Fungal damage in durable foodstuffs with special reference to storage in the tropics

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1. What are fungi?

Fungi are micro-organisms generally classified as plants, although there is growing support for the separation of the fungi and other micro-organisms into an entirely new kingdom. For this reason Fungi are perhaps best described as plant like micro-organisms that do not possess chlorophyll.

Fungi may be simple in structure, as are the yeasts which consist of a single cell or chains of cells that reproduce by "budding" and which generally give rise to rather slimy, pink, pale brown or cream coloured colonies. Typical moulds on the other hand, are more complex, multicellular fungi, of variable appearance and colour. Most reproduce by means of microscopic asexual spores which vary considerably in size and shape and are formed either on stalks or in special vesicles which grow from, or are embedded in, the vegetative part of the fungus (mycelium). This consists of many strands (hyphae) which typically grow together to form a 'mycelial mat'. It is this mycelium that is largely responsible for the formation of exzymes, fungal toxins, etc. Many fungi also have a sexual stage in their life cycle. (Note: Except in certain very high moisture content situations yeasts are relatively unimportant and this supplement refers mainly to the mould fungi).

Whereas fungi that attack growing plants are fairly restricted in number and are true pathogens, fungi in stored products are much more numerous and are saprophytic, or only weakly pathogenic by nature. This also applies to the 'field fungi'. The A/ternaria and Cladosporium groups are common examples causing a blackening or weathering effect on unharvested, mature crops of maize, sorghum and other grains. The Fusarium group is also important at this stage. Once in store the field fungi tend to decline, their place being taken by such typical storage groups as Aspergillus and Penicillium. These and other storage moulds often fall within the 'fungi imperfect)', so called because they reproduce only by asexual means. For descriptive details of the fungi and other micro-organisms that cause damage in stored produce please see T.S.T.S. OLL.

In common with other living things, when fungi grow they breathe (a process known as respiration) using oxygen from the air which reacts with the substrate on which they are growing to produce carbon dioxide, moisture and heat. By-products of the normal growth process are often of a complex chemical nature and may be beneficial but are sometimes undesirable.

2. Where do fungi occur?

Fungi are found everywhere. Spores occur in dust and are therefore present in the air we breathe. All stored commodities are contaminated with fungal spores and these, unlike insects, cannot be excluded by careful handling and treatment. Even processed foods that been subjected to high temperatures during their preparation (e.g. oil-seed cake) though perhaps significantly free of living moulds immediately following processing, are rapidly recontaminated during packaging and storage. It is, therefore, most important to prevent fungal spores from germinating and growing into visible colonies.

3. When do fungi grow?

Fungal spores require moisture to germinate and if this is excluded (i.e. if grain is stored at its safe moisture content or below) moulds generally will not grow. The maximum safe storage moisture content may be defined as the amount of absorbed water held within a commodity which is in equilibrium with an atmospheric relative humidity of 70%. Moisture content is closely bound to temperature. Under certain circumstances, temperature differences can cause the re-distribution of moisture leading to local mould growth. Other factors that affect fungal development and the production of spores are availability of oxygen, light, acidity, and salt/sugar content.

4. Why is it important to prevent fungal growth?

Fungi do a tremendous amount of damage by causing:

  1. Direct loss when the grain is too mouldy to eat.
  2. Caking in grain and flour, rendering the material difficult to handle.
  3. Changes in colour, texture and flavour rendering the produce unacceptable by:
  1. fermentation (carbohydrates converted to acids and gas)
  2. putrifaction (protein breakdown), and
  3. rancidity (fats converted to acids).

The latter is especially important in oilseeds (e.g. groundnuts, oil palm kernels) and oily products (rice-bran, copra etc) where the enzymatic conversion of oil to free fatty acid results in uneconomic processing or in financial penalties on world markets.

  1. Charring or spontaneous combustion if mould growth is unchecked as this releases heat and grain is a poor conductor of heat. Quite dramatic temperature rises can occur especially within large bulks or stacks. Normally the temperature rise Is restricted to a maximum level of about 60°C at which point moulds are generally inhibited. However, in oilseeds, enzymatic heating can continue even leading eventually to spontaneous combustion.
  2. Reduction in germination capacity of grain.
  3. Poisoning in man and animals due to the production of mycotoxins (poisonous fungal breakdown products) by certain species. Aflatoxin, produced by the common storage fungus Aspergillus flavus is known, for example, to cause liver collapse in certain domestic animals.
  4. Lung diseases such as asthma and skin allergies if spores are present in the atmosphere in very high concentrations (e.g. handling mouldy straw or prolonged exposure when emptying underground pits).
  5. Deterioration and weakening of fibres used for packaging or protecting grain (e.g. jute sacks, tarpaulins). This can lead to spillage or water entry. Container sealing materials such as stitching and adhesives are also liable to mould attack.
  6. Deterioration of store fabrics, especially wood, causing rotting and disintegration.

