Survey on ochratoxin A in Brazilian green coffee destined for exports

Investigação de ocratoxina A em café verde brasileiro destinado à exportação

Survey on ochratoxin A in Brazilian green coffee destined for exports

Investigação de ocratoxina A em café verde brasileiro destinado à exportação

A. P. B. Gollücke^{I, II, *}; M. H. Taniwaki^III; D. Q. Tavares^II

^ICatholic University of Santos. Av. Conselheiro Nébias, 300. Santos-SP Brazil 11015-002

^IIDepartment of Food Planning and Nutrition, State University of Campinas. CP. Campinas-SP Brazil 13081-970. E-mail: [e-mail]

^IIIFood Technology Institute. Av. Brasil, 2880. Campinas-SP Brazil 13073-001

SUMMARY

The presence of ochratoxin A (OTA) in foods has led some countries to establish regulatory limits. Although coffee is not a major source of OTA in human consumption, the European Community (EC) may establish limits in the near future, with possible economic impact on producing countries. This study measured the OTA content with HPLC in 37 samples of Brazilian green coffee exclusive destined to the export market and also verified a possible relation between coffee defects and OTA content. The results showed an OTA concentration ranging from < 0.16ng/g (detection limit) to 6.24ng/g (average of 3.20ng/g) for 37 samples. Of the five samples observed for defects, toxin content of sound beans ranged from 0.22 to 0.80ng/g (average 0.46ng/g) and of defective beans from 0.42 to 17.46 (average 4.52ng/g). Morphological differences among sound and defective beans showed no susceptibility for mould invasion under optical microscopy observation. One black bean depicted the presence of mould and spores on observation under Scanning Electron Microscope (SEM). According to this investigation, Brazilian green coffee for export complies with most limits in place.

Keywords: mycotoxin; Brazil; coffee.

RESUMO

A presença de ocratoxina A (OTA) em café, detectada nos últimos anos, tem levado alguns países a estabelecer limites regulatórios. Embora o café não seja uma fonte importante de OTA no consumo humano, a União Européia poderá estabelecer limites, causando impacto econômico em países produtores desses grãos. O presente estudo analisou OTA utilizando CLAE em 37 amostras de café verde brasileiro destinado exclusivamente à exportação e verificou a possível relação entre grãos defeituosos e concentração de OTA. Os resultados mostraram que a concentração de OTA esteve entre < 0,16ng/g (limite de detecção) e 6,24ng/g (média 3,2ng/g). Das cinco amostras avaliadas quanto à relação defeitos X OTA, o nível da toxina esteve entre 0,22 e 0,80ng/g (média 0,46ng/g) entre grãos sadios e 0,42 e 17,46ng/g (média 4,52ng/g) entre grãos defeituosos. Diferenças morfológicas entre grãos sadios e defeituosos observados em Microscopia Ótica não demonstraram susceptibilidade à invasão fúngica entre os defeituosos. Entre os 15 grãos observados em Microscopia Eletrônica de Varredura (MEV), apenas um (preto) revelou presença de fungos e esporos. Os resultados desta investigação apontam que o café verde brasileiro destinado à exportação encontra-se dentro dos limites para OTA já estabelecidos.

Palavras-chave: micotoxina; Brasil; café.

1 – INTRODUCTION

Ochratoxin A (OTA) is a nephrotoxic and possible carcinogenic mycotoxin mostly found in cereals and cereal products in Europe. It is produced by some species of Aspergillus and Penicillium as a secondary metabolite. Ochratoxin A inhibits protein synthesis both in vitro and in vivo through competition with phenylalanine and it was also found to increase lipid peroxidation, leading to further cell and mitochondrial damage [2]. Due to health concerns, the FAO/WHO Joint Expert Committee on Food Additives and Contaminants (JECFA) established a provisional tolerable weekly intake of 100ng/kg body weight (bw) [4]. Moreover, the EC Scientific Committee on Food (SCF) recommends that levels of OTA should be reduced as much as possible, i.e., 'below 5ng/kg bw/day'.

Reports have shown OTA incidence in other foodstuffs such as wine [8], beer [5], cocoa [8], dried wine fruits [8] and green and roasted coffee [9, 15]. Although a minor contributor to the dietary intake of OTA, coffee has received special attention in the last few years [11]. As a consequence, some countries such as Italy, Switzerland, Finland and Greece have set regulatory limits with maximum OTA values ranging from 4 to 20ppb (ng/g). The EC might establish a limit in the near future.

