A case study: EC Project

Relevance to a HACCP approach to Mycotoxin Control.

By David Aldred and Monica Olsen

Introduction

In Newsletter II, we introduced an EC Project, running from February 2000-July 2003, which is looking at the prevention of the occurrence of ochratoxin A (OTA) in cereals. This Project has the acronym "OTA PREV", and has particular relevance to our discussions since it is specifically applying HACCP type principles to the problem of OTA. Further, it is attempting to take into account the entire cereal food chain "from field to table", and is therefore groundbreaking in its approach. The Project is multidisciplinary and is made up of four separate "tasks", which are summarized here with comments on their relevance in the development of HACCP plans:

Task 1

Task 1 is concerned primarily with data collection. The data collected is comprised of two different categories:

(i) Occurrence of OTA producing fungi in Europe.

This part of Task 1 has involved screening cereal samples from across Europe for OTA producing fungi. Samples have been taken from both the at-harvest and post-harvest environments. Traditional isolating techniques and chemical characterization, as well as state-of-the-art molecular "fingerprinting" techniques (AFLP) have been used to identify the isolates and determine whether they are OTA producers.

(ii) Farmer’s questionnaire

A questionnaire was compiled and distributed to agricultural experts in member states to obtain detailed information on farming practices across the Community. The questionnaire was designed to highlight areas relevant to the potential for OTA production within the various farming methods.

Results

Sample screening has revealed a large number of strains of one particular fungus capable of producing OTA. This fungus is called Penicillium verrucosum, and is found commonly in the cereal environment at- and post-harvest. Strains of this species have been obtained from UK, Germany, Austria, Norway, Sweden and Denmark, where they are already known. In 2001 however, the Project also obtained OTA producing strains of this species from Italy, Spain, France and Portugal for the first time. The project has striven to isolate other potential ochratoxin A producers (A. ochraceus, A. niger, A. carbonarius). These other species were occasionally isolated but they were not found to be OTA producers in this situation. This makes P. verrucosum one of the most important fungi in the European cereal industry, at least in the post-harvest storage and processing situations, since it is not usually significant in the growing crop.

Studies undertaken in Denmark, Sweden and UK have identified that P. verrucosum contamination occurs either during or after harvest. Contamination sources appear to be harvesting, processing and storage equipment, such as combines, dryers and silos.

Results of the questionnaire have been obtained from 12 countries and the report is available at the project web site.

Relevance to the development of a HACCP plan.

The information gained in task 1 can be used in a number of ways to aid the development of a HACCP plan:

1. Information gathered on farming practices across the EC will be essential in the preparation of detailed commodity flow diagrams (CFD’s) specific to both product types and locations. The drawing up of specific and detailed CFD’s probably represents the most crucial step in the preparation of HACCP plans since they are pivotal in the identification of critical control points (CCP’s).

2. Information on the occurrence of OTA producing fungi is of great value in hazard identification. For example, the identification of P. verrucosum as the sole OTA producer effectively establishes this fungus as the OTA hazard in the system (as we have previously discussed, mycotoxins are classed as biological hazards). This allows for a far more accurate hazard identification to be performed related to the geographical distribution of this particular fungus, and its presence within particular processing systems. However, the geographical distribution of a fungal species is likely to be a dynamic parameter which is subject to change, and this needs to be taken into account in the development of any HACCP plan.

3. The identification of harvesting, storage and processing equipment as sources of P. verrucosum contamination is again of vital importance when assessing the points of control within the commodity manufacturing and processing steps (as detailed in the CFD). This immediately leads to the identification of sensitive steps in the commodity supply chain which may themselves represent critical control points (CCP’s) or, more likely, areas where pre-requisite programs such as Good Agricultural Practice (GAP), Good Storage Practice (GSP) and Good Manufacturing Practice (GMP) will need to be adhered to carefully.

