N.K.S. Gowda
ICAR-National Institute of Animal Nutrition and Physiology Bangalore – 560 030 India nksgowda@rediffmail.com
Mycotoxins are fungal secondary metabolites present in animal feeds and human food stuffs. The extent of fungal contamination depends on environmental factors like excess moisture, hot and humid climate and insect damage. Most common mycotoxins of significance are aflatoxin, ochratoxin and fusarial toxins(Fumonisins, Zearalenone, Trichothecenes). Aflatoxins produced by Aspergillus fungi affect all categories of livestock and poultry being more susceptible. They are hepatocarcinogenic and on cumulative consumption cause poor performance and reduced immunity. Aflatoxins are mutagenic and carcinogenic in nature. Hence suitable preventive and counteractive measures need to be undertaken. Suggested measures include storing feeds at low moisture level and use of fungicides (organic acids, urea, ammonia) is effective to arrest fungal growth. Use of non-toxigenic strains of fungi as biocompitator to control the growth of toxigenic fungi is found effective. Some anti-fungal metabolites (cyclic dipeptides, caproic acid, lactic acid, fungicin) produced by bacteria have been proved effective. Genetic modification of mold susceptible plants for increased production of anti-fungal proteins and phenolics also reduces the fungal colonization. Dry heating and sun drying of aflatoxin contaminated feed degrade the toxin. Silicated binders and charcoal are effective in counteracting the aflatoxins. Chemicals like sodium bisulphite, ammonia and sodium hypochlorite are effective in breaking the cyclic structure of aflatoxins. Yeast and lactic acid bacteria are known to bind aflatoxins, rendering them unavailable for gut absorption. Certain microbes degrade aflatoxins and biotransform them to less toxic metabolites. Nutritional strategies like supplementation of hepatotrophic nutrients, methionine, vegetable oil and antioxidants like BHT, BHE, Vitamin E, Selenium and curucumin are also known to reduce the toxic effects of aflatoxins. Of late, there is a greater interest in use of microarray technique for early forecasting of fungal contamination of feeds and understanding the mechanism of aflatoxicosis at cellular level. This would help in evolving better strategies for aflatoxin prevention and counteraction.
INTRODUCTION
Mycotoxins are the toxic products of fungal metabolism occur in a wide variety of commodities like animal feeds and human food products. Mycotoxins on ingestion can cause health hazards both in livestock and human beings and hence there is a greater economic implication. The severity of mycotoxin contamination is determined by major environmental factors like excessive moisture in the field and in storage, hot and humid climate and insect infestation. Mycotoxin contamination of feed affect dairy cattle, poultry, sheep swine and reduces growth efficiency, impairs resistance to infection and reproduction. Economic losses due to mycotoxicosis are derived directly from livestock morbidity and mortality and wastage of contaminated feed. On a global scale, it is estimated that around 25% of the world’s crops are affected by mycotoxins annually and this has a direct bearing on economy due to loss of feed, reduced animal productivity and costs involved in monitoring the level of mycotoxins. The mycotoxins that are of significance in animal feed are : Aflatoxin, Ochratoxin and Fusarial toxins (Fumonisin, Zearalenone, Trichothecenes, Deoxynivalenol).
Aflatoxins and biological action
The aflatoxins are highly toxic and carcinogenic compounds produced by Aspergillus fungi at an optimum temperature of 25-320 C, moisture of greater than 12-16% and a relative humidity of 85%. Commonly affected feeds are maize, groundnut cake, cottonseed cake and copra cake and causes toxicity in poultry, cattle, sheep and swine. Animal consuming aflatoxin contaminated feed display poor performance, reduced immunity, liver damage, kidney and intestinal haemorrhage and liver tumor. Among the afltoxins B1 is more prevalent and toxigenic. This is metabolized to Aflatoxin M1 in liver and is excreted in milk of dairy cattle and also as residue in egg / meat. Epoxide derivative of aflatoxin B1 binds with DNA and disrupts transcription and translation activities, thus initiating carcinogenesis. Oxidative nature of the toxic derivative releases free radicals and cause cell damage (Fig.1). Advancement in molecular techniques like microarray and PCR has helped to understand the precise mechanism of action of aflatoxin. Recent gene expression studies have shown that down regulation of mitochondrial carnitine palmitoyltransferase (CPT) system, down regulation of fatty acid metabolism pathway, up- regulation of cell proliferation pathway and down regulation of B cell activation are respectively
responsible for decreased body weight gain, fatty liver / increased liver weight, carcinoma and lowered immunity in birds fed aflatoxin. Supplementation of curcumin through turmeric powder ameliorated most of the ill effects induced by aflatoxin. Adverse effects of aflatoxicosis are much severe when there is a concurrent contamination with other toxins like aflatoxin,ochratoxin and T-2 toxin.
