Mycotoxins are fungal metabolites frequently associated with grains, cereals, and other human and animal foods obtained from these sources. However, based on the latest studies and recently implemented norms, other products associated with mycotoxins contamination have begun to be taken into account, especially regarding toxins which are considered a human risk, as is the case with Ochratoxin A (OTA). Ochratoxin A is produced by various species of Aspergillus and Penicillium. Several aspects distinguish this mycotoxin from the others. Chemically, it is the only mycotoxin that has a Chlorine- atom as a substitute. From a toxicological point of view, it is considered nephrotoxic, teratogenic and immunotoxic, as well as a possible human carcinogen (Group 2B – IARC).¹

In temperate and cold climates, OTA is mainly produced by Penicillium verrucosum or P. nordicum. In tropical and subtropical regions, the main OTA producing mould is Aspergillus ochraceus, although the production of this toxin in warm regions has also been associated to two other Aspergillus sp: A. niger var niger and A. carbonarius. ²

The great variety of foods in which OTA has been detected ranges from cereals and oleaginous crops, to coffee, dried fruits, wines, cheese, processed meat, smoked or salted fish and even medicinal herbs. As the sources of exposure are so diverse, the presence of OTA has been recorded in most of the studied population.

The half-life of orally ingested OTA in human plasma is approximately 35 days, based on data inferred from experimental monkeys. This information has led to several studies evaluating the degree of exposure in a normal diet. In 1998, a Tolerable Daily Intake (TDI) of up to 5 ng/kg b.w./day was recommended based on results by SCOOP (Scientific Cooperation on Question relating to Food – European Comission).

Ideally, however, daily intake should not exceed 1.2 to 1.4 ng/kg b.w./day. ^2,4,5


Ochratoxin A in beverages for human consumption

The number of reports and studies regarding Ochratoxin A has increased since 1995, especially since the first study carried out in order to establish the level of OTA intake by the population of EC State Members. Although this study was based on a limited amount of data obtained, it proved very useful as a tool for basic information and future decisions. Since this initial report, numerous others have been published evaluating the frequency of OTA occurrence, OTA levels in biological fluids such as blood, plasma, urine, breast milk, as well as its occurrence in new sources of exposure (raisins, wine and spices). ⁴

The report by SCOOP Task 3.2.7 (2002), which compiled on the dietary intake of OTA by the EU population, describes the contribution of each type of food to the mean European OTA intake, and shows that even though cereals contribute to 44% of the total amount of OTA consumed by the EU population, the contribution provided by beverages amounts to the following:

• - wines 10 %
• - coffee 9 %
• - beer 7 %

OTA intake through breast milk is considered in a separate section, but only with data from four countries.

Based on this report, new maximum limits of OTA were established for different products. (seeTable 1).


Occurrence of OTA in beverages.

Beer: Although this beverage is not directly included in the European regulation -as mycotoxin controls are performed on cereals, it is considered that the quality of the end product can be determined-, some countries consider beer as a product derived from cereals, which corresponds to a maximum tolerance level of 3.0 μg/kg. However, Italy proposes a maximum tolerance level of 0.2 μg/kg.

In view of the global importance of beer consumption, there have been many reports investigating the levels of OTA in different countries, but information on OTA intake through beer was only gathered in the EU.

Table 2 shows OTA levels reported in different beverages, taking into consideration the areas in which occurrence was registered. It shows that, with the exception of a report from South Africa, the concentrations of OTA found in beer are considerably low.⁶ Table 3 shows the contribution of different beverages to the daily intake of OTA in European countries.

Wine: European legislation and the OIV (Office International de la Vigne et du Vin) recommend an OTA tolerance level of no more than 2.0 μg/kg for all types of wine: white, red, rosé, sparkling, etc.





As is the case of beer, wine is only drunk by certain consumer groups, and it is important to consider this when evaluating OTA intake through these beverages.⁴

A revision of published reports evidences a clear relation between the type of fermentation and the presence of OTA in wines. As a general rule, white wines usually have lower OTA levels than rosé wines, which in turn have lower levels than red wines. This evidences a clear relationship between the process of maceration and OTA solubilization in the grape must. On the other hand, sweet wines elaborated with a process that involves partial dehydration in the sun tend to have higher OTA levels compared to dry wines. ^{7, 8} The occurrence of OTA in wines also varies according to the region (see Table 2).