Task 2

Task 2 of the Project represents fundamental laboratory based studies investigating the microbial ecology of the OTA producing fungi, primarily P. verrucosum. These studies are providing data on the environmental conditions under which the fungi grow and produce OTA. Main environmental factors under investigation are moisture level (measured as water activity, a_W), temperature and gas composition (CO₂ concentration). Laboratory based studies have also investigated the interactions between the main fungi inhabiting the stored cereal ecosystem, and the relative dominance of relevant species under different environmental conditions (niche occupation and niche overlap). Further laboratory studies have investigated the use of novel antimicrobial agents such as antioxidants, essential oils and products derived from bacteria and fungi, as potential methods for preventing fungal growth and OTA production in contaminated grain.

Data from storage experiments on wheat, barley and rye, repeated during three seasons, have been used to develop a mathematical model to predict the safe storage time of cereals at different water activities and temperature.

Studies have also been conducted on temperature and moisture distribution in different types of out-door silos with the objective of evaluating and suggesting improvements in common types of silo systems used for grain storage.

Results

These studies have provided a large amount of previously unavailable data on the fundamental behavior of OTA producing fungi and other species likely to be present in the grain ecosystem. It has been found that there are complex relationships between the fungi inhabiting grain, with P. verrucosum and A. ochraceus co-existing under high moisture conditions (a_W = 0.995), but with P. verrucosum becoming dominant in drier conditions. Further, P. verrucosum has been shown to be less sensitive to high atmospheric CO2 concentrations than competing fungi. These findings generally seem to indicate that the primary OTA producing organism, P. verrucosum, is likely to be dominant under prevailing conditions in the grain ecosystem.

The mathematical model, developed from the storage experiments, can be used to predict the safe storage time of cereals at different water activities and temperatures. With the goal of reducing the cost connected with drying of cereals, it has become more and more common to use buffer storage. The model shows that if buffer store at high moisture content (above 20% moisture content) is used, there may only be a few safe days, or even less. It is recommended not to buffer store grain at higher moisture content than 18% before final drying.

The data from the storage experiments have also been used to predict the probability of ochratoxin A levels above the maximum limit (in the EC regulation 472/2002) at different levels of colony forming units (CFU/g). The risk is clearly increased when levels of more than 1000 CFU/g are found in wheat.

The observations in the two-year study of common types of out-door grain silos have been summarised as four major recommendations. The design of the top ventilation of the silo is of great importance. Among marketed systems, ventilation hoods, if possible mounted sheltered from the wind, seem to be the best solution. Leakage of water seems to cause more problems than expected, with extensive moisture increase in the grain and subsequent mould growth and toxin production. It is therefore recommended to control the silo condition before harvest and tighten apertures from which water penetration may be possible. Mould growth and toxin production mostly occurred in the upper layer of the grain. For this reason the upper layer should be inspected and mouldy grain removed before unloading the silo. Finally, it is recommended that the silo is equipped with a system for aeration of the grain to reduce the risk of condensation. To control the cooling process, it is necessary that the silo is equipped with a grain temperature measuring system.

Screening experiments using antioxidants, essential oils and microbial extracts revealed potential preservatives that were capable of more than 90% inhibition of fungal growth and OTA production.

Relevance to the Development of a HACCP Plan.

These studies provide the type of fundamental information which will support the risk assessment exercises carried out on the steps in the CFD’s. Used together with the information gained in Task 1 relating to the sources of contamination of relevant organisms, they reveal vital information on the conditions under which P. verrucosum will be present, active and producing OTA. In particular they provide data on moisture levels, temperatures, gas composition, duration of storage and likely fungal interactions critical for fungal growth and OTA production, and therefore underpin the identification of critical control points and critical limits.

Information gained on typical moisture behavior in out-door silos similarly allows for critical limits to be set for permissible moisture levels.

Studies on chemical preservative systems provide information on their possible use in, for example, certain situations where grain is stored damp. Preservative use could then represent a critical control, and the concentrations required would form the basis of critical limits.

Task 3

Task 3 of the Project involves the development of rapid monitoring methods for OTA. This has included developing state-of-the-art technologies such as molecular imprinted polymers (MIP’s) which can be used as very specific detection tools for chemical substances such as mycotoxins. In addition to MIP technology, Task 3 has involved the development of an enzyme linked immunoassay (ELISA) with the ultimate aim of producing a rapid one step rapid test kit, such that an OTA analysis will take just a few minutes.