Limits of aflatoxin
The presence of Aflatoxin M1 in food products meant for human consumption is not desirable and the residual concentration should not exceed 0.5 ppb. Aflatoxin B1 level of 20 ppb in the diet of dairy cattle is appropriate for reducing the risk of aflatoxin M1 in milk. In many countries there are strict guidelines for maximum tolerable limits of aflatoxins, beyond which the commodity is unsafe and not accepted (Table 1).
Table 1. Suggested limits for aflatoxin.
| Particular | US FDA | EU(Very stringent) | BIS/FSSAIIndia |
| Compound feed AFB1 | 20 | 5 | 20 |
| Milk, AFM1 | 0.5 | 0.05 | 0.50 |
| Cereal grains AFB1 | 20 | 20 | 50 |
| Oilseeds and meal | 20 | 20 | 50 |
CONTROL AND COUNTERACTION OF AFLATOXINS
Preventive measures
Aflatoxins affect mainly liver and kidney and are also carcinogenic and mutagenic. Therefore effective control and detoxification measures need to be undertaken. Toxin producing fungi may invade at pre-harvesting period, harvest-time, during post harvest handling and in storage. According to the site and time of infestation, the fungi can be divided into three group:
(a) Field fungi (b) Storage fungi (c) Advanced deterioration fungi. Field fungi are generally plant pathogenic fungi; namely Fusarium. The storage fungi are Aspergillus and Penicillium. The advanced deterioration fungi, normally do not infest intact grains but easily attack damaged grains and requires high moisture content, that include Aspergillus clavatus, Aspergillus fumigatus. Prevention and effective plan for reducing fungal growth and toxin production is very
important. The recommended practices include 1. Development of fungal resistant varieties of plants, 2. Suitable pre-harvest, harvest and post harvest techniques, 3. Store commodities at low temperature as for as possible 4. Use fungicides and preservatives against fungal growth and 5. Control of insect damage in grain storage with approved insecticides.
The secondary prevention of fungal growth include limiting the growth of infested fungi by re-drying the product, removal of contaminated seeds and inactivation of mycotoxins produced. The tertiary measures could be to prevent the transfer of fungi and their health hazardous toxins into the food/feed and to the environment. This include complete destruction of the contaminated product or diversion for fermentation to produce ethanol or detoxification / destruction of mycotoxins to the minimum level. Among the mycotoxins, aflatoxin is the most well-known and thoroughly studied and its prevention and control has been most successfully practiced in many countries.
- Fungal growth inhibition
The inhibition of fungal growth can be achieved by physical, chemical and biological treatments. After the crop is harvested, drying and proper storage and suitable transportation of the commodities are of prime importance. Factors contribute to the growth of fungi and toxin production include high moisture content, humid climate, warm temperature (25-40 °C), insect infestation and grain damage.
Physical methods
- Drying seeds and commodities to the safe moisture level (< 9-11%).
- Maintenance of the container or store house at low temperature and humidity.
- Keep out insects and pests from the storage.
- Gamma-irradiation of large-scale commodities.
- Dilution of the contaminated feed with safe feed.
Chemical methods
- Use of fungicides (acetic acid, propionic acid, benzoic acid, citric acid and their sodium salts, copper sulfate): 0.2–0.4 % in feed.
- Use of fumigants – ammonia: 0.2-0.4%
- Addition of herbal extracts (garlic, onion, clove oil, turmeric powder, thyme) : 0.25- 0.5%
Biological methods
Anti-fungal enzymes, chitinase and β-1,3 glucanase found in plant seeds may act as defense against pathogenic fungi, since chitin and glucan are major polymeric components of many fungal cell walls. Such polysaccharides in fungal cell wall could be enzymatically hydrolysed into smaller products resulting in killing of mycelia or spore of fungi. It is foreseen that seeds rich in such anti-fungal enzymes likely to resist the infestation of fungi. Use of non- toxigenic biocompetitive Aspergillus strains to out-compete the toxigenic isolates has been found effective in reducing pre harvest contamination with aflatoxin in peanut and cotton, however, the aflatoxin contamination process is so compelx that a combination of approaches will be required to eliminate toxin production. Application of non-toxigenic strains of Aspergillus flavus and Aspergillus parasiticus to soil in maize plots, favoured the reduction in colonization of toxigenic fungi in subsequent years, when the weather conditions were suitable for fungal growth and resulted in 65-80% decline in aflatoxin production as compared to control. Inoculation of chitosan, Bacillus subtilis and Trichoderma harzianum to pre harvest maize along with Aspergillus flavus inhibits aflatoxin production. Many anti-fungal metabolites (cyclic dipeptides, phenylactic acid, caproic acid, reuterin, lactic acid, acetic acid, fungicin) have been isolated from different cultures of lactic acid bacteria. Aflastin A, an anti-microbial compound produced by Streptomyces Spp,. MRI 142 strain of bacteria is known to inhibit aflatoxin production by Aspergillus parasiticus. Iturin, an anti-fungal peptide produced by Bacillus subtilis had inhibitory effect an Aspergillus parasiticus.
