In this article we will discuss about: 1. Introduction to Aflatoxins 2. Occurrence and Distribution of Aflatoxins 3. Fluorescence Production 4. Properties 5. Health Hazards 6. Factors Favouring Aflatoxin Production 7. Analysis for Aflatoxins in Foods and Feeds 8. Control and Management 9. Biological Control.
- Introduction to Aflatoxins
- Occurrence and Distribution of Aflatoxins
- Fluorescence Production of Aflatoxins
- Properties of Aflatoxins
- Health Hazards Caused by Aflatoxins
- Factors Favouring Aflatoxin Production
- Analysis for Aflatoxins in Foods and Feeds
- Control and Management of Aflatoxins
- Biological Control of Aflatoxins
1. Introduction to Aflatoxins:
Mycotoxin (Gk. mykes, mushrooms-fungus) is a toxin produced by an organism of the fungus includes mushrooms, moulds and yeasts. Aflatoxins are naturally occurring mycotoxins that are produced as secondary metabolites by some strains of Aspergillus flavus and A. parasiticus, plus related species A. nomius and A. niger on a varity of food stuff causing health hazards to animals consuming them.
2. Occurrence and Distribution of Aflatoxins:
Aflatoxin in swine has been reported in Georgia USA in 1940. The death of swine was traced as a result of feeding mould maize. Similar incident occurred in 1950 at Alabama. In 1960 more than 100,000 young turkeys on poultry farms in England died in the course of a few months from an apparently new disease that was termed Turkey X disease.
It was soon found that the difficulty was not limited to turkeys. Ducklings and young pheasants were also affected and heavy mortality was experienced. A careful survey of the early outbreaks showed that they were all associated with feeds, namely Brazilian peanut meal.
An intensive investigation of the suspect peanut meal was undertaken and it was quickly found that this peanut meal was highly toxic to poultry and ducklings with symptoms typical of Turkey X disease.
Speculations made during 1960 regarding the nature of the toxin suggested that it might be of fungal origin. In fact, the toxin-producing fungus was identified as Aspergillus flavus in 1961 and the toxin was given the name Aflatoxin by virtue of its origin (A. flavus > Afla).
This discovery has led to a growing awareness of the potential hazards of these substances as contaminants of food and feed causing illness and even death in humans and other mammals.
In India Aflatoxin levels were found in range of 1000 to 5000 ppb (parts per billion) in 12% of groundnut samples in 1965 during a study conducted in Andhra Pradesh, 50% of groundnut cake samples were positive for aflatoxin in 1976 in Madhya Pradesh, similarly a study conducted by National Institute of Nutrition (N.I.N) (Hyderabad) recorded aflatoxin contamination in maize grain up to 1560 ppb which resulted death of 100 people by acute hepatitis in Tribal belts in western India in 1986.
Biology of Aspergillus flavus and Aspergillus Parasiticus:
The two fungi A. flavus Link ex Fr. and A. parasiticus Spear, are closely related and grow as saprophytes on organic material left on and in the soil. They are distributed worldwide, with a tendency to be more common in countries with tropical climates that have extreme ranges of rainfall, temperature and humidity.
Members of the genus Aspergillus are characterized by the production of non-septate conidiophores, which are quite distinct from hyphae and which are swollen at the top to form a vesicle on which numerous specialized spore-producing cells, known as phialides or sterigmata are borne either directly (uniseriate) or on short outgrowths known as metulae (biseriate).
Some time difficulty may arise especially to determine because the primary sterigmata are tiny and are easily obscured by spores or other sterigmata. Colonies of A. flavus are green- yellow to yellow-green or green on Czapek’s agar. They usually have biseriate sterigmata; reddish-brown sclerotia are often present, conidia are finely roughened, variable in size and oval to spherical in shape.
Colonies of A. parasiticus dark green on Czepak’s agar, remain green with age. Sterigmata are uniseriate, sclerotia are usually absent; conidia are coarsely echinulate, uniform in shape, size and echinulation. There are about 18 different types of aflatoxins identified major members are four plus two additional metabolic products, designated as B1; B2, G1; G2, and M2.
3. Fluorescence Production of Aflatoxins:
The aflatoxins fluorescence strongly in ultraviolet light (365 nm). Designation of aflatoxins B1, and B2 resulted from the exhibition of blue fluorescence of the relevant structures under ultraviolet light, G1 and G2 form yellow green fluorescence under ultraviolet light.