Another area of research in Task 3 is the development of rapid molecular methods to detect OTA producing fungi. One particular line of research is the detection of fungal genes that are activated during OTA production, which has involved work with monoclonal antibodies.

Results

MIP’s developed for OTA in this Project appear to have high affinity for the toxin, which is a crucial first step in the design of a detection system.

An ELISA assay and the competitive Lateral Flow Device (LFD) for P. verrucosum and A. ochraceus have been developed and will become much faster than traditional methods, which take a week to complete. The development of ELISA methods for ochratoxin A, with a working range of 0.02 to 16 microgrammes/kg has provided a very suitable method for rapid monitoring which is an essential part of the HACCP concept. In addition, the promising new results using a quantitative LFD reader indicate an additional rapid and sensitive method, which may also be used in a field system.

The project has also developed PCR primer pairs that appear to be highly specific for A. ochraceus and Penicillium verrucosum. The primers may find use in the development of rapid identification protocols for ochratoxigenic fungi.

Relevance to the Development of a HACCP Plan.

Work within Task 3 has particular relevance to the development of a HACCP plan. , While parameters such as moisture level and temperature are important critical controls, specific mycotoxin analysis and detection of specific fungal species rarely, if ever, figure as monitoring parameters for critical controls. The reason for this is straightforward enough. Up until now these parameters have been far too complex, time consuming and expensive to represent test systems that could be used realistically in food production systems. The grain industry in particular works on a "just in time" basis and therefore any analyses that cannot be carried out rapidly and on-line have no place in the production environment. And yet, the ability to analyse specifically for the toxic chemical itself, and for the presence of the specific fungus responsible, undeniably represent the most useful tests possible, with regard to this particular hazard. The value of task 3 is therefore in the potential to develop critical controls and monitoring methods that relate directly to the hazard of concern, the mycotoxin itself, and the fungi responsible. This can only be feasible if the tests concerned are rapid, simple, economical, accurate and sensitive. These are specifically the factors that task 3 is attempting to address.

Task 4

Task 4 is concerned with the latter stages in the cereal commodity chain, including the processing stages such as flour milling, bread making, malting and brewing, that lead to consumer products. OTA analysis during the milling process has involved looking at fractions such as the bran and offal (impurities). Analyses were also carried out during baking and the extrusion processes that lead to a number of consumer products. Investigations of the malting process involved conducting experiments on naturally contaminated grain, and grain specifically contaminated with P. verrucosum.

Results

Results for Task 4 are summarized below:

1. Cleaning and scouring of wheat resulted in an insignificant reduction in OTA levels.

2. White flour showed significant reduction (approx. 50%) in OTA levels, due mainly to the removal of bran and offal fractions.

3. Bread baking had little influence on OTA levels.

4. Extrusion resulted in a small reduction in OTA which appeared to be related to extrusion temperature.

5. Significant quantities of OTA could potentially be produced during germination and kilning during malting. OTA production seemed particularly related to process temperature. This is significant as process temperatures vary considerably within the malting industry.

6. There is a significant reduction of ochratoxin A during brewing and approximately 20% of the initial ochratoxin A in the malt remains in the beer.

Relevance to the Development of a HACCP Plan.

Again, the relevance of the results of these experimental studies is in the conducting of risk analyses on the steps in the CFD’s for each of the relevant product types. Knowledge of the effects of the processing steps on OTA production and levels is vital for identifying the CCP’s, critical limits and corrective actions. For example, from the information obtained in Task 4, some steps in malting could represent very sensitive processes with CCP’s relating particularly to moisture, temperature and duration of particular steps. Similarly, the removal of bran and offal fractions in white flour production has been shown to reduce OTA levels, and could therefore represent a useful control measure. Conversely, other processes such as bread baking and product extrusion have been shown to have only a small effect on OTA levels, and therefore do not have a significant bearing on the OTA hazard.

Applying HACCP type principles to the latter processing stages of the cereal supply chain, in all of its various forms, is closest to the areas where HACCP is already well established. The identification of CCP’s at this end of the supply chain will be extremely product specific and complex, depending on the product type and local practices. For some products, particularly those that undergo the most processing steps, a number of CCP’s may be involved which cumulatively work together to result in a final product with an acceptable OTA level.