5. In what practical situations can fungal damage occur?

Fungi can develop if conditions favour their growth at any of the following times:

  1. In the field, prior to harvest, when the crop is maturing.
  2. After harvest during the drying period.
  3. In store. This can occur for two unrelated reasons and it is important to distinguish between them:
  1. due to the premature storage of inadequately dried material, and
  2. due to the re-absorption of water while in store (especially during the wet season), as follows:
  1. exposure to high relative humidity,
  2. through a leaking store roof, cover or container,
  3. water uptake through the floor or walls of a store,
  4. development of temperature gradients in grain leading to condensation.
  1. In transit.
  2. During processing.

6. Hints on preventing fungal damage

  1. Harvest the crop at maturity. Grain harvested prematurely takes longer to dry and is therefore more susceptible to mould damage. It is also likely to shrivel. The actively growing crop has a natural resistance to invasion by storage fungi (e.g. Aspergillus and Penicillium spp.) but this is to some extent lost at maturity or if the grains are damaged by rodents, insects, etc.
  2. Dry the produce as quickly as possible bearing in mind the need for a 'curing' period in some crops such as groundnuts. Remember that hot sunlight is not always the best. A method that protects the commodity from rain, dew and damp soil but allows dry air to pass freely over the produce is ideal. In certain extreme climates where the air is continuously humid artificial drying may be essential.
  3. Avoid physical damage to the produce at all stages of handling. Harvesting and shelling are two occasions when damage is likely to occur. Broken groundnut pods, for example, are more easily invaded by moulds than undamaged pods. The grain skin is also resistant to fungal invasion and should be kept intact if possible.
  4. Check the moisture content of the commodity before it is stored. Make sure that it is quite dry first. if there is any doubt, or if drying has proved to be a problem, do not store the produce in solid walled containers. 'Pigeon hole' stacking can be used for bagged produce suspected of being damp as it allows air to circulate through the stack.
  5. Avoid storing warm grain as the heat will be retained and encourage rapid mould and insect multiplication. Also, if the store structure is cool, condensation may occur where the warm grain touches the cool surfaces.
  6. Ensure that all stores, silos, etc. are in good repair before use.
  7. When storing bagged produce, keep it well away from the walls of the store and use dunnage to raise the sacks away from the floor.
  8. Allow newly constructed concrete stores or floors dry out throughly before use. If possible build a water vapour barrier (e.g. polyethylene sheet, bitumenastic layer) into the floor and walls during construction.
  9. Allow for controlled ventilation around the produce within a store so that air of high relative humidity can be excluded during the wet season. Ventilate only if the internal air is moist and the outside air is dry, or if the produce needs cooling.
  10. Ideally, produce and store should be maintained at an even temperature.
  11. If produce is thoroughly dry, polyethyelene bags or sheets can be used to exclude moisture during storage. However, care must be taken to avoid exposure to direct sunlight or condensation will occur beneath the plastic surface. This is also likely to occur in a store where the internal temperature fluctuates excessively. To prevent condensation under these circumstances the top and sides of the stack should be insulated with a layer (several if possible) of sacks or similar material, placed outside the plastic sheet.
  12. If practical, cover all metal silos to prevent direct sunlight from falling on the walls. If this is not possible a coat of white paint will help to reflect the heat and keep the produce cool.
  13. Apply adequate pest control measures to prevent insect 'hot spots' from developing.
  14. Do not load or unload grain in the open if it is raining and avoid placing sacks of produce on wet ground.
  15. Ensure that all railway wagons, lorries, small boats and other mobile containers are adequately covered and in good repair, especially if movement during the rainy season is likely.
  16. It is almost impossible to avoid some fungal damage in traditional underground pit stores. However, any form of lining which prevents grain from coming into direct contact with the soil will help. Also, pits should be completely filled; a little grain in a large pit will usually become very mouldy.

7. The chemical control of storage fungi.

Chemicals for the prevention of mould growth in grain stored for human consumption are not at present available. The very broad spectrum of moulds found in stored products is one problem and toxicity to man another. However, propionic and other organic acids have been successfully used in temperate countries to protect highs moisture content barley and other grains destined for animal feed. While it may be possible in the future to use propionic acid in hot climates where drying is a problem, considerable research under tropical conditions is needed first. Taint and smell are two aspects requiring investigation if propionic acid is to be applied to grain for human consumption. Furthermore, the application rate is critical and complete coverage of the grain is necessary before mould growth commences if adequate control is to be achieved.

