Aflatoxicosis and cancer effects of Aflatoxin
Aflatoxin adsorption property of lactic acid bacteria could be used as a detoxification method which is most appropriate for food and dairy products.
Seven strains of lactic acid bacteria (Lactobacillus casei, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus bulgaricus, Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium bifidium) plus two mixed commercial cultures FD–DVS YCX11 50U (Lactobacillus bulgaricus + Streptococcus thermophilus) and FD–DVS ABT2 50U (Lactobacillus acidophilus+ Streptococcus thermophilus +Bifidobacterium bifidum) were tested for their AFB1 and AFM1 adsorption rate, Factors affecting such adsorption were studied.
The bioavailability of the adsorbed toxin was determined by feeding rats with L. casei-AFB1, L. casei-AFM1 complexes, and both free toxins and rats' liver tissues were examined. Adsorption reaction was carried out in 1 ml of phosphate buffer saline containing 1 µg of the toxin and about 1.7x1011 CFU of bacteria at 37°C for 2 hrs and pH of 7.3.
Out of the tested strains, L. casei showed the highest toxin removal rate of 34.1% and 27.7% for AFB1 and AFM1, respectively. These rates were increased by reducing the toxin into 0.5 µg /ml, lowering the reaction pH into acidic pH and using acid or heat killed cells. Moreover, the rate was increased by adapting L. casei to toxin adsorption by repeated exposure to toxin, the rate increased from 34.1% to 50% of AFB1. Toxin concentration higher than 0.5 µg / ml reduced the adsorption and caused changes in cell morphology. Aflatoxin bound by bacteria showed no toxicity effect on rats. Liver tissues of rats fed on the toxin complex were normal structure as compared to tissues of rats fed on free toxins which showed macroviscular fatty change, hydropic degeneration and congested hepatic sinusoids.
The toxin complex was not absorbed since it did not adhere to intestinal wall. Yoghurt and sweet cultured milk were processed from AFB1 and AFM1 contaminated milk using L. casei culture, giving a close rate of AFB1 removal as from PBS ”phosphate buffer saline” , but AFM1was at lower rate than from PBS buffer.
Aflatoxins are group of mycotoxins with mutagenic, carcinogenic, and immune suppressive properties. They have been classified by International Agency for Research on Cancer (IARC) as a class one human carcinogen (Kankaanp. et al., 2000). The toxin contamination is widespread and reaching humans or animals through their foods or by inhalation.
When absorbed into blood from small intestine, the toxin accumulates in various tissues of the body including the tissues of liver and respiratory, renal and gastrointestinal nervous, reproductive and immune system.
Of all tissues, liver contains the highest concentration, about 10 fold higher than in muscles (Liginsky et al., 1970).
AFB1 appeared in liver proteins after 6 h from injection, and hepatic enzymes are released after 24h. AFB1 was found in nucleus of liver cells, interacting with DNA, inhibiting RNA production (Clifford et al., 1976) and producing liver tumors which are hepatocellular carcinoma (Bulter et al., 1969). Moreover the AFB1 ingestion or inhalation by animal farm caused the decrease in milk & egg production, and is excreted in milk, eggs, and animal tissues (El-Nezami et al., 2000). A variety of detoxification methods for human foods and animal feeds have been reported. These included physical methods (bentonite, hydrated sodium calcium aluminosilicate, and clay adsorbents), chemical methods (5% NaOCl, 10% Chlorine gas, ammonia, and solvent extraction), phytochemicals (iso-thiocyanate, flavonoids, allicin, and chlorophyllin), and biological methods (Flavobacterium aurantiacum), However, a practical, large scale, cost effective and safe method for a complete detoxification of aflatoxin – containing human food or animal feeds are currently not available (El Nezami et al.,1998b). The problem becomes more difficult with human foods since the safe treatments or additives that could be permitted are difficult to find. For example, milk and dairy products such as ripened cheese are usually contaminated with aflatoxin M1, and sometimes with aflatoxin B1, cannot accept any of the above additive or treatments. Probiotic bacteria, microorganisms that confer health benefits when consumed by humans, are mostly lactic acid bacteria (LAB) and their safety has been proven by their safe use for ages. Beside their health benefits, probiotics protect against food mutagens such as heterocyclic amines, nitroso-compounds and aflatoxin (Hascard et al., 