While M1 and M2 were first isolated from milk of lactating animals fed aflatoxin preparations. A. flavus typically produces B1 and B2, whereas A. parasiticus produce G1 and G2 as well as B1 and B2. Four other aflatoxins M1, M2, B2A and G2A (Fig. 20.4) were isolated from cultures of A. flavus and A. parasiticus.
4. Properties of Aflatoxins:
Studies revealed (Table 20.1) that aflatoxins are produced primarily by some strains of A. flavus and by most, if not all, strains of A. parasiticus, plus related species, A. nomius and A. niger. Moreover, these studies also revealed that there are four major aflatoxins: B1, B2, G1, G2 plus two additional metabolic products, M1 and M2, that are of significance as direct contaminants of foods and feeds.
The aflatoxins M1 and M2 were first isolated from milk of lactating animals fed aflatoxin preparations; hence, the M designation. Whereas the B designation of aflatoxins B1 and B2 resulted from the exhibition of blue fluorescence under UV-light, while the G designation refers to the yellow- green fluorescence of the relevant structures under UV-light.
These toxins have closely similar structures and form a unique group of highly oxygenated, naturally occurring heterocyclic compounds. Food products contaminated with aflatoxins include cereal (maize, sorghum, pearl millet, rice, wheat etc.), oilseeds (groundnut, soybean, sunflower, cotton), spices (chilies, black pepper, coriander, turmeric, zinger), tree nuts (almonds, pistachio, walnuts, coconuts) and milk.
Aflatoxins are potent toxic, carcinogenic, mutagenic, immunosuppressive agents, produced as secondary metabolites by the fungus Aspergillus flavus and A. parasiticus on variety of food products. Among 18 different types of aflatoxins identified, major members are aflatoxin B1; B2, G1 and G2 (Fig. 20.4). Aflatoxin B1 (AFB1) is normally predominant in amount in cultures as well as in food products.
Pure AFB1 is pale-white to yellow crystalline, odorless solid. Aflatoxins are soluble in methanol, chloroform, acetone, acetonitrile. Aflatoxin M1 and M2 are major metabolites of aflatoxin B1 and B2 respectively, found in milk of animals that have consumed feed contaminated with aflatoxins.
Aflatoxins are normally refers to the group of difuranocoumarins and classified in two broad groups according to their chemical structure; the difurocoumarocyclopentenone series (AFB1, AFB2, AFB2A, AFM1, AFM2, AFM2A and aflatoxicol) and the difurocoumarolactone series (AFG1, AFG2, AFG2A, AFGM1, AFGM2, AFGM2A and AFB3).
The aflatoxins display potency of toxicity, carcinogenicity, mutagenicity in the order of AFB1 > AFG1 > AFB2 > AFG2 as illustrated by their LD50 values for day-old ducklings.
Structurally the dihydrofuran moiety, containing double bond, and the constituents liked to the coumarin moiety are of importance in producing biological effects. The aflatoxins fluorescence strongly in ultraviolet light (ca. 365 nm); B1 and B2 produce a blue fluorescence where as G1 and G2 produce green fluorescence.
5. Health Hazards Caused by Aflatoxins:
In 1967, twenty people of Taiwan became ill with apparent food poisoning due to moldy rice that was later confirmed to contain Aflatoxin B1. In 1978 Significant levels of aflatoxin were found in the livers 23 of children who had died of Rye’s syndrome in Thailand. Children who died in Czechoslovakia and New Zealand in 1977 have also been found to have aflatoxin in their liver at autopsy.
The out break of hepatitis that effected 400 people in western states of India in 1974 of whom 100 died by consuming corn contaminated with A. flavus containing about 15 mg/kg aflatoxin. Aflatoxicosis is primarily a hepatic disease. The susceptibility of individual animals to aflatoxins varies considerably depending on species, age, sex, and nutrition.
In fact, aflatoxins cause liver damage, decreased milk and egg production, recurrent infection as a result of immunity suppression (e.g., salmonellosis), in addition to embryo toxicity in animals consuming low dietary concentrations. While the young of a species are most susceptible, all ages are affected but in different degrees for different species.