8. What should be done with mouldy foodstuffs?

This is a vexed question as no-one wishes to waste food. Indeed, some foods like 'blue' cheese are purposely infected with fungi. However, since the discovery over the last decade that a wide range of storage fungi can produce substances that cause disease when fed to animals there has been considerable speculation as to whether man is at risk through eating badly stored foodstuffs which have been accidentally contaminated with fungi. There is strong circumstantial evidence to support such a suggestion, and fungal invaded grain, pulses and especially oilseeds should therefore be avoided if at all possible. It is worth noting here that cleaning grain to remove surface fungal growth (this is, for example, practised in some rural communities with mouldy pit-stored grain), is unlikely to remove any toxin present within the grain and therefore is not recommended. Similarly, cooking does not necessarily destroy mould toxins and aflatoxin is a good example of this. Careful sorting to remove visibly damaged grain is therefore recommended, and in certain commodities, especially oilseeds such as groundnuts, the seeds should be cut open and examined for hidden fungal growth within the grain.

The old maxim that mouldy grain can be safely fed to animals therefore no longer applies. If mouldy grain is used for animal feed it may cause death and at best is likely to give poor results (e.g. a reduction in expected weight increase). If used it should be considerably diluted with fungus free material. Extreme care is needed with oilseed cake in particular, as this is very readily contaminated with aflatoxin, a substance that is toxic in very small quantities. Appearance is not a good criterion when judging oilseed cake, for although aflatoxin may have been present in the original seeds, visible mould damage will not be seen in the finished produce unless re-wetting has occurred.

 

AFLATOXIN ANALYSIS: OVERVIEW

Aflatoxin is one of the most well-known mycotoxin in tropical and sub-tropical areas. Crop in these regions or more subject to contamination than those in temperate regions, since optimal conditions for toxin formation are prevalent in areas with high humidity and temperature. Toxin-producing fungi can infect growing crops. As a consequence of insect or other damage, and may produce toxins prior to harvest, or during harvesting and storage.

The ingestion of food containing aflatoxin may have serious adverse health effects in man. Aflatoxins are demonstrated liver toxin and liver carcinogens in some animals including non-human primates. Dose response relationships have been established in studies on rat and rainbow trout, with 10/tumour incidence estimated to occour at feed level of AF B1 of 1 µ/kg and 0.1 µ/kg respectively. In some studies, carcinoma of the colon and kidney have been observed in rats treated with aflatoxin. AF 3(1) causes chromosomal aberrations and DNA breakage in plant and animal cells after microsomal activation, gene mulation in several bacterial test systems. In high doses, it may be teratogenic.

The acute toxicity and carcinogenicity of aflatoxin are greater in male than in female rats; hormonal involvement may be responsible for this sex-linked difference. Nutritional status in animals, particularly with respect to lipotropes, proteins, vitamin A, and lipids (including cyclopropenoid fatty acid) can modify the expression of acute toxicity or carcinogenicity or both.

Liver cancer is more common in some regions of Africa and Southeast Asia than other parts of the world when local epidemiological information is considered together with experimental animal data. It appears that increased exposure to aflatoxins may increase the risk of primary liver cancer.

In view of the evidence concerning the effects, particularly the carcinogenic effects of aflatoxin in several animal species, and in view of the association between aflatoxin exposure levels and human liver cancer, incidence observed in some parts of the world, exposure to anatoxins should be kept as low as practically achievable. The tolerance levels for food products established in several countries should be understood as management tools, intended to facilitate the implementation of aflatoxin control programmes, and not as exposure limits that necessarily ensure health protection.

Aflatoxins are now recognized to be involed in the aetiology of certain human and animal diseases. An awareness of the level of contamination of Aflatoxin in natural products can only be obtained by developing good analytical methodologies for detecting aflatoxin in foods, mixed feeds and Ingredients, animal tissue, blood, urine and milk.

Aflatoxin detection methods can be divided into three categories

  1. rapid presumptive tests to identify samples from agriculture products such as corn, peanut lots that may contain toxin,
  2. rapid screening procedures to determine the presence or absence of toxin,
  3. quantitative methods to determine aflatoxin levels.

The presumptive test for aflatoxin in corn in the black light test or Bright Greenish-Yellow fluorescent test (BOY) based on the fluorescence under ultraviolet light (365 nm.) associated with Aspergillus flavus and A. parasiticus.

Rapid screening tests have included mini column methods that can be done in a laboratory with minimal facilities, and thin layer chromatography (TLC).

Quantitative methods to determine aflatoxin levels involves extraction, purification of extract, and measurement of the toxin by TLC using visual comparisons with a standard or densitometry or high pressure liquid chromatogaphy.

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