samples were positive for OTA contamination ranging from 0.2 to 4.9 ng g1 with overall mean of 1.1 1.4 ng g1. These two reports concluded that green coffee samples of African origin had significantly higher levels of OTA than those from America or Asia. In conclusion, this survey provided valuable data on the occurrence of OTA in a large numbers of samples of green coffee from batches destined for export. Although the incidence of OTA contamination was high (74%), the overall mean level (2.17 2.45 ng g1) was low. Furthermore, the level of OTA in the majority of green coffees (94%) analyzed were below 5 ng g1, which complies with current regulatory limits. Nevertheless, an effective and integrated management system in the form of a Hazard Analysis CriticalControl Point (HACCP) throughout the entire production chain may help in reducing fungal and OTA contamination and improve the overall quality of green coffee beans
References Abarca ML, Bragulat MR, Castella G, Cabanes FJ. 1994. Ochratoxin A production by strains of Aspergillus niger var. niger. Appl Environ Microbiol. 60:2625–2650. Anonymous. 2003. Harvest and post harvest practices for quality coffee preparation. Coffee guide. Coffee Board of India: Bangalore. p. 100–109. Anonymous. 2006. Database on coffee. Economic and market intelligence unit. Coffee Board of India: Bangalore. p. 1–10, July/September 2006. Bacha H, Hadidane R, Creepy EE, Regnault C, Ellouze F, Dirheimer G. 1988. Monitoring and identification of fungal toxins in food products, animal feed and cereals. J Stored Prod Res. 24:199–206. Bayman P, Baker JL. 2006. Ochratoxins: A global perspective. Mycopathologia 162:215–223. Celeste ML, Lurdes B, Pena A, Noronha Silveira I. 2006. Determination of ochratoxin A in coriander by HPLC/ fluorescence deflection. Quim Nova 29:436–439. Chelkowski J, Trojanowska K, Wiewiorowska M. 1983. Microbiological evaluation of cereal grain quality, connected with mycotoxin occurrence. Nahrung. 27:311–318.
56
T.N. Gopinandhan et al.
De Moraes MHP, Luchese RH. 2003. Ochratoxin A on green coffee: influence of harvest and drying processing procedures. J Agric Food Chem. 51: 5824–5828. Filternborg O, Frisvad JC, Svendsen JA. 1983. Simple screening method for molds producing intracellular mycotoxins in pure cultures. Appl Environ Microbiol. 45:581–585. Frank JM. 1999. HACCP and its mycotoxin management control potential: Ochratoxin A in coffee production. Proceeding of the 7th International Committee on Food Microbiology and Hygiene held at Veldhoven, The Netherlands; p. 122. Gopinandhan TN, Velmourougane K, Panneerselvam P, Keshamma E, Raghuramulu Y. 2007. Occurrence of ochratoxin A in green and commercial coffee samples. J Food Sci Technol. 44:247–249. Guldborg M. 1997. Ochratoxin A in Danish beer. Brygmesteren. 54:16–17. Heenan CN, Chaw KJ, Pitt JI. 1998. Ochratoxin A production by Aspergillus carbonarius and A. niger isolates and detection using coconut cream agar. J Food Mycol. 1:67–72. Hesseltine CW, Vandegraft EE, Fennell DJ, Smith MJ, Shotwell OL. 1972. Aspergilli as ochratoxin producers. Mycologia. 64:539–550. Joosten HMLJ, Goetz J, Pittet A, Schellenberg M, Bucheli P. 2001. Production of ochratoxin A by Aspergillus carbonarius on coffee cherries. Int J Food Microbiol. 65:39–44. Le-Bars J, Le-Bars P. 2000. Mycotoxigenesis in grains: application to mycotoxic prevention in coffee. Coffee biotechnology and quality. Kluwer Academic: Dordrecht. p. 355–368. Leoni LAB, Valente Soares LM, Oliveira PLC. 2000. Ochratoxin A in Brazilian roasted and instant coffees. Food Addit Contam. 17:867–870. Levi CP, Trenk HL, Mohr HK. 1974. The occurrence of ochratoxin A in green coffee beans. J Assoc Off Anal Chem. 57:866–870. Lombaert GA, Pellaers P, Chettiar M, Lavalee D, Scott PM, Lau BPY. 2002. Survey of Canadian retail coffees for ochratoxin A. Food Addit Contam. 19:869–877. Macdonald S, Wilson P, Barnes K, Damant A, Massey R, Mortby E, Shephered MJ. 1999. Ochratoxin A in dried wine fruit: method development and survey. Food Addit Contam. 16:253–360. Majerus P, Bresch H, Otteneder H. 2001. Ochratoxin A in wines, fruit juices and seasonings. Arch Lebensmittelhyg 51:95–97. Majerus P, Max M, Klaffke H. 2000. Ochratoxin A in Sussholz, Lakritze und daraus hergestellten Erzeugnissen. Deutsche Lebensmittel-Rundschau 96:451–454. Matissek R, Rater M. 2000. Ochratoxin A in cocoa and human health aspects. Proceedings of the 13th International Cocoa Research Conference; Kota Kinahalu, Malaysia; 2000 Oct 9–14; p. 9. MAFF. 1996. Surveillance of ochratoxin A in green (unroasted) coffee beans. Food Surveillance Information Sheet 80. Ministry of Agriculture, Fisheries and Food, London.