According to estimates of the Institute for Scientifc Information on Coffee (ISIC) implemented by FAO, if the EC establishes a regulation for OTA on the proposed level (5ppb), 7% of coffee batches worldwide would exceed this amount [1]. The rejected shipments would lead to economic losses to producing countries of over one billion dollars and an extra 500 million dollars to the EC alone on laboratory costs.

Brazil is the largest coffee producer and exporter with a 22% average market share in the last seven years [13]. Research on OTA occurrence in green, roasted and soluble coffee produced in Brazil has been carried out. In 132 samples of Brazilian green coffee, 27 were contaminated with OTA at levels of 0.7 to 47.8ng/g [7]. Samples of soluble coffee and roast and ground coffee contained OTA levels between 0.31 and 1.78ng/g and 0.99 and 5.87ng/g, respectively [12]. Most of studies on the incidence of OTA in Brazilian coffee have shown that the contamination in coffee is not significant [6, 7, 14, 16]. However, to date, no investigation has been reported on Brazilian green coffee exclusively destined for export, which would be directly impaired by the regulatory limits of importing countries. The objectives of the present work were to quantify OTA in green coffee samples obtained from export batches and to search for morphological susceptibility of defective beans to microbial invasion.

2 – MATERIAL AND METHODS

2.1 – Samples

Thirty seven samples (1kg each) of Brazilian green coffee were obtained from export companies in Santos (Brazil), comprising 6 states, 2 harvest years, arabica and robusta, 'organic' and government stock samples as shown in Table 1. Samples were collected according on warehouses at random from 100bags/each at different parts of the warehouse in July 2001. Samples were obtained from the 1999/2000 and 2000/2001 harvests for all origins except for Bahia (dry process) (2000/2001) and government stock (1987/1988).

2.2 – Sub-sampling

Of the total sampling lot, five samples were submitted to classification by a coffee expert at an export company in Santos (Brazil) and the beans were separated into black, sour and sound. Black and sour beans are considered coffee defects, probably a result of over-ripe cherries, deficient drying in field and/or undesirable fermentation, which could lead to infection of OTA producing fungi and consequently OTA formation [13]. Black and sour beans of each sample were grouped together composing a defective sub-sample while the sound ones composed the sound sub-sample. It was not possible to classify and separate more than five samples due to lack of enough defected beans to conduct the analysis (minimum 25g).

2.3 – Extraction and cleanup of OTA in coffee

The extraction and cleanup procedures of OTA in the coffee samples were performed according to Pittet et al. [10]. Twenty five grams of the finely blended samples were mixed with 200mL of methanol/3% aqueous sodium hydrogen carbonate (50:50). The suspension was blended for 3 min at medium speed with a Polytron homogenizer and then filtered through a 55mm Whatman GF/B glass microfiber filter under low vacuum. Five milliliters of the filtrate were transferred to a cylinder and the volume then completed to 100mL with phosphate-buffered saline (PBS). The whole extract was applied to the immunoaffinity column at 2-3mL/min with the aid of a vacuum pump. The column was then washed with 10mL distilled water, followed by 4mL methanol. The eluate was collected and evaporated to dryness under a nitrogen stream at 40°C. The residue was resuspended in 1mL of the High Performance Liquid Chromatograph (HPLC) mobile phase. The peak areas were plotted on a standard curve and the concentrations were obtained.

2.4 – HPLC

The HPLC equipment used was a Shimadzu SCL-10AVP (Shimadzu Corporation, Japan) chromatograph with an automatic injector. Toxin detection was obtained with a Shimadzu RF-10AXL fluorescence detector operating at excitation and emission wavelengths of 330nm and 470nm respectively. A SupelcosilÔ LC-18 (5mm particle size, 4.6mm x 250mm) column (Supelco, USA) was used with a Hypersil guard column (5mm particle size, 4.6mm x 25mm). For the mobile phase a solution in the proportion of 42% acetonitrile/58% 4mM sodium acetate/acetic acid (19:1) was applied. The injection volume of the extract was 50mL at a flow rate of 1.0mL/min. The OTA concentration in samples was determined using peak area and then plotting it on a standard curve.