2001). Certain strains from LAB have been reported to adsorb aflatoxins B1, B2, G1, G2, M1, M2 and Zearalenone from liquid media, milk and from the intestine forming a stable complex. Viable and heat or acid killed bacteria bind the toxin and the rate of binding depends on the microorganism and the strain and is linear with toxin, and bacterial concentration. The adsorption is a physical process, bacterial cell wall binds the toxin with non – covalent weak bonds accompanied with some electrostatic attraction through lactinine like protein, polysaccharides and peptidoglycan (Haskard et al., 2000).Toxin binding is a fast process, its optimum temperature and pH were 35 - 37°C, 6 – 7.5 respectively (Ambrosini et al., 1999). The adsorption rates varied from 5.6% by Lactobacillus lactis to 88% in vitro and 92% in vivo (chicks) by Lactobacillus rhamnosus GG (El-Nezami et al.,2000, Peltonen et al.,2001). L. rhamnosus GG and L. rhamnosus Lc705 removed AFM1, from skim and whole milk with lower percentages than from phosphate buffer medium, with a range from 18.8 to 69.6% (Pierides et al., 2000).Number of treatments affects the bacterial adsorption of the toxin. Treating the bacteria with 1M of HCl, boiling at 100°C, and autoclaving increased toxin binding and the acid treatment was the most effective. Exposure of the cells to ethanol, U.V. irradiation, sonication and alkali showed either no effect or reduced binding (El- Nezami et al., 1998b). Heat as well as acid alter surface properties of bacteria leading to higher toxin adsorption and lower desorption rates. The complex, though stable, is reversible and of extracelluler nature, its stability depends on strain, treatment and the environmental conditions (Peltonen et al., 2001). Actually, conditions, in duodenum enhanced bacteria to bind the toxin and improved the complex stability, and the toxin is not released back into the duodenum contents. The complex was found to be stable under the luminal conditions for one hour (Ambrosini et al.,1999).The toxin can be removed from the complex partly (~30%) by excessive washing with buffer solution and almost completely (90%) with organic solvents. However, autoclaving, temperature exposure from 4 to 37°C and pH 2 -10 did not release the toxin (Gratz et al., 2005). Most important properity of this method is that binding the toxin by bacteria reduced their adhesion into the intestinal epithelium, preventing its accumulation in the intestine causing its release out of the body (Kankaanp et al., 2000).
For example, the complex reduced capability of L. rhamnosus GG adhesion into the intestinal from 30 to 5 %. There was a 74% reduction in the uptake of AFB1 by the intestinal tissue in the presence of L. rhamnosus GG. In vitro LAB reduced AFB1 by 54% in the soluble fraction of luminal fluid in one minute. Therefore this properity allowed the detoxification without the need for the removal of the bacterial-toxin complex from the food which is an impractical step. Actually, the toxin changed bacterial cells morphology and this might lead to a change in adhesion sites. (Kankaanpää et al., 2000).
Therefore, probiotics beside their immune modulating effect are good prospect for physical detoxification of foods. Actually they are regularly used in food processing and if they are not part of the process, heat killed bacteria can do the detoxification without altering the taste or acidity of the food. However, that this method renders the toxin unavailable for absorption in the intestine thus alleviating the toxin harmful effect is yet to be proven. Moreover, the method requires screening the available probiotic bacteria for selecting the proper microorganism and the strain which have high adsorption rate, and in the same time fits processing of a certain food. After the selection, the factors within the food affecting complex formation should be studied. Therefore, research was carried out to:
1. Test number of the available probiotics for their rate of AFB1 and AFM1 binding and study the affecting factors.
2. Use of the selected probiotic bacteria for manufacturing yogurt and sweet cultured milk from AFB1 and AFM1 contaminated milk.
3. Determine adhesion of AFB1–bacterial complex, AFM1–bacterial complex to mucus cells.
4. Study the morphological changes of probiotic on alter binding toxin.
5. Determine the bioavailabity of the toxin when adsorbed by bacteria by comparing the effect of feeding rats on pure AFB1 and AFM1 as well as their bacterial – toxin complex on rats' liver tissues.
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