Table 1: Summary of the European regulation for OTA levels in beverages.
Product	Level (μg/kg. = ppb)	Document
Dietary foods for special medical purposes for children	0.5	EC 683/2004 O. J. L 106 – 15/04/04
Roasted coffee beans and ground coffee made from roasted beans with the exception of soluble coffee.	0.5	EC 123/2005 O. J. L 25 – 28/01/05
Soluble coffee (instant coffee)	10.0
Wine (white, red, rosé) and other wines, or grape must-based beverages	2.0
Grape juice, grape juice ingredients in other beverages including grape nectar and concentrated grape juice as reconstituted.	2.0
Grape must and concentrated grape must as reconstituted, intended for direct human consumption.	2.0
Beer and cocoa.	Still under discussion

Table 2: OTA occurrence in beverages.4,6,9,10
Beverage	Region	N – (% positive) (N: number of samples tested)	Mean OTA concentration
Beer	Europe	496 – (36 %)	0.032 μg/L
Beer	South Africa	29 – (45 %)	3 – 2340 μg/L
Beer	Canada	41 – (63 %)	0.061 μg/L
Beer	Germany	250 – (75 %)	0.01 – 0.29 μg/L
Wine	Southern Europe	625 - (72,3 %)	0,636 μg/L
Wine	Northern Europe	835 – (50,3 %)	0,181 μg/L
Wine	Italy	184 – (86 %)	1.565 μg/L
Wine	Argentina-Chile	84 – (-)	<0.008 μg/L
Green coffee	Europe	1704 – (36 %)	3.641 μg/L
Processed coffee	Europe	1205 – (47,3%)	1.092 μg/L

Table 3: Contribution of beverages to the daily OTA intake of the European population.4
Beverage	Region	Contribution to daily OTA intake (ng/kg pc/day)
Beer	Europe	0.01 (Italy), - 0.14 (Denmark)
Wine	Europe (all the population)	0.02 (Portugal), - 0.86 (Italy)
Wine	Europe (consumers)	0.003 (France, children), - 2.94 (Italy)
Coffee	Europe (all the population)	0.06 (Italy), - 0.42 (Finland)

Coffee: OTA contamination in coffee is assessed at two stages: green coffee and processed coffee. Several authors have reported that the contamination in green coffee values between 0.2 and 360 μg/kg.¹¹

The results shown in the Task report 3.2.7 for green and processed coffee are summarized in Table 2. No significant differences were found between the mean values of processed coffee when the geographical location of each country (North and South) was considered. However, OTA values were significantly higher in the green coffee of southern countries, especially Greece and France (16.14 μg/kg and 6.55 μg/kg respectively). This decrease observed in processed coffee evidently amounts to the fact that the processes used in southern countries are much more drastic than those applied in northern countries, as well as to the commercially available mixtures of coffee varieties.

The main factors that influence OTA contamination in the coffee production chain are failures in agricultural and in manufacturing practices. FAO, together with other international organizations related to coffee, has initiated a global program to reduce the level of this toxin in coffee (http://www.coffee-ota.org/), which will mainly be applied in coffee producing countries.¹²


Romer Labs® solutions for the detection of OTA in beverages

Romer Labs®, offers solutions for OTA detection in beverages that can be applied to all types of analytical and industrial needs: immunoaffinity columns, solid phase columns, liquid calibrators and ELISA tests.

OchraStar^TM: Immunoaffinity columns were developed to clean-up complex matrices and to obtain lower detection levels. Besides using the classical methods applicable to beers, wines and other liquid matrices, Romer Labs® has developed a specific technique to detect OTA in roasted coffee using HPLC-FLD.

MycoSep®/MultiSep® 229 Ochra: Romer Labs® single step columns reduce the clean-up time to just 30 seconds, thus providing a solution for the analysis of large amounts of samples, highly useful at an industrial scale. For green coffee samples, the MycoSep® 229 column provided a recovery of 98% for levels ranging between 2.6 and 91 μg/kg, whereas for red wine the recovery was 97 % in the same range.¹³

Biopure: Ready-to-use liquid calibrators are recommended for best analysis performance. These are backed by the relevant Certificates of Analysis indicating, amongst other details, the certified value with its associated uncertainty. Biopure also offers certified pure crystalline Ochratoxin A.





