PART III PROCESS SAFETY
4. Control
environmental contamination and resistance of target organisms. However, a possible source of mycotoxins is from fungal BCAs. These may produce toxins and are sprayed onto crops pre- and (potentially) post-harvest. Metarrhizium anisopliae is one such and produces the highly toxic cytochalasins amongst other things (Paterson, 2004b). These compounds have been detected in agricul- tural commodities (CAST 2003) indicating the need to carefully regulate the use and production of these products. In addition, enniatins and beauvaricins are metabolites of insect and plant pathogens and have been detected in wheat and maize infected with Fusarium with negative implications for using the insect pathogens as biological control agents. The fact that BCAs are often for use in developing countries compounds the disproportionate mycotoxin problem that already exists.
3.7. Warfare
There are reports of mycotoxins having been used as biochemical weapons.
Recent events have indicated that they are still actively being considered. CAST (2003) recommends that assessments are required of mycotoxins as biological weapons. Obviously, levels of mycotoxins in the environment used for this pur- pose will be at higher concentrations than occur naturally. Drinking water is an obvious target (Paterson and Lima, 2005).
sample preparation are of paramount importance and it is crucial to obtain a representative sample. The distribution of toxins in a commodity is heterogeneous, with pockets of high concentration coupled with low or no amounts in other parts.
It is extremely difficult to determine with confidence what the true concentration is. An analogous sampling problem exists for isolating the producing fungi (Paterson, et al., 2004).
Analysis is normally by chromatography. Initially, thin layer chromatographic (TLC) methods were employed (see aflatoxins) (CAST, 2003). However, this has been superseded, in many cases, by high performance liquid chromatography (HPLC). Gas chromatography is often used for Fusarium mycotoxins. So-called hyphenated techniques are employed such as diode array and mass spectroscopic detection. The use of immunoaffinity columns for analysis of most of the main mycotoxins has greatly improved analysis in terms of quantification, purification and convenience. Immunological test kits such as ELISA are available but they are not as accurate as the chromatography-based methods. TLC is optimal for the detection of previously unknown “new” compounds because of the more univer- sal detection methods employed (CAST, 2003).
4.1. Good agricultural practice (GAP)
The prevention of mycotoxin formation is achieved by influencing environmen- tal conditions through management of agricultural practices prior to harvest (CAST, 2003). In the case of peanuts, it is known that the formation of aflatox- ins in the plant takes place when ambient temperatures of 25 to 32 ˚C occur simultaneously with low humidity in the soil. Therefore, it is possible to control the biosynthesis of aflatoxins in peanuts by controlled irrigation at critical peri- ods of the day. The use of recommended crop management practices and har- vesting crops when they are mature decreases, for instance, aflatoxins.
Something as prosaic as adjustment of combine harvesters to prevent excess damage to kernels can be effective (CAST, 2003). Careful adjustments to the machine may actually eliminate contaminated kernels, although sound ones may still contain high levels of mycotoxins and so the removal of the damaged ones will not eliminate mycotoxins completely. Care is required to clean storage bins, auger pits, maintain clean lorries, trailers and combine harvesters. Overall decreases of total fumonisins of 60% by screening and gravity settling corn can be obtained.
4.2. Good management practices (GMP)
After harvest, two overriding factors for storage are water activity (aw) and tem- perature. Others are cleaning, insect control, use of antifungal agents and main- taining integrity of the seed coat. These parameters, if not kept at the desired levels, may lead to the growth of fungi and the accumulation of mycotoxins in stored crops (CAST, 2003). The source of this inoculum could be the crop itself,
mainly when it is of a poor quality, or colonised plant debris, when storage facil- ities are not properly cleaned.
Harvested grain, coffee beans, fruits, and oilseeds crops should be dried imme- diately. The final safe moisture depends on crop and climatic conditions where stored. Corn which is contaminated with A. flavus and subjected to increased moisture by 18 % for 4-6 h can increase aflatoxin levels (Sinha and Bhatnagar, 1998). Early harvesting of corn followed by drying may avoid increased aflatoxin contamination (but drying may not be economical). Grain needs to be dried to 15
% or less before storing. Drying is critical to coffee (Paterson et al., 2001) and peanuts to avoid OTA and aflatoxins respectively. Contamination of coffee with OTA fungi from soil apparently occurs in the drying process if beans are kept for more than 4 days at an awabove 0.8 (about 16 % humidity in grain) (Sinha and Bhatnagar, 1998).
If the product is dry and kept dry, no further biodeterioration will occur.
However, insects, rodent activity, moisture migration, condensation and/or water leaks may re-hydrate the grain, for example, and lead to fungal growth and pos- sible toxin production. Insects and rodents need to be controlled as they create conditions suitable for fungal growth, which once started, the water of metabo- lism can be sufficient for more fungal growth and mycotoxin production. Low toxicity antifungal agents are used to supplement good management practices but are not a substitute. Air treatment in storage facilities may prevent growth and contamination, e.g., propionic acid, mixtures of propionic and acetic acid, or ozone (CAST, 2003).
4.3. Removal of mycotoxins after harvesting
Another way of controlling mycotoxins is by removal after harvest. This may be achieved from granulated products, such as cereals, bean or others, since con- taminated kernels have distinctive characteristics. Damaged kernels may exhibit differences in colour, density, shape or size; they may also exhibit fissures, mould growth, or may be broken. Peanut kernels containing aflatoxin are usually shriv- elled and discoloured and can be separated from sound ones by sieving, electronic sorting and hand picking. These contaminated kernels have a lower density, as does contaminated corn, and thus removal is possible by flotation and density sorting. These procedures are reported to remove more than 90% of aflatoxins (Sinha and Bhatnagar, 1998).