Clinical signs of aflatoxicosis in animals include gastrointestinal dysfunction, reduced re-productivity, reduced feed utilization and efficiency, anemia, and jaundice. Nursing animals may be affected as a result of the conversion of aflatoxin B1 to the metabolite aflatoxin M1 excreted in milk of dairy cattle.
Fig. 20.6. A rat live fed with high doses of aflatoxins B1. Notice the induced tumors in the liver.
The induction of cancer by aflatoxins has been extensively studied. Aflatoxin B1, aflatoxin M1 and aflatoxin G1 have been shown to cause various types of cancer in different animal species.
However, only aflatoxin B1 is considered by the International Agency for Research on Cancer (IARC) as having produced sufficient evidence of carcinogenicity in experimental animals to be identified as a carcinogen (Fig. 20.5 and 20.6).
Thus aflatoxins were proved to be potent toxic .carcinogenic, mutagenic, immunosuppressive agents. Their toxicity is illustrated by their LD50 values for day old ducklings (Table 20.2).
Effect of A. Flavus and Aflatoxins Contamination:
Deteriorate in grain quality due to A. flavus growth and become unfit for marketing and consumption. In groundnut, seed and non-emerged seedling decay and afla-root disease was observed due to fungus attack. Aflatoxins contamination in grain poses a great threat to human and livestock health as well as international trade.
According to FAO estimates, 25% of the world food crops are affected by mycotoxins every year. And also crop loss due to aflatoxins contamination costs US producers more than $100 million per year on average including $ 26 millions to peanuts ($ 69.34/ha).
6. Factors Favouring Aflatoxin Production:
Fungal growth and aflatoxin contamination are the consequence of interactions among the fungus, the host and the environment. The appropriate combinations of these factors determine the infestation and colonization of the substrate, and the type and amount of aflatoxin produced.
However, a suitable substrate is required for fungal growth and subsequent toxin production, although the precise factor(s) that initiates toxin formation is not well understood.
Water stress, high-temperature stress, and insect damage of the host plant are major determining factors in mold infestation and toxin production. Similarly, specific crop growth stages, poor fertility, high crop densities, and weed competition have been associated with increased mold growth and toxin production.
Aflatoxin formation is also affected by associated growth of other molds or microbes. For example, pre-harvest aflatoxin contamination of peanuts and corn is favored by high temperatures, prolonged drought conditions, and high insect activity; while post harvest production of aflatoxins on corn and peanuts is favored by warm temperatures and high humidity.
7. Analysis for Aflatoxins in Foods and Feeds:
Sampling and Sample Preparation:
Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample preparation, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level.
In this regard, specific plans have been developed and tested rigorously for some commodities such as corn, peanuts, and tree nuts; sampling plans for some other commodities have been modeled after them. A common feature of all sampling plans is that the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample.
Thin layer chromatography (TLC), also known as flat bed chromatography or planar chromatography is one of the most widely used separation techniques in aflatoxin analysis. Since 1990, it has been considered the AOAC official method and the method of choice to identify and quantitative aflatoxins at levels as low as 1 rg/g (Table 20.3). The TLC method is also used to verify findings by newer, more rapid techniques.
Liquid chromatography (LC) is similar to TLC in many respects, including analytic application, stationary phase, and mobile phase. Liquid chromatography and TLC complement each other.
For an analyst to use TLC for preliminary work to optimize LC separation conditions is not unusual. Liquid chromatography methods for the determination of aflatoxins in foods include normal-phase LC (NPLC), reversed-phase LC (RPLC) with pre- or before-column derivatization (BCD), RPLC followed by post column derivatization (PCD), and RPLC with electrochemical detection.
Thin layer chromatography and LC methods for determining aflatoxins in food are laborious and time consuming. Often, these techniques require knowledge and experience of chromatographic techniques to solve separation and interference problems.
Through advances in biotechnology, highly specific antibody-based tests are now commercially available that can identify and measure aflatoxins in food in less than 10 minutes.
These tests are based on the affinities of the monoclonal or polyclonal antibodies for aflatoxins. The three types of immunochemical methods are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoaffinity column assay (ICA).
Manonmani (2006) developed a rapid method for assessment of aflatoxigenic fungi in food using an indigenously designed primers for aflatoxin regulatory gene aflR in PCR shows positive amplification with DNA of aflatoxigenic A. flavus and A. parasiticus can detect up to 100 cfu of target toxigenic fungi.