Nakajima M, Tsubouchi H, Miyabe M, Ueno Y. 1997. Survey of aflatoxin-B and ochratoxin-A in commercial green coffee beans by HPLC. Food Agric Immunol. 9:77–83. Otteneder H, Majerus P. 2000. Occurrence of ochratoxin A in wines: influence of the type of wine and its geographical origin. Food Addit Contam. 17:793–798. Pardo E, Marim S, Ramos AJ, Sanchis V. 2004. Occurrence of ochratoxigenic fungi and ochratoxin A in green coffee from different origins. Food Sci Technol Int. 10:45–50. Patel S, Hazel CM, Wintertone AGM, Gleade AE. 1997. Survey of ochratoxin A in UK retail coffees. Food Addit Contam. 14:217–222. Perez de Obanos A, Gonzalez-Penas E, Lopez de Cerain A. 2005. Influence of roasting and brew preparation on the ochratoxin A content in coffee infusion. Food Addit Contam. 22:463–471. Pitt JI. 1987. Penicillium virdicatum, Penicillium verrucosum and production of ochratoxin A. Appl Environ Microbiol. 53:266–269. Pittet A, Tornare D, Huggett A, Viani R. 1996. Liquid chromatographic determination of ochratoxin-A in pure and adulterated soluble coffee using an immunoaffinity column clean up procedure. J Agric Food Chem. 44:3564–3569. Ramesh VB, Vasanthi S. 2005. Natural occurrence of ochratoxin in Indian coffee. Indian J Nutr Diet. 42:106–113. Reddy AGS. 2000. Production and productivity of coffee in Kerala: a perspective. Indian Coffee 2: 23–24. Romani S, Sacchetti G, Chaves Lopez C, Pinnavaia GG, Dalla Roas M. 2000. Screening on the occurrence of ochratoxin A in green coffee beans of different origins and types. J Agric Food Chem. 48:3616–3619. Steiner W, Brunschweiler K, Leimbacher E, Schneider R. 1993. Aflatoxine B1 and G1, Cyclopiazonsaure, Kojisaure und ochratoxin A in Trockenfeigen im BGY– Fluoreszenz. Mitteilungen aus dem Gebiete dem Lebensmitteluntersuchung und Hygiene. 84:523–536. Studer-Rohr I, Dietrich D.R. Schlatter J, Schlatter C. 1995. The occurrence of ochratoxin A in coffee. Food Chem Toxicol. 33:341–355. Taniwaki MH. 2006. An update on ochratoxigenic fungi and ochratoxin A in coffee. In: Hocking, AD. Pitt, JI, Samson RA, Thrane U. editors. Advances in Food Mycology, New York: Springer. p. 189. Taniwaki MH, Pitt J.I, Urbano, GR Teixeria AA, Leitao MFF. 1999. Fungi producing ochratoxin A in coffee. In: Proceeding of the 18th ASIC Coffee Conference; Helsinki, Finland; p. 239. Taniwaki MH, Pitt JI, Teixeria AA, Iamanaka BT. 2003. The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods. Int J Food Microbiol. 82:173–179. Thirumala DK, Mayo MA, Gopal R, Reddy SV, Delfosse P, Reddy DVR. 2000. Production of polyclonal antibodies against ochratoxin A and its detection in chillies by ELISA. J Agric Food Chem. 48:5079–5082. Tsubouchi H, Yamamoto K, Hisada K, Sakabe Y, Udagawa S. 1987. Effect of roasting on ochratoxin A level in green coffee beans inoculation with Aspergillus ochraceus. Mycopathologia 97:111–115.
Food Additives and Contaminants: Part B Vander Stegen GVD, Jorissen U, Pittet A, Saccon M, Steiner W, Vincenzi M, Winkler M, Zapp J, Schlatter C. 1997. Screening of European coffee final products for occurrence of ochratoxin A. Food Addit Contam. 14:211–216. Varga J, Kevi F, Rinyu E, Teren J, Kozakiewicz Z. 1996. Ochratoxin production by Aspergillus species. Appl Environ Microbiol. 60:4461–4464.
57
Yani A. 2004. Fungal infection and ochratoxin contamination in green coffee beans collected from farmers and collectors in Bengkulu province. Thesis. Bogor: Bogor Agricultural University. Yashpal S, Geeta S. 1999. Natural occurrence of ochratoxin A and toxigenic Aspergillus ochraceus strains in dry fruit slices of quinces from Jammu and Kashmir. Indian Phytopathol. 52:148–150.