Plant breeding, genetic engineering and microarray
Genetic modification of mold susceptible plants holds some promise in ensuring food safety. This involves increasing production of compounds like anti-fungal proteins, hydroxamic acids, phenolics that reduce fungal contamination. This may be accomplished by introducing a novel gene to express the target compound, or enhancing the expression of such compounds by the existing genes, thereby capitalizing on the plant’s own defense mechanisms. Enzymes that catalyze production of anti-fungals could be targeted for their expression and such an approach is
being actively pursued by researchers. Enhanced expression of an α-amylase inhibitor in Aspergillus could result in reduced aflatoxin synthesis. Hybrid varieties of cereals with Bt (Bacillus thermophilus) genes have shown reduced aflatoxin production, probably due to higher resistance of plants against pest and insects.
A cluster of genes are responsible for aflatoxin production through pathway-specific transcriptional regulator. A total of 20 genes in the aflatoxin biosynthetic cluster and 3 additional genes outside the aflatoxin biosynthetic cluster responsible for aflatoxin production have been identified. Identification of critical genes governing aflatoxin formation could lead to use of non- aflatoxigenic bio-competitive strains of Aspergillus flavus through use of gene disruption techniques. The advances in molecular biology could aid in early detection of mycotoxin production in food/feed material. DNA-chip with microarray system containing oligonucleotide primers that are homologues to genes of several fungal species responsible for the expression of mycotoxins can be employed to forecast the mycotoxin production in advance and accordingly critical anti-fungal strategies can be employed. Such PCR based molecular techniques are of value in assessing the potential for mycotoxin production. The time gap between expression of a set of genes and actual mycotoxin production is about 4-5 days. This early forecasting of extent of mycotoxin production will help in adopting immediate preventive anti-fungal measures.
- Counteraction / Detoxification of aflatoxins
Aflatoxins in foods and feeds can be removed, inactivated or detoxified by physical, chemical and biological means. The treated products should be health safe from the chemicals and their essential nutritive value should not be deteriorated.
Physical methods
Physically, aflatoxin contaminated seeds can be removed by hand picking or photoelectric detecting machines, but this is labor oriented and expensive. Heating and cooking under pressure can destroy nearly 70% aflatoxin. Dry roasting can reduce about 50-70% of aflatoxin and sunlight drying of aflatoxin contaminated feed could reduce the toxin level by more than 70%. The addition of binding agents able to fix mycotoxins may reduce the bioavailability of these compounds in animals, and limit the presence of toxin residues in animal products. In
case of aflatoxin B1 (AFB1), hydrated sodium calcium aluminosilicates (HSCAS) and phyllosilicates derived from natural zeolites have a high affinity, both in vitro and in vivo. Zeolites, which are hydrated aluminosilicates of alkaline cations are able to adsorb AFB1. Bentonites have been shown to be effective for the adsorption of AFB1. Other clays, such as kaolin, sepiolite and montmorillonite, bind AFB1 but less effectively than HSCAS and bentonite. Activated charcoal is also effective against AFB1. Combination of yeast, zeolite and activated charcoal is found effective in reducing the toxicity of aflatoxin B1. The limitation of using binders is the reduction in bioavailability of certain trace minerals and vitamins in the diet. Some of these binders are not biodegradable and could pose environmental problem.
Chemical methods
A variety of chemical agents such as acids, bases (ammonia, caustic soda), oxidants (hydrogen peroxide, ozone, sodium hypochlorite), reducing agents (Bisulphites), chlorinated agents and formaldehyde have been used to degrade mycotoxins in contaminated feeds particularly aflatoxins. However, these techniques are not totally safe, are expensive and not well accepted by consumers.