2.5 – Analytical quality control

For recovery tests, uncontaminated samples were spiked with two different concentrations of standard solution corresponding to 8ng/g and 80ng/g and analysed in duplicate using the same procedure as described for samples. The average recovery was 77.9% and 90.9% respectively. The detection limit of the method was obtained analysing coffee samples with minimal concentration of OTA (< 1ng/g). The standard deviation (0.058) of ten repetitions was multiplied by the corresponding number listed on the t (Student) table for 99% of confiability. For ten repetitions and nine degree of freedom the t Student value was 2.821. The detection limit of method (DTM) was 0.16ng/g. This is somewhat lower than to that reported (0.2ng/g) by Pittet et al. [10]. An identity confirmation was carried out adding 0.1mL of concentrated hydrochloric acid (37%) to the OTA residue, and the vial was closed and kept overnight at room temperature. After evaporating the reaction mixture to dryness and dissolving the residue in 30mL of HPLC solvent, 20mL of it were analysed for OTA methyl ester formation according to a procedure described by ZIMMERLI & DICK [17]. A positive confirmation of identity was provided by the disappearance of the OTA peak at a retention time of 6min and the appearance of a new peak (OTA methyl ester) at 8min.

2.6 – Optical and Scanning Electron Microscopy (SEM)

In total, 15 beans classified as sound, black and sour were observed separately under optical microscopy and SEM. For optical analysis, beans were placed in the microtomy (no previous inclusion), and cut into sections of about 5mm. Observations were made without previous staining; morphology was enhanced by means of polarization lenses. As for SEM preparation, the coffee beans were cut in half, de-fatted with acetone for 3 hours and placed in an incubator overnight. After spattering with gold the beans were observed under SEM at 10kV of acceleration.

3 – RESULTS AND DISCUSSION

3.1 – Quantitative determinations

From the 37 original coffee samples analysed, 36 presented OTA content ranging from < 0.16ng/g to 0.85ng/g and only one sample showed a content of 6.24ng/g (average 3.20ng/g). Twenty samples (54%) did not presented OTA level as they were below the detection limit of 0.16ng/g and 97.5% of samples showed OTA concentration below 5ng/g (Figure 1). Although the sampling lot was composed of raw coffee from several producing regions in Brazil, two different coffee treatments, and more than one harvest and two varieties, no correlation was found among any of these variables versus OTA levels.

The results of the OTA investigation of sound and defective sub-samples are presented in Table 2. Except for one, all sub-samples, sound or defective showed higher contents of OTA than the original samples, most likely due to the heterogenicity of contamination, which is often a concern in OTA investigations. Average OTA level of sound sub-samples was 0.46ng/g against 4.52ng/g of the defective sub-sample. HEILMANN, REHFELDT & ROTZOLL [3] observed correlation of OTA levels and coffee defects with the use of a colour sorting machine to separate defective beans and measuring OTA levels. LEONI et al. [7] however, found no correlation between OTA contamination and defective beans using total defects counting according to the Brazilian classification system.

3.2 – Optical and Scanning Electron Microscopy

Visually, sound beans had the typical green colour and good appearance. Black ones, known to be a result of over-riping or deficient drying, presented a dark colour. Sour beans, a consequence of the same process failures, presented a brown, shiny aspect. The results of optical microscopy (Jenaval Zeiss Optical Microscope) depicted no significant morphological differences in the endosperm of sound, sour and black beans as shown in Figures 2 to 4. (Differences in colour represent the best polarized light used in each observation). The cell walls indicated by arrows in Figure 3 show the characteristic shape and thickness of Coffea, while the middle lamellae is defined by the brilliant cresyl blue dye. In Figure 4 the arrows point to tissues of endosperm whose cells have lost their organization. Diffusion of cell contents occurred due to the high level of moisture. Furthermore, no mould, bacteria or yeast contaminations were found using this technique. With SEM (Jeol JSM-T300 Scanning Electron Microscope), the morphology of sound and sour coffee beans also showed an organized endosperm with cells containing the contracted cytoplasm (Figures 5 and 6). The dotted arrow in figure 5 shows the thick cell walls in Coffea while cytoplasm is also depicted (solid arrow) though not preserved. The black beans showed a somewhat compressed endosperm and badly defined cellular contents (Figure 7). Oil is emerging from cells, as pointed to by arrows, also observed with sound and sour beans. Of the 15 coffee beans, only one (black) depicted the presence of moulds and spores under SEM (not shown).

4 – CONCLUSIONS

There seems to be a correlation between higher quality of export coffee beans and lower concentration of OTA, since the results of the present study exclusive on export coffee show consistent lower OTA content than previous data on raw coffee produced in the country [6, 7, 14, 16]. However, defective beans characterised as sour and black, which are less present in export coffee, do not necessarily correspond to higher OTA contamination. It was not possible to correlate the visual aspect of sour and black beans with obvious damage to the endosperm structure, which could indicate susceptibility to mould invasion. In this investigation OTA levels showed no correlation with the green coffee origin nor to the coffee variety and/or after-harvest process. According to the present survey, Brazilian raw coffee for export complies with most regulatory limits in place.

5 – REFERENCES

Recebido para publicação em 04/07/2003. Aceito para publicação em 28/10/2004 (001161).