Survey on ochratoxin A in Indian green coffee destined for export T.N. Gopinandhana*, G.S. Kannanb, P. Panneerselvamc, K. Velmourouganed, Y. Raghuramulue and Jayaramae a
Analytical Laboratory, Coffee Board, No. 1 Dr. B.R. Ambedkar Veedhi, Bangalore-560 001, Karnataka, India; bFaculty of Agriculture and Animal Husbandry, Gandhigram Rural University, Gandhigram-624 302, Dindigul, Tamilnadu, India; cDivision of Soil Science and Agricultural Chemistry, Indian Institute of Horticultural Research, Bangalore, India; dCrop Production Division, Central Institute for Cotton Research (CICR), Indian Council of Agricultural Research, Post Box No. 2, Shankar Nagar, Nagpur, India; eCentral Coffee Research Institute, Coffee Research Station, Chikmagalur-577 117, Karnataka, India (Received 7 October 2007; final version received 29 January 2008) Ochratoxin A (OTA) is a toxic metabolite, produced by Aspergillus spp. and Penicillium verrucosum, that is nephrotoxic and possibly carcinogenic to humans. The aim of this study was to evaluate OTA contamination in batches of green coffee destined for export. Analysis of 80 green coffee samples indicated that, although a high incidence (74%) of OTA contamination (0.2–13.5 ng g1) was recorded, the overall mean OTA level (2.17 2.45 ng g1) was low. The highest recorded OTA concentration was 13.5 ng g1 in a robusta cherry sample and only five samples had OTA above 5 ng g1 level. The mean OTA level was higher in cherry (range: 1.63 0.97–4.8 3.90) than parchment (0.56 0.35–1.10 0.28), indicating a correlation between processing method and OTA contamination. Keywords: coffee; ochratoxin A; Aspergillus; high performance liquid chromatography
Introduction Ochratoxin A (OTA) is an extra-cellular mycotoxin (Filternborg et al. 1983) produced by several toxigenic species of two common fungal genera, Aspergillus and Penicillium. OTA has been shown to be produced by Aspergillus ochraceus and related species belonging to section circumdati (Hesseltine et al. 1972; Varga et al. 1996), A. carbonarius, A. niger in section nigri (Abarca et al. 1994; Varga et al. 1996; Heenan et al. 1998) and Penicillium verrucosum (Pitt 1987). OTA has been extensively documented as a global contaminant of a wide variety of commodities, including cereals (Chelkowski et al. 1983), figs (Steiner et al. 1993), beer (Guldborg 1997), dried vine fruit (MacDonald et al. 1999), quinces (Yashpal and Geeta 1999), cocoa (Matissek and Raters 2000), spices (Thirumala et al. 2000), liquorice (Majerus et al. 2000), grape juice (Majerus et al. 2001), wine (Otteneder and Majerus 2000) and coriander (Celeste et al. 2006). The first report on the occurrence of OTA in coffee appeared in 1974 (Levi et al. 1974). Since then, several reports have confirmed the presence of OTA in green coffee beans (Tsubouchi et al. 1987; Nakajima et al. 1997; Romani et al. 2000; de Moraes and Luchese 2003), roasted coffee (Studer-Rohr et al. 1995; Leoni et al. 2000) and instant coffee (Patel et al. 1997; Lombaert et al. 2002). OTA has also been detected in *Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2008 Taylor & Francis DOI: 10.1080/19393210802236984 http://www.informaworld.com
the final coffee brew prepared under normal methods (van der Stegen et al. 1997; Perez de Obamos et al. 2005). OTA is the main mycotoxin reported in coffee (Le-Bars and Le Bars 2000) with the contamination level ranging from 0.1 to 80 ng g1 in green coffee (Nakajima et al. 1997). As far as coffee is concerned, three Aspergilli, Aspergillus ochraceus, A. carbonarius and A. niger, have been reported to be responsible for OTA accumulation (Taniwaki et al. 1999; Bayman and Baker 2006). However, A. ochraceus is believed to be the major source of OTA in green coffee (Frank 1999). Several studies have shown a high occurrence of A. ochraceus isolates in green coffee (Taniwaki et al. 2003) and a high percentage (75–90%) of isolates with a capacity to produce OTA has been detected (Taniwaki et al. 2003). Extensive sampling of green coffee from all origins and both types of coffees (arabica and robusta) has shown that OTA contamination may be more frequent in some areas but no producing country is entirely free from contamination (Taniwaki 2006). In India, coffee is grown mainly in the southern states of Karnataka, Kerala and Tamilnadu. Karnataka accounts for 59% of the planted area (29% arabica and 30% robusta) and 72% of Indian production. Kerala with 22% (1% arabica and 21% robusta) and Tamilnadu with 8% (6.5% arabica and
52
T.N. Gopinandhan et al.