Biological / microbiological methods
The biological decontamination of mycotoxins using yeast Saccharomyces cerevisiae and lactic acid bacteria has received much attention. Yeast and lactic acid bacterial cells are known to bind different toxins on the cell wall surface. This will be of immense value in reducing the mycotoxin hazards (Table 2), and effective binding strains of these microbes could eventually be used to minimize aflatoxin exposure and improving overall health in animals. Esterified glucomannan obtained from the cell wall of yeast Saccharomyces cerevisiae are able to bind AFB1 and provide protection against aflatoxins. About 500 gm of glucomannans from yeast cell- wall have the same adsorption capacity as 8 kg of clay. This binder reduces the AFM1 content of milk by 58% in cows given a diet contaminated with AFB1 at a concentration of 0.05% of dry mater. Probiotic strain of Lactobacillus acidophilus CU028 has shown to bind aflatoxin. Probiotic fermented milk containing Lactobacillus casei and Lactobacillus rhamnosus strains alone or in combination with chlorophyllin exhibited protective effect against aflatoxin B1–
induced hepatic damage. Acid treated lactic acid bacteria were able to bind high dosage of aflatoxin in gut conditions.
Biotransformation
Dual cultivation of Aspergillus niger, Mucor racemosus, Alternaria alternata, Rhizopus oryzae and Bacillus stearothermophilus with toxigenic strain of Aspergillus flavus results in 70- 80% degradation of aflatoxins. Certain microbes are also able to metabolize mycotoxins (Corynebacterium rubrum) in contaminated feed or to biotransform them(Rhizopius, Trichosporon mycotoxinivorans, Rhodotorula rubra, Geotrichum fermentans). However, these biological processes are generally slow and have a varied efficiency. Ruminants are considered to be relatively resistant to aflatoxins, due to biodegrading and biotransforming ability of rumen microbes compared to monogastric animals. This would be a great asset in biological detoxification of aflatoxins and with the help genetic engineering techniques, benefits of this can be better realized.
Table 2. Aflatoxin binding ability of different strains of yeast and bacteria
Isolates | Number of aflatoxin B1 binding strains | |||
| Percentage of binding | ||||
| <15 | 15-39 | 40-59 | >60 | |
| Yeast | ||||
| Saccharomyces cerevisiae | 1 | 8 | 3 | 3 |
| Condida krusei | 4 | 5 | 1 | 1 |
| Candida parapsilosis | ||||
| Trichosporon mucoides | 1 | – | – | – |
| Candida catenulanta | 1 | – | – | – |
| Bacteria | ||||
| Lactobacillus plantarum | – | – | 4 | 1 |
| Lactobacillus fermentum | – | – | – | 1 |
| Pediococcus acidilactici | – | – | 1 | – |
- Dietary manipulations
Hepatotropic nutrients and anti-oxidants
Various nutritional strategies have been employed to alleviate the adverse effects of aflatoxins. Addition of specific amino acids like methionine in excess of their requirement protect the chicks from growth depressing effects of AFB1, possibly through an increased rate of detoxification by glutathione, a sulfur amino acid metabolite. Supplementation of phenyl alanine has shown to alleviate toxicity of ochratoxin. Addition of vegetable oil (safflower oil, olive oil) to aflatoxin contaminated feed improves the performance of chicks. Aflatoxins cause toxicity through release of free radicals and lipid peroxidation. Hence, antioxidants could aid in the overall detoxification process in liver and hence may help in alleviation of aflatoxicosis. Butylated hydroxy toluene (BHT) is effective in preventing the adverse effects of AFB1. Vitamin E and Selenium supplementation also has shown to overcome negative effects of afla toxin. Of late, there is a growing interest in the use of phytochemicals (curcumin, flavonoids, resveratrol, Allixin, polyphenolics) as antioxidants in increasing the activity of antioxidant enzymes (SOD, catalase, glutathione peroxidase) and neutralizing the free radicals, thus, ameliorating the mycotoxin toxicity.
CONCLUSION
Aflatoxins are common in nature, hence minimizing the contamination is not a easy task due to the interaction of fungus with environment and feed material. This involves constant attention during the entire process of grain harvest, storage, feed manufacturing and animal production. Most effective methods (physical, chemical, biological, biotechnological) to improve seed production, cultivation, harvest and storage need to be adopted. Use of binders and understanding their mechanism of action is the current concept and research areas in use of microbes for decontamination and biotransformation of aflatoxins is gaining momentum. Biotechnological intervention in terms of developing transgenic fungal resistant crops and biological control using non-toxigenic, competitive fungal species holds a better promise in managing the problem of aflatoxicosis. Advancement in molecular techniques using fungal oligonucleotide probes with PCR based microarray analysis would help in early forecasting / detection of potential aflatoxin production, suggesting for critical control strategies.