8. Control and Management of Aflatoxins:
Aflatoxins are considered unavoidable contaminants of food and feed, even where good manufacturing practices have been followed. The FDA has established specific guidelines on acceptable levels of aflatoxins in human food and animal feed by establishing action levels that allow for the removal of volatile lots from commerce.
The action level for human food is 20 ppb total aflatoxins, with the exception of milk which has an action level of 0.5 ppb for aflatoxin M1. The action level for most feeds is also 20 ppb.
However, it is very difficult to accurately estimate aflatoxins concentration in a large quantity of material because of the variability associated with testing procedures. No presentable data is available to form an experimentally valid basis for regulation of aflatoxin levels in food and feed hence, the true aflatoxin concentration in a lot cannot be determined with 100% certainty.
In US and Europe aflatoxins are only mycotoxins jurisdictionally regulated (Table 20.2, and Table 20.3). In Asian countries regulation is primarily introduced to protect export market. On the other hand domestic regulatory measures on aflatoxin received little attention. In India mycotoxin legislation have been introduced but its implementation is inadequate.
Because aflatoxin contamination is unavoidable, numerous strategies for their detoxification have been proposed. These include physical methods of separation, thermal inactivation, irradiation, solvent extraction, adsorption from solution, microbial inactivation, and fermentation. Chemical methods of detoxification are also practiced as a major strategy for effective detoxification.
Structural Degradation Following Chemical Treatment:
A diverse group of chemicals has been tested for the ability to degrade and inactivate aflatoxins. A number of these chemicals can react to destroy (or degrade) aflatoxins effectively but most are impractical or potentially unsafe because of the formation of toxic residues or the perturbation of nutrient content and the organoleptic properties of the product.
Two chemical approaches to the detoxification of aflatoxins that have received considerable attention are ammoniation and reaction with sodium bisulfite.
Many studies provide evidence that chemical treatment via ammoniation may provide an effective method to detoxify aflatoxin-contaminated corn and other commodities. The mechanism for this action appears to involve hydrolysis of the lactone ring and chemical conversion of the parent compound aflatoxin to numerous products that exhibit greatly decreased toxicity.
On the other hand, sodium bisulfite has been shown to react with aflatoxins (B1, G1 and M1) under various conditions of temperature, concentration, and time to form water-soluble products.
Modification of Toxicity by Dietary Chemicals:
The toxicity of mycotoxins may be strongly influenced by dietary chemicals that alter the normal responses of mammalian systems to these substances.
A variable array of chemical factors, including nutritional components (e.g., dietary protein and fat, vitamins, and trace elements), food and feed additives (e.g., antibiotics and preservatives), as well as other chemical factors may interact with the effects of aflatoxins in animals.
Alteration of Bioavailability by Aflatoxin Chemisorbents:
A new approach to the detoxification of aflatoxins is the addition of inorganic sorbent materials, known as chemisorbents, such as hydrated sodium calcium aluminosilicate (HSCAS) to the diet of animals. HSCAS possesses the ability to tightly bind and immobilize aflatoxins in the gastrointestinal tract of animals, resulting in a major reduction in aflatoxin bioavailability.
9. Biological Control of Aflatoxins:
Ciegler (1966) after screening about 1000 microorganisms found Flavobacterium aurantiacum NRRL B-164 ability to remove aflatoxin B1 form solution.
Milk corn oil and peanut butter were artificially contaminated with 600, 700 and 700 micro gram of aflatoxin B1 respectively when viable cellos of Flavobacterium aurantiacum were added to each of these foodstuffs aflatoxin concentration were reduced to about 0.
When viable cells were mixed with soybean, corn and peanut seeds contaminated with aflatoxin were completely removed from corn and peanut and about 86% from soybean. Displacement of toxigenic strains of A. flavus with a toxigenic strains is one of the effective strategy.
This strategy is possible because of great variability of phenotypes of A. flavus in agricultural fields and common occurrence of a toxigenic strains. Use of bio-fungicides can be a better choice for controlling aflatoxin fungi.
Neem extracts have been reported to block aflatoxin biosynthesis up to 98% (Bhatnagar and McCormic, 1988). Essential oils of Thymus eriocalyx and Gracinia indica (extract) have been reported inhibitory against aflatoxin producing fungi and also inhibit aflatoxin production.