1.5% robusta) of planted area accounts for 21 and 7% of Indian coffee production, respectively. There are 60 licensed curing factories located in different parts of India; 40 of them are in Karnataka, 13 in Tamilnadu and six are in Kerala (Anonymous 2006). In India, 75% of arabica and 15% of robusta are processed by the wet method. While dry processing is predominately followed in processing India’s robusta and a proportion of arabica coffee. With the wet method, only ripe fruits are passed through a pulping machine, which removes the fruit skin. Later, the mucilage surrounding the bean is removed either by natural fermentation or by aquawashers. After removal of the mucilage, the coffee beans are subjected to sun-drying for 6–7 days by spreading them evenly on drying yards. The coffee thus prepared is called parchment coffee. In contrast, the dry method is relatively simple. The harvested fruits are subjected to sun-drying by spreading them on drying yards for 12–15 days. Coffee dried by this method is called cherry coffee (Anonymous 2003). Although surveys of OTA in green coffee from several origins have been published, only a few studies have reported its occurrence in green coffee samples of Asian origin with a limited number of samples from India (Nakajima et al. 1997; Romani et al. 2000; Yani 2004; Pardo et al. 2004; Perez de Obanos et al. 2006; MAFF 2006). Furthermore, data on the presence of OTA in Indian coffees are limited and, to our knowledge, only two studies (Ramesh and Vasanthi 2005; Gopinandhan et al. 2007) have recorded OTA contamination in coffee samples of Indian origin. Ramesh and Vasanthi (2005) examined 158 green coffee samples collected from different coffee-growing
regions in Karnataka state and reported that OTA was present in 60% of the samples analyzed, but the majority of the samples (88%) contained OTA below 5 ng g1. In our previous study (Gopinandhan et al. 2007), a total of 129 green coffee samples, randomly collected from curers, traders and local auction platforms, were tested for OTA. The results indicated that 81% of samples had OTA level below 5 ng g1. Only three samples had OTA above 5 ng g1and the highest level of OTA was 11.7 ng g1 in a robusta cherry sample. To date, there are no reports on OTA levels on Indian green coffee exclusively destined for export. With this objective in mind, the present survey was designed to assess OTA level in green coffee samples intended for export and collected from various curing factories located in different parts of the country.
Material and methods For the present study, 48 green coffee samples were collected from four curing works in each of the Chikmagalur, Hassan and Kodagu districts in Karnataka state. Four green coffees (one arabica parchment, one arabica cherry, one robusta parchment and one robusta cherry) were taken from each curing works, making a total of 48 samples (i.e. 4 samples 12 curing works). Seventeen green coffees (two robusta parchment and 15 robusta cherry) were collected from two curing works in two districts of Kerala state and 15 coffees (10 arabica parchment and five arabica cherry) from two curing works in two districts of Tamilnadu state. Details of sampling locations and sample types collected are given in Table 1.
Table 1. Composition of samples according to location and coffee type. Location
No. of curing works
No. of sample
Type
Karnataka State Chikmagalur
4*
16
Hassan Coorg
4* 4*
16 16
Arabica parchment (4 samples) Arabica Cherry (4 samples) Robusta parchment (4 samples) Robusta cherry (4 samples) As above As above
Kerala State Wayand
1
7
Palkad
1
10
Tamilnadu State Dindigal
1
8
Salem
1
7
Total
16
80
Robusta parchment (2 samples) Robusta cherry (5 samples) Robusta cherry Arabica Arabica Arabica Arabica
parchment (5) cherry (3) parchment (4) cherry (3)
*From each curing works, four samples (one arabica parchment, one arabica cherry, one robusta parchment and one robusta cherry) were collected, making a total of 16 samples.
Food Additives and Contaminants: Part B
53
Sampling the green coffee
Extraction and clean-up
To study the natural distribution of OTA in batches of export coffee, a total of 50 bags of 60 kg each were randomly selected from the identified lot. From each bag, a composite sample of 100 g of beans was taken, pooled and 1 kg of green coffee taken from this pool. Samples were drawn from all sides (center, top, bottom, right side and left side) of the bags using a sleeve-type seed trier. In Karnataka state, one arabica parchment, one arabica cherry, one robusta parchment and one robusta cherry (1 1 kg each) were collected from each curing works. In Kerala state, two robusta parchment (2 1 kg) and five robusta cherry (5 1 kg) samples from a curing works in the Wynad district and 10 robusta cherry (10 1 kg) samples from a curing works in the Palkad district were collected. In Tamilnadu state, five arabica parchment (5 1 kg) and three arabica cherry (3 1 kg) from a curing works in the Dindigul district and four arabica parchment (4 1 kg) and three arabica cherry (3 1 kg) from a curing works in the Salem district were collected. Samples were collected during the 2006–2007 harvest seasons. Arabica and robusta samples were collected during January and April 2007, respectively.