AgraQuant® Ochratoxin ELISA test kit: To determine the level of OTA in different matrices, including beverages, Romer Labs® has also developed a direct competitive ELISA test, which covers the necessary range for achieving fast and reliable assessments. The kit is also validated for green coffee, red wine (with a specific application according to the content of ethanol of the wine) and beer. This method provides rapid low cost results, especially when applied to the monitoring system recommended by HACCP programs for the various agroalimentary chains.¹⁴

Conclusions:

Greater demands by markets and regulators compel the beverage industry to incorporate new determinations, such as OTAdetection and quantification. It has become necessary to establish fast and reliable methods that can be applied industrially, as well as to validate them for the quantification of OTA in beverages. Romer Labs® offers the widest range of analytical alternatives and its experts provide advice on choosing the best analytical option for OTA detection in beverages.

References

¹ Murphi P., Hendrich S., Landgren C., Bryant C. Food Mycotoxins: An Update. J. Food Scie. Vol. 71, Nr. 5, 2006 – R51-R65.

² Ringot D., Chango B., Schneider Y-J., Larondelle Y. Toxicokinetics and toxicodynamics of ochratoxin A, an update. Chemico-Biological Interactions 159, 2006, 18-46

³ Marquardt R. R., Frohlich A. A. A review of recent advances in understanding ochratoxicosis. J. An. Scie. 70, 1992, 3968-3988.

⁴ SCOOP – Task 3.2.7 Assessment of dietary intake of Ochratoxin A by the population of EU Member States. 2002 .

⁵ JECFA 47. WHO FOOD ADDITIVES SERIES: 47 Safety evaluation of certain mycotoxins in food - Ochratoxin. 2001 World Health Organization, Geneva, 2001IPCS

⁶ Odhav B., Naicker V. Mycotoxin in South African traditionally brewed beer. Food Add. Cont. 19 (vol1) – 2002- pp 55-61

⁷ Zamora Marín F. La Ocratoxina A; un problema emergente. h t t p: // www. enologo.com /tecnicos/ eno34/ eno34.html

⁸ Gambuti A., Strollo D., Genovese A., Ugliano M., Ritieni A., Moio L. Influence of Enological Practices on Ochratoxin A Concentration in Wine. Am. J. Enol. Vitic. 56,2 (2005) 158-162

⁹ Ratola N., Martins L., Alves A. Ochratoxin A in wines-assessing global uncertainty associated with the results. Analytica Chimica Acta 513 (2004) 319–324.

¹⁰ Pacin A., Resnik S., Vega M., Saelzer R., Ciancio Bovier E., Rios G., Martinez N., Occurrence of ochratoxin A in wines in the Argentinian and Chilean markets. ARKIVOC 2005 (xii) 214-223.

¹¹ Taniwaki M., Pitt J., Teixeira A., Iamanaka B. The source of ochratoxin A in Brazilian coffee and its formation in relation to processing methods. Int. J. Food Micr. 82 (2003) 173– 179

¹² Paulino de Moraes M.H., Luchese R.H. Ochratoxin A on Green Coffee: Influence of Harvest and Drying Processing Procedures. J. Agric. Food Chem. 2003, 51, 5824-5828

¹³ Buttinger G., Fuchs E., Knapp H., Berthiller F., Schuhmacher R., Binder EM., Krska R. Performance of new cleanup column for the determination of ochratoxin A in cereals and foodstuffs by HPLC-FLD. Food Add. Cont. Vol 21, Nº 11 (November 2004) pp 1107-1114.

¹⁴ Zheng Z., Hanneken J., Houchins D., King R., Lee P., Richard J. Validation of an ELISA test kit for the detection of ochratoxin A in several food commodities by comparison with HPLC. Mycopathologia (2005) 159: 265–272

Author: Patricia Silvina KNASS (Bqca.), is a Technical Advisor in Latin America for Romers Labs Diagnostic GmbH. Her education began at the National University of Misiones (Argentina), in the field of Biochemistry. Her master thesis title is Critical Control Point Identification for Aflatoxin and Ochratoxin in two different swine farms in Argentina (work in progress). She worked as a Technical Advisor in Latin America; was the General Manager for agriNEA (Argentina); researcher at the Mycotoxin Laboratory of the National University of Misiones (Argentina). She’s currently working for Romer Labs (Austria).