• [1] BOUTRIF, E.; BESSY, C. Global significance of mycotoxins and phycotoxins. In: Mycotoxins and Phycotoxins in Perspective at the Turn of the Millennium Edited by W. J. De Koe, R. A. Samson, H. P. Van Egmond, J. Gilbert e M. Sabino. p. 3-16, 2001.
• [2] DIRHEIMER, G. Mechanistic approches to ochratoxin toxicity. Food Additives and Contaminants, London, v.13, suplem., p. 45-48, 1996.
• [3] HEILMANN, W.; REHFELDT, A.G.; ROTZOLL, F. Behaviour and reduction of ochratoxin A in green beans in response to various processing methods. European Food Research Technology, 209, p. 297-300, 1999.
• [4] JECFA, 1995. Technical Report Series n. 859 (Geneva: Joint FAO/WHO Expert Committee on Food Additives)
• [5] JØRGENSEN, K. Survey on pork, poultry, coffee, beer and pulses for ochratoxin A. Food Additives and Contaminants, London, v. 15, n. 5, p. 550-554, 1998.
• [6] LEONI, L.A.B.; FURLANI, R.P.Z.; VALENTE SOARES, L.M.; OLIVEIRA, P.L.C. Ochratoxin A in Brazilian roasted beans and instant coffee. Food Additives and Contaminants London, v.17, n.10, p.867-870, 2000.
• [7] LEONI, L.A.B.; FURLANI, R.P.Z.; VALENTE SOARES, L.M.; OLIVEIRA, P.L.C. Ochratoxin A in Brazilian Green Coffee. Ciênc. Tecnol. Aliment. Campinas, v. 21, n. 1, p.105-107, 2001.
• [8] MAFF. Ministry of Agriculture, Fisheries and Foods, 1999. Survey on retail products for ochratoxin A. Food Surveillance Information Sheet n. 173
• [9] PPATEL, S.; HAZEL, C.M.; WINTERTON, A.G.M.; GLEADLE, A.E. Survey of ochratoxin A in UK retail coffees. Food Additives and Contaminants, London, v. 14, n. 3, p. 217-222, 1997.
• [10] PITTET, A.; TORNARE, D.; HUGGETT, A.; VIANI, R. Liquid chromatographic determination of ochratoxin A in pure and adulterated soluble coffee using an immunoaffinity column cleanup procedure. Journal of Agricultural and Food Chemistry, Washington, v. 44, n. 11, p. 3564-3569, 1996
• [11] PITTET, A. Natural occurrence of mycotoxins in foods and feeds: a decade in review. In: Mycotoxins and Phycotoxins in Perspective at the Turn of the Millennium Edited by W. J. De Koe, R. A. Samson, H. P. van Egmond, J. Gilbert e M. Sabino. p. 153-172. 2001
• [12] PRADO, G.; OLIVEIRA, M.S.; ABRANTES, F.M. Incidência de ocratoxina A em café torrado e moído e café solúvel consumido na cidade de Belo Horizonte, MG. Ciênc. Tecnol. Aliment. Campinas, v. 20, n. 2, p. 192-196, 2000.
• [13] SILVA, J.M. Curso de classificação e degustação de café verde. 2001. Edited by Centro do Comércio de Café do Estado de Minas Gerais. Varginha. p. 66-69.
• [14] TANIWAKI, M.H.; PITT, J.I.; TEIXEIRA, A.A. & IAMANAKA, B.T. The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods. International Journal of Food Microbiology, v. 82, p. 173-179, 2003.
• [15] TRUCKSESS, M.W.; GILER, J.; YOUNG, K.; WHITE, K.D.; PAGE, S.W. Determination and survey of ochratoxin A in wheat, barley and coffee- 1997 Journal of AOAC International, Baltimore, v. 82, n. 1, p. 85-89, 1999.
• [16] URBANO, G.R.; TANIWAKI, M.H.; LEITÃO, F.F.; VICENTINI, M.C. Occurrence of ochratoxin A-producing fungi in raw Brazilian coffee. Journal of Food Protection, v. 64, n. 8, p. 1226-1230, 2001.
• [17] ZIMMERLI, B.; DICK, R. Determination of ochratoxin A at the ppt level in human blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column cleanup: methodology and Swiss data. Journal of Chromatography, v. 666, p. 85-89, 1995.

*

A quem a correspondência deve ser enviada.

Publication Dates

• Publication in this collection
  09 Mar 2005
• Date of issue
  Dec 2004

History

• Accepted
  28 Oct 2004
• Received
  04 July 2003