OTA was determined using the immunoaffinity column clean-up procedure of Pittet et al. (1996). In brief, OTA was extracted from 25 g of finely milled green coffee with 500 ml of a methanol/3% aqueous sodium hydrogen carbonate (1:1, v/v) solution. Extraction was carried out for 3 min in a 500-ml glass jar using a high-speed blender. A 50-ml aliquot of sample extract first centrifuged at 5000 rpm for 10 min and the supernatant filtered through a GF/B microfiber filter under vacuum. Then, 4 ml of filtrate was transferred to an amber-colored, graduated cylinder and diluted to 100 ml with phosphate-buffered saline. All the diluted extract was applied to an immunoaffinity column at a flow rate of 2–3 ml min1. After washing the column with 10 ml of Milli-Q water, OTA was eluted with 4 ml of HPLC-grade methanol. To ensure the complete removal of bound toxin, the methanol was left in column for at least 3 min by reversing the flow of methanol (back flushing) two or three times. The eluate was then evaporated to dryness under a stream of nitrogen at 40 C and the residue was redissolved in 500 ml of HPLC mobile-phase.
Sample preparation The whole green coffee sample (1 kg) was ground in a Romer analytical sample mill. To overcome the problem of the non-homogeneity of OTA distribution, the ground green coffee was mixed thoroughly and two 25-g aliquot taken from the mixed, homogenized sample for OTA analysis.
Chromatographic conditions for OTA analysis OTA in green coffee was quantitated by reverse-phase HPLC with fluorescence detection (Shimadzu–LC 10A Series). The HPLC analysis was performed using 45% acetonitrile–55% 4 mM sodium acetate/acetic acid (19:1 v/v) on a Spherisorb ODS II of 5 mm, 250 4.6 mm I.D., connected to a ODS Hypersil precolumn of 5 mm, 25 4.6 mm I.D. Separation was performed at ambient temperature and a flow rate of 1 ml min1; excitation maximum was 330 nm and emission 470 nm.
Chemical and reagents The OTA and phosphate-buffered saline tablet were obtained from Sigma–Aldrich (Bangalore, India). Analytical grade methanol, sodium acetate trihydrate, sodium hydrogen carbonate and HPLCgrade methanol, acetonitrile, glacial acetic acid and toluene were purchased from S.D. Fine Chemicals (Bangalore, India). The Ochratest immunoaffinity column (Vicam) was purchased from M/s. Special Instrument Consortium (Chennai, India). Water was obtained from a Milli-Q water purification unit. All solvents were filtered through a 0.45 mm nylon filter and degassed by sonication in a water bath. A stock solution of OTA was prepared by dissolving the crystalline material in HPLC-grade toluene/acetic acid (99:1, v/v) and the concentration was verified spectrophotometrically (Bacha et al. 1988).
Calibration curve The calibration curve was obtained using the linearsquares regression procedure of the peak area versus concentration. Linearity for OTA in the working standard solutions at three determinations of six concentrations levels (0.1, 1, 5, 10, 20 and 30 ng g1) was good, as shown by the correlation coefficient (r2) of 0.9913.
Recovery test Recovery testing were performed in triplicates by spiking standard solution at 5 and 10 ng g1 into OTA-free samples and analysis as described for samples. The average recovery was 89 and 82%, respectively, for the two spiking levels. The lower
54
T.N. Gopinandhan et al.
Table 2. OTA contamination in green coffee samples from Karnataka State. OTA (ng g1) Location Chikmagalur
Hassan
Coorg
Type
No. of samples
Frequency
Range
Mean (S.D.)
Arabica parchment Arabica cherry Robusta parchment Robusta cherry Arabica parchment Arabica cherry Robusta parchment Robusta cherry Arabica parchment Arabica cherry Robusta parchment Robusta cherry
4 4 4 4 4 4 4 4 4 4 4 4
2 4 3 4 0 2 2 4 1 3 2 4
0.2 – 0.9 0.5 – 2.3 0.5 – 1.1 0.9 – 2.3 0 1.1 – 1.8 0.5 – 0.8 0.7 – 4.5 0.6 0.9 – 3.6 0.8 – 1.4 0.5 – 2.9
0.55 1.25 0.83 1.47 0 1.45 0.65 2.07 0.6 2.26 1.10 1.50
( 0.49) ( 0.80) ( 0.30) ( 0.60) ( 0.49) ( 0.21) ( 1.67) ( 1.35) ( 0.42) ( 1.05)
Table 3. OTA contamination in green coffee sample from Kerala and Tamilnadu States. OTA (ng g1) Location Kerala Wayand Palkad Tamilnadu Dindigul Salem
Type
No. of samples
Frequency
Range
Mean (S.D.)
Robusta parchment Robusta cherry Robusta cherry
2 5 10
2 5 8
0.9 – 1.3 0.7 – 7.8 0.5 – 13.5
1.10 ( 0.28) 3.46 ( 2.82) 5.63 ( 4.43)
Arabica parchment Arabica cherry Arabica parchment Arabica cherry
5 3 4 3
3 3 4 3
0.4 – 1.3 1.2 – 4.3 0.4 – 1.4 0.5 – 3.7
0.86 3.03 0.97 1.93
Table 4. Mean OTA levels in green coffees of different origins.