A separation of kernels based on different size is also useful for those situations where broken kernels occur, as in the case of fumonisins in corn. These myco- toxins are usually present in amounts ten times higher in broken kernels than in whole ones. Other products, such as almonds, may not be broken but have fis- sures. From these fissures oil may leak, which is detected by its fluorescence under UV light. For cereals, cleaning and polishing (scouring) may be useful for the removal of OTA (see case study below) or DON (Skudamore and Banks, 2004).
4.4. Removal during processing
The amount of mycotoxins still present in raw material, after the trimming process, may be further reduced during processing. Physical, chemical or biolog- ical processes often employed in food processing may be useful in the removal or deactivation of mycotoxins: these include (a) the transformation of mycotoxins during heat treatments (e.g. roasting coffee beans), (b) exposure to sun light, (c) adsorption with clarifying agents (e.g. during refining of edible oils), (d) extrac- tion of immiscible solutions, (e) cross reaction with food additives (e.g. sulphur compounds used in fruit juices production) or (f) reduction during alcohol pro- duction by Saccharomyces cerevisiae.
Aflatoxins and OTA are rather stable when exposed to dry heat and less stable when exposed to humid heat (Sinha and Bhatnagar, 1998). However, at high tem- peratures they can be transformed into other products, some of them less toxic, although there may be others formed that are of unknown toxicity. Boiling rice at normal pressure reduces aflatoxins up to 50 %. Increasing the temperature increases the level of aflatoxin transformation: a pressure-cooking can degrade aflatoxins by 70 %. Frying can reduce aflatoxins by 60 % when oil temperature is at 150 ˚C for 20 minutes. OTA is partially removed from coffee beans during roasting. Degradation of OTA to a maximum of 90 % is possible, depending to the conditions of operation while roasting (e.g. temperature usually between 200 and 250 ˚C and resident time from 5 to 20 minutes).
Aflatoxins present in edible oil after its extraction from seeds are mainly dis- persed in fine particles (Sinha and Bhatnagar, 1998). The removal of these parti- cles is usually aided by the addition of adsorbents, which consequently remove mycotoxins.
OTA appears to be associated with the solid phase in the vinification process of wine (Fernandes et al., 2003). The resulting juice for fermentation contains a considerably lower concentration than the original material. However, OTA, when present in grapes, may be carried over to the juice and persist after fermen- tation. Wine is usually a cloudy suspension that is clarified with the aid of chem- ical adjuvant (fining agents). OTA binds to these agents and so is partially removed.
Solvent extraction is employed in various food processes, such as the refin- ing of edible oils or the removal of caffeine to produce decaffeinated coffee.
During these processes, mycotoxins are co-extracted with the solvent. As exam- ples, a partial removal of aflatoxins is obtained from edible oil while refining and OTA is removed from beans in the decaffeination of coffee (Pittet et al., 1996). Water is a good solvent for OTA in certain food matrixes, such as cof- fee: so brewing coffee is the perfect way of extracting OTA from the coffee grains to the cup!
“Phase changes” can often be employed in food processing e.g. cheese pro- duction where two immiscible phases (cheese and cheese whey) are obtained from milk. Aflatoxin M1is distributed between these phases; since the cheese fraction is much smaller than the whey fraction (10 litres milk for 1 kg cheese)
aflatoxin M1 usually has a higher concentration in cheese than in milk (van Egmond, 1989). However, aflatoxins are generally insoluble in water and so may also dissolve more readily in the lipid rich cheese fraction.
Enzymes and microorganisms are used frequently in food processing and may transform mycotoxins into other compounds with less or unknown toxic- ity (CAST, 2003). This is the case with proteases. They can hydrolyze the peptide bond of OTA yielding phenylalanine and the much less toxic ochratoxin alpha. S. cerevisiae transforms patulin into other compounds during alcohol production in cider production. Patulin is also degraded by (a) sulphur dioxide, and (b) sulphur containing proteins. This approach should be considered as a method for reducing patulin in other commodities. Indeed many of the above processes may find uses in other commodity systems but have simply not been tried.
4.5. Degradation of mycotoxins
The technology for the chemical degradation of aflatoxin in animal feedstuffs (Coker, 1999) is most developed for remediating contaminated material.
Numerous oxidising agents, aldehydes, acids, and bases (inorganic and organic) have been investigated as chemical detoxification agents. Patented procedures exist that use ammonia, calcium hydroxide, methylamine and a mixture of cal- cium hydroxide and methylamine. Most interest is in the use of ammonia in the anhydrous form and in aqueous solution. There have been many studies on the efficiency of the ammonia detoxification and on the nutritional and toxicological properties of the resulting feeds. Ammoniation is an accepted method for animal feeds in some states of USA, Brazil, Mexico, Senegal, South Africa and Sudan (CAST, 2003).
4.6. Others
Considerable effort has been applied to breeding resistance to fungi into crops.
For example, groundnuts and maize have been made resistant to A. flavus.
However, trials were unsuccessful in the field (CAST, 2003). Biocontrol of fungi by the use of nontoxigenic fungal competitors is attractive in theory but in prac- tice there is little evidence that it works, or that it is intrinsically safe.
Food additives that absorb some mycotoxins in the animal gut and hence decrease bioavailability to host animals are available (CAST, 2003). Commercial absorption materials (e.g. clays) have been included in animal feed to absorb afla- toxins and make it less accessible to the animal’s digestive system. An example is the absorption of aflatoxin by adapted chicken feed (CAST, 2003). It is unclear whether this works effectively. It obviously adds cost to the feed and the absorbent would presumably always have to be added as it would not be neces- sarily be known whether the feed actually contained aflatoxins. In the authors opinion there is nothing to suggest the mycotoxin problem has been solved by this procedure.