The EU is the second largest agricultural exporter with trade based on cereals, in particular wheat, and other agricultural products as identified in a study published by the European Commission in relation to the EC Agricultural Policy for the 21st Century. About 25% of the world's food crops are affected by mycotoxins every year. The presence of ochratoxin Ain edible tissue and particularly in pig kidneys, as a result of consumption of contaminated feed, has been demonstrated in Denmark and Sweden by Krogh and Rutquist already in 1977, respectively. As a consequence prevention strategies and early detection systems of mycotoxin-contaminated raw materials form an essential need. Consequently, monitoring and surveillance programmes have been set-up. Ochratoxin A (OTA) is a mycotoxin of considerable concern for human health and is classified as a possible human carcinogen. The EC, Scientific Committee for Food (SCF) has concluded that the intake of OTA should be reduced as far as possible, e.g. below 5 ng/kg body weight/day. Cereals normally correspond to 50-80% of average consumer intake. Consequently, prevention of OTA formation by specific moulds in cereals would have a significant impact on levels of human exposure.
The over-all objective for this project is the protection of the consumer's health by decreasing the amount of ochratoxin A in cereals produced in Europe. This will be achieved by identifying the key elements in an effective HACCP programme for ochratoxin A for cereals, and by providing tools for preventive and corrective actions. The project includes the whole food chain from primary production to the final processed food product. The objectives and expected achievements are divided into 4 different tasks, all important steps in a HACCP managing programme for ochratoxin A in cereals: identification of the critical control points; establishment of critical limits for the critical control points; developing rapid monitoring methods, and establishment of corrective actions in the event of deviation of a critical limit. The outcome from all tasks, which consist of 11 workpackages, will serve as a pool of knowledge for HACCPbased ochratoxin management programmes, which will increase food safety and support the EU cereal industry.
During the second reporting period of Task 1, the examination of ochratoxin positive cereal samples has continued. The number of countries, in which Penicillium verrucosum has been identified as the toxin producer, has increased considerable during 2001. Now, P. verrucosum has not only been found in cereals in Denmark, Sweden, Norway, United Kingdom, Germany and Austria, but also in Italy, Spain, France and Portugal. Still P. verrucosum is the only ochratoxin producer found. More than 100 isolates of P. verrucosum from these environments have been fingerprinted phenotypically and examined for production of ochratoxin A, and many different clones have been found. Replies on the questionnaire, aiming to compile a detailed summary of the different farming methods used across the Community have been received from 12 countries. The data is currently being tabulated and assessed.
Task 2 is providing new knowledge concerning the microbial ecology of the ochratoxin A-producing fungi. The results indicate that niche occupation and niche overlap be- - 235 - tween P. verrucosum and other species varies considerable with water availability and temperature. A. ochraceus occupied the same niche as P. verrucosum at high water activity (aw=0.995), but was more competitive under drier conditions. Studies on the effect of gas composition on germination showed that A. ochraceus is more sensitive to high concentrations of CO2 than P. verrucosum. Overall, P. verrucosum is very tolerant up to 50% CO2. The storage experiments with winter wheat, harvested in August 2000, have been completed. The experiments were supplemented with tests using irradiated grain. The results indicate that an increase in respiration rate occurred just before or at the same time as growth of P. verrucosum or of other moulds could be demonstrated. At water activities above 0.85, an obvious increase of ochratoxin A usually could be detected at the same time or just after P. verrucosum started to grow. Irradiation of grain caused faster growth of moulds and higher production of ochratoxin A. The first year's collection of data from out-door silos, which will be used to describe the heat and moisture transfer in critical parts of a silo, showed that, independent of silo type, there was often a gradual moistening from 13 to15% moisture content of the upper surface of the grain during the storage period. Detailed screening of anti-oxidants, essential oils and antimicrobial extracts from mushrooms have been completed and the best candidates have been tested on irradiated and natural wheat and maize. A few candidates give more than 90% inhibition on the growth of fungi and of the ochratoxin production. In addition, the results from miniscale storage experiments with irradiated barley contaminated with toxigenic moulds and inoculated with lactic acid bacteria (LAB) suspensions, showed that certain LAB restrict efficiently the growth of Penicillium verrucosum.
In Task 3 rapid monitoring methods are being developed. A new set of molecular imprinted polymers (MIPs) has been produced. The MIPs showed excellent affinity to ochratoxin A, and do not bind towards other tested chemical as the initial material did. An extraction procedure for removal of ochratoxin A from grain samples has been developed. Currently, a protocol for detection of ochratoxin Ain spiked and real samples is being tested. Very sensitive prototype ELISAs have been produced and are being evaluated. The assay is well within the range of the recent established EU legislation of 3-5 (g/kg. To establish a molecular detection system for the ochratoxin producing fungi, the strategy is now targeted at using suppression subtractive hybridisation-PCR to target the genes that are up-regulated by cells producing ochratoxin A. Preliminary results indicate that this approach is partially successful.
Task 4 deals with the establishment of corrective actions during milling and cereal processing and during malting and brewing. Two 100-kg batches of wheat were inoculated with P. verrucosum to produce samples with a concentration of ochratoxin A at 5 and 50 (g/kg. The results showed that cleaning and scouring of wheat only resulted in a small loss of ochratoxin A. However, the bran and offal fraction contained much higher concentrations of ochratoxin A, so that this resulted in significantly reduced toxin levels in the white flour by about 50%. There was only a small additional loss during baking. Extrusion appeared to produce a small reduction that appeared to be related to temperature rather than moisture content or screw speed. Malting experiments have been carried out using barley with natural contamination and barley artificially contaminated with P. verrucosum. The growth of P. verrucosum during malting was negligible and only minor amounts of OTA were found. However, high amounts of OTAwere produced during germination and in most cases also during kilning (drying), when artificially contaminated samples were malted. The process temperature during malting had a pronounced effect on the growth of P. verrucosum and the formation of ochratoxin A.