Type
Mean OTA (ng g1, S.D.)
Arabica parchment Arabica cherry Robusta parchment Robusta cherry Robusta parchment Robusta cherry Arabica parchment Arabica cherry
0.56 ( 0.35) 1.63 ( 0.97) 0.85 ( 0.32) 1.66 ( 1.11) 1.10 ( 0.28) 4.80 ( 3.90) 0.92( 0.40) 2.46 ( 1.57)
Origin Karnataka
Kerala Tamilnadu
limit of detection (LOD), based on a signal-to-noise ratio of 3, was 0.1 ng g1.
Results and discussion OTA levels in green coffee samples collected from different export batches are summarized in Tables 2 and 3. Of the 80 green coffee samples, 74% contained detectable amount of OTA with concentrations
( 0.45) ( 1.62) ( 0.43) ( 1.62)
ranging from 0.2 to 13.5 ng g1. The highest concentrations of OTA were 13.5 ng g1 in a robusta cherry sample, followed by 4.3 and 1.4 ng g1 in arabica cherry and parchment of both arabica and robusta samples, respectively. The frequency of OTA in different types of coffee were 10 of 21 (48%) in arabica parchment, 15 of 18 (83%) in arabica cherry, 9 of 14 (64%) in robusta parchment and 25 of 27 (93%) in robusta cherry samples. Similarly, the frequency of OTA in coffee from different states was 65% in Karnataka, with a very low average of 1.37 ng g1. In contrast, the frequency of OTA was high in green coffees from Kerala (88%) and Tamilnadu (87%), with mean values of 4.3 and 1.64 ng g1, respectively. The mean OTA level was higher in cherry (range: 1.63 0.97–4.8 3.90) than parchment (0.56 0.35– 1.10 0.28) samples (Table 4). The frequency distribution of OTA contamination for green coffee samples is shown in Figure 1. Of the 80 samples examined, 21 were below the detection limit of 0.1 ng g1 and over 90% of samples were in the range 0.2–5 ng g1. Only five samples had OTA level above 5 ng g1 and all were robusta cherry samples from Kerala state.
Food Additives and Contaminants: Part B 25
23 21
Sample Number
20
18
15
10 5
5
0
< 0.1
0.1-1.0
1.1-2.0
4
4
5
2.1-3.0 3.1-4.0 4.1-5.0 5.0-15.0
OTA content range (ng g−1)
Figure 1. Frequency distribution of OTA in green coffee samples. Numbers on the histogram indicate the total number of samples in each range.
Table 5. Statistical analysis of influence of processing on the level of OTA contamination in green coffee beans. Type of coffees Arabica parchment Robusta parchment Arabica cherry Robusta cherry S.E. CD (p ¼ 0.05)
Overall mean OTA (ng g1) 0.82a 0.91a 1.97b 3.30c 0.105 0.230
Data marked with same superscript are not significantly different at a p value of 0.05 level.
Post-harvest practices, particularly drying, affect OTA contamination (de Moraes and Luchese 2003). The high occurrence of OTA in coffee from Kerala state could be linked to the post-harvest practices followed in that region. The majority of coffee-growers in Kerala, dry their coffee on low-quality drying surfaces, such as bare or cow dung-plastered soil (Reddy 2000), thereby, affecting the overall quality. This is in accordance with the findings of de Moraes and Luchese (2003) who reported that coffee dried on a compacted soil floor had a higher level of OTA contamination (563 ng g1) compared to samples dried on a cement floor (10 ng g1). Statistical analyses to compare OTA contamination between sample types (parchment versus cherry), show that mean OTA concentration in cherry samples (1.97–3.30 ng g1) was significantly higher (p ¼ 0.05) than parchment (0.82–0.91 ng g1) samples (Table 5), indicating processing had a significant influence on accumulation of OTA in green coffee beans. This result is in good agreement with the findings of Ramesh and Vasanthi (2005) who reported that cherry samples had higher levels of OTA contamination than parchment coffee. Frank (1999)
55
reported that parchment coffee is less susceptible to infection by Aspergillus species and OTA contamination. Since, the fruit pulp is an excellent substrate for the growth of OTA-producing strains (Joosten et al. 2001), its removal during wet processing eliminates a suitable substrate for proliferation of moulds. In the present study, the level of OTA contamination was in the range 0.2–13.5 ng g1 with an overall mean OTA level of 2.17 2.45 ng g1. In comparison, Pardo et al. (2004) analyzed 14 green coffee samples of Asian origin and reported OTA concentrations ranging from 1.6 to 31.5 ng g1 with an overall mean of 6.0 7.9 ng g1. Similarly, Romani et al. (2000) showed that 11 of 18 Asian green coffee samples were positive for OTA contamination ranging from 0.2 to 4.9 ng g1 with overall mean of 1.1 1.4 ng g1. These two reports concluded that green coffee samples of African origin had significantly higher levels of OTA than those from America or Asia. In conclusion, this survey provided valuable data on the occurrence of OTA in a large numbers of samples of green coffee from batches destined for export. Although the incidence of OTA contamination was high (74%), the overall mean level (2.17 2.45 ng g1) was low. Furthermore, the level of OTA in the majority of green coffees (94%) analyzed were below 5 ng g1, which complies with current regulatory limits. Nevertheless, an effective and integrated management system in the form of a Hazard Analysis CriticalControl Point (HACCP) throughout the entire production chain may help in reducing fungal and OTA contamination and improve the overall quality of green coffee beans
References Abarca ML, Bragulat MR, Castella G, Cabanes FJ. 1994. Ochratoxin A production by strains of Aspergillus niger var. niger. Appl Environ Microbiol. 60:2625–2650. Anonymous. 2003. Harvest and post harvest practices for quality coffee preparation. Coffee guide. Coffee Board of India: Bangalore. p. 100–109. Anonymous. 2006. Database on coffee. Economic and market intelligence unit. Coffee Board of India: Bangalore. p. 1–10, July/September 2006. Bacha H, Hadidane R, Creepy EE, Regnault C, Ellouze F, Dirheimer G. 1988. Monitoring and identification of fungal toxins in food products, animal feed and cereals. J Stored Prod Res. 24:199–206. Bayman P, Baker JL. 2006. Ochratoxins: A global perspective. Mycopathologia 162:215–223. Celeste ML, Lurdes B, Pena A, Noronha Silveira I. 2006. Determination of ochratoxin A in coriander by HPLC/ fluorescence deflection. Quim Nova 29:436–439. Chelkowski J, Trojanowska K, Wiewiorowska M. 1983. Microbiological evaluation of cereal grain quality, connected with mycotoxin occurrence. Nahrung. 27:311–318.
56
T.N. Gopinandhan et al.
De Moraes MHP, Luchese RH. 2003. Ochratoxin A on green coffee: influence of harvest and drying processing procedures. J Agric Food Chem. 51: 5824–5828. Filternborg O, Frisvad JC, Svendsen JA. 1983. Simple screening method for molds producing intracellular mycotoxins in pure cultures. Appl Environ Microbiol. 45:581–585. Frank JM. 1999. HACCP and its mycotoxin management control potential: Ochratoxin A in coffee production. Proceeding of the 7th International Committee on Food Microbiology and Hygiene held at Veldhoven, The Netherlands; p. 122. Gopinandhan TN, Velmourougane K, Panneerselvam P, Keshamma E, Raghuramulu Y. 2007. Occurrence of ochratoxin A in green and commercial coffee samples. J Food Sci Technol. 44:247–249. Guldborg M. 1997. Ochratoxin A in Danish beer. Brygmesteren. 54:16–17. Heenan CN, Chaw KJ, Pitt JI. 1998. Ochratoxin A production by Aspergillus carbonarius and A. niger isolates and detection using coconut cream agar. J Food Mycol. 1:67–72. Hesseltine CW, Vandegraft EE, Fennell DJ, Smith MJ, Shotwell OL. 1972. Aspergilli as ochratoxin producers. Mycologia. 64:539–550. Joosten HMLJ, Goetz J, Pittet A, Schellenberg M, Bucheli P. 2001. Production of ochratoxin A by Aspergillus carbonarius on coffee cherries. Int J Food Microbiol. 65:39–44. Le-Bars J, Le-Bars P. 2000. Mycotoxigenesis in grains: application to mycotoxic prevention in coffee. Coffee biotechnology and quality. Kluwer Academic: Dordrecht. p. 355–368. Leoni LAB, Valente Soares LM, Oliveira PLC. 2000. Ochratoxin A in Brazilian roasted and instant coffees. Food Addit Contam. 17:867–870. Levi CP, Trenk HL, Mohr HK. 1974. The occurrence of ochratoxin A in green coffee beans. J Assoc Off Anal Chem. 57:866–870. Lombaert GA, Pellaers P, Chettiar M, Lavalee D, Scott PM, Lau BPY. 2002. Survey of Canadian retail coffees for ochratoxin A. Food Addit Contam. 19:869–877. Macdonald S, Wilson P, Barnes K, Damant A, Massey R, Mortby E, Shephered MJ. 1999. Ochratoxin A in dried wine fruit: method development and survey. Food Addit Contam. 16:253–360. Majerus P, Bresch H, Otteneder H. 2001. Ochratoxin A in wines, fruit juices and seasonings. Arch Lebensmittelhyg 51:95–97. Majerus P, Max M, Klaffke H. 2000. Ochratoxin A in Sussholz, Lakritze und daraus hergestellten Erzeugnissen. Deutsche Lebensmittel-Rundschau 96:451–454. Matissek R, Rater M. 2000. Ochratoxin A in cocoa and human health aspects. Proceedings of the 13th International Cocoa Research Conference; Kota Kinahalu, Malaysia; 2000 Oct 9–14; p. 9. MAFF. 1996. Surveillance of ochratoxin A in green (unroasted) coffee beans. Food Surveillance Information Sheet 80. Ministry of Agriculture, Fisheries and Food, London.
Nakajima M, Tsubouchi H, Miyabe M, Ueno Y. 1997. Survey of aflatoxin-B and ochratoxin-A in commercial green coffee beans by HPLC. Food Agric Immunol. 9:77–83. Otteneder H, Majerus P. 2000. Occurrence of ochratoxin A in wines: influence of the type of wine and its geographical origin. Food Addit Contam. 17:793–798. Pardo E, Marim S, Ramos AJ, Sanchis V. 2004. Occurrence of ochratoxigenic fungi and ochratoxin A in green coffee from different origins. Food Sci Technol Int. 10:45–50. Patel S, Hazel CM, Wintertone AGM, Gleade AE. 1997. Survey of ochratoxin A in UK retail coffees. Food Addit Contam. 14:217–222. Perez de Obanos A, Gonzalez-Penas E, Lopez de Cerain A. 2005. Influence of roasting and brew preparation on the ochratoxin A content in coffee infusion. Food Addit Contam. 22:463–471. Pitt JI. 1987. Penicillium virdicatum, Penicillium verrucosum and production of ochratoxin A. Appl Environ Microbiol. 53:266–269. Pittet A, Tornare D, Huggett A, Viani R. 1996. Liquid chromatographic determination of ochratoxin-A in pure and adulterated soluble coffee using an immunoaffinity column clean up procedure. J Agric Food Chem. 44:3564–3569. Ramesh VB, Vasanthi S. 2005. Natural occurrence of ochratoxin in Indian coffee. Indian J Nutr Diet. 42:106–113. Reddy AGS. 2000. Production and productivity of coffee in Kerala: a perspective. Indian Coffee 2: 23–24. Romani S, Sacchetti G, Chaves Lopez C, Pinnavaia GG, Dalla Roas M. 2000. Screening on the occurrence of ochratoxin A in green coffee beans of different origins and types. J Agric Food Chem. 48:3616–3619. Steiner W, Brunschweiler K, Leimbacher E, Schneider R. 1993. Aflatoxine B1 and G1, Cyclopiazonsaure, Kojisaure und ochratoxin A in Trockenfeigen im BGY– Fluoreszenz. Mitteilungen aus dem Gebiete dem Lebensmitteluntersuchung und Hygiene. 84:523–536. Studer-Rohr I, Dietrich D.R. Schlatter J, Schlatter C. 1995. The occurrence of ochratoxin A in coffee. Food Chem Toxicol. 33:341–355. Taniwaki MH. 2006. An update on ochratoxigenic fungi and ochratoxin A in coffee. In: Hocking, AD. Pitt, JI, Samson RA, Thrane U. editors. Advances in Food Mycology, New York: Springer. p. 189. Taniwaki MH, Pitt J.I, Urbano, GR Teixeria AA, Leitao MFF. 1999. Fungi producing ochratoxin A in coffee. In: Proceeding of the 18th ASIC Coffee Conference; Helsinki, Finland; p. 239. Taniwaki MH, Pitt JI, Teixeria AA, Iamanaka BT. 2003. The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods. Int J Food Microbiol. 82:173–179. Thirumala DK, Mayo MA, Gopal R, Reddy SV, Delfosse P, Reddy DVR. 2000. Production of polyclonal antibodies against ochratoxin A and its detection in chillies by ELISA. J Agric Food Chem. 48:5079–5082. Tsubouchi H, Yamamoto K, Hisada K, Sakabe Y, Udagawa S. 1987. Effect of roasting on ochratoxin A level in green coffee beans inoculation with Aspergillus ochraceus. Mycopathologia 97:111–115.
Food Additives and Contaminants: Part B Vander Stegen GVD, Jorissen U, Pittet A, Saccon M, Steiner W, Vincenzi M, Winkler M, Zapp J, Schlatter C. 1997. Screening of European coffee final products for occurrence of ochratoxin A. Food Addit Contam. 14:211–216. Varga J, Kevi F, Rinyu E, Teren J, Kozakiewicz Z. 1996. Ochratoxin production by Aspergillus species. Appl Environ Microbiol. 60:4461–4464.
57
Yani A. 2004. Fungal infection and ochratoxin contamination in green coffee beans collected from farmers and collectors in Bengkulu province. Thesis. Bogor: Bogor Agricultural University. Yashpal S, Geeta S. 1999. Natural occurrence of ochratoxin A and toxigenic Aspergillus ochraceus strains in dry fruit slices of quinces from Jammu and Kashmir. Indian Phytopathol. 52:148–150.
