**13. Decontamination, detoxification, exposition**

Aflatoxin produced by *Aspergillus flavus, A. parasiticus* and *A. nomius* crops contamination, is a worldwide food safety concern. Several strategies, including chemical, physical and bio‐ logical control methods have been investigated to manage these potent toxic secondary me‐ tabolites in foods. Among them, biological control seems nowadays to be the most promising approach for the aflatoxins control. From the food safety point of view, fermenta‐ tion with microorganisms, a technique quite commonly used in food production (e.g. fer‐ mentation with lactic acid bacteria, alcoholic fermentation, conventional fermentation of the protein from vegetables as common in South Asia, etc.) should be preferred. In optimal con‐ ditions, this procedure can result in a mycotoxin-free food or feed.

The reaction of aflatoxins to various physical conditions and reagents have been studied ex‐ tensively because of the possible application of such reactions to the detoxification of afla‐ toxins contaminated material. Aflatoxins in dry state are stable to heat up to the melting point. However, in the presence of moisture and at elevated temperatures there is destruc‐ tion of aflatoxin and this can occur with aflatoxin in oilseed meals, roasted peanuts or in aqueous solution at pH 7. Although the reaction products have not been examined in detail it seems likely that such treatment leads to opening of the lactone ring with the possibility of decarboxylation at elevated temperatures. At a temperature of about 100°C, ring opening followed by decarboxylation occurs, and reaction may proceed further, leading to the loss of the methoxy group from the aromatic ring.

LC/MS has been used as a confirmation technique for the already well established, reliable and robust LC-FL methodology (Kussak, Nilsson, Andersson, Langridge, 1995; Abbas, Williams, Windham, Pringle, Xie, Shier, 2002; Blesa, Soriano, Molto, Marin, Manes, 2003; Abbas, Cart‐ wright, Xie, Shier, 2006) and has also been used to confirm positive results of TLC and ELISA based screening analyses. All the aflatoxins exhibit good ESI ionisation efficiency in the posi‐

iano, Molto, Marin, Manes, 2003; Ventura, Gomez, Anaya, Diaz, Broto, Agut, Comellas, 2004; Kussak, Nilsson, Andersson, Langridge, 1995) and typically, for aflatoxins B1, B2, G1 and G2, the formation of sodium adduct ions can easily be suppressed by the addition of ammonium ions to the mobile phase leading to a better mass spectroscopy (MS) sensitivity (Cavaliere, Foglia, Pastorini, Samperi, Lagana, 2006). Reports about the utility of atmospheric pressure chemical ionization (APCI) interfaces and ionization efficiencies in this mode seem to be highly depend‐ ent on the aflatoxin studied and the APCI interface geometry (Abbas, Williams, Windham,

This method has been proved to be more sensitive for the simultaneous determination of aflatoxins B1, B2, G1, G2, M1, M2, and moreover smaller sample volumes of serum can be used for the analysis. Aflatoxins are in free equilibrium with the albumin combined form and it is reported in the literature the effect of pH and/or serum concentration of fatty acids on the formation of the adducts. Moreover, a recent study showed that green tea polyphenols might modulate the formation of the adducts between aflatoxin B1 and albumin (Tang,

Advanced spectrometric methods, such as LC-MS/MS, permit quantification and recogni‐ tion of the free aflatoxins in the sera with fewer problems on recovery, sensitivity and chem‐ ical identification (Santini, Ferracane, Meca, Ritieni, 2009; Huang, Zheng, Zengxuan, Yongjiang, Yiping, 2010) evaluating the aflatoxin exposure directly from their free forms.

Aflatoxin produced by *Aspergillus flavus, A. parasiticus* and *A. nomius* crops contamination, is a worldwide food safety concern. Several strategies, including chemical, physical and bio‐ logical control methods have been investigated to manage these potent toxic secondary me‐ tabolites in foods. Among them, biological control seems nowadays to be the most promising approach for the aflatoxins control. From the food safety point of view, fermenta‐ tion with microorganisms, a technique quite commonly used in food production (e.g. fer‐ mentation with lactic acid bacteria, alcoholic fermentation, conventional fermentation of the protein from vegetables as common in South Asia, etc.) should be preferred. In optimal con‐

The reaction of aflatoxins to various physical conditions and reagents have been studied ex‐ tensively because of the possible application of such reactions to the detoxification of afla‐ toxins contaminated material. Aflatoxins in dry state are stable to heat up to the melting

and sodium adduct ions (Blesa, Sor‐

tive ion mode with abundant protonated molecules [MH]+

360 Aflatoxins - Recent Advances and Future Prospects

Pringle, Xie, Shier, 2002; Abbas, Cartwright, Xie, Shier, 2006).

Tang, Xu, Luo, Huang, Yu, Zhang, Gao, Cox, Wang, 2008).

**13. Decontamination, detoxification, exposition**

ditions, this procedure can result in a mycotoxin-free food or feed.

In alkali solution reversible hydrolysis of the lactone moiety occurs. Recyclization has been observed after acidification of a basic aflatoxin containing solution.

In the presence of acids, aflatoxin B1 and G1 are converted in to aflatoxin B2A and G2A due to acid-catalyzed addition of water to the double bond in the furan ring. In the presence of ace‐ tic anhydride and hydrochloric acid the reation proceeds further to give the acetoxy deriva‐ tive. Similar adducts of aflatoxin B1 and G1 are formed with formic acid-thionyl chloride, acetic acid-thionyl chloride and trifluoroacetic acid.

Many oxidizing agents, e.g. sodium hypochlorite, potassium permanganate, chlorine, hy‐ drogen peroxide, ozone and sodium perborate react with aflatoxin and change the aflatoxin molecule in some way as indicated by the loss of fluorescence. The mechanisms of these re‐ actions are uncertain and the reaction products remain unidentified in most cases. Reduc‐ tion of aflatoxin B1 and B2 with sodium borohydride yielded aflatoxin RB1 and RB2, respectively. These arise as a result of opening of the lactone ring followed by reduction of the acid group and reduction of the keto group in the cyclopentene ring. Hydrogenation of aflatoxin B1 and G1 yields aflatoxin B2 and G2 respectively. Further reduction of aflatoxin B1 using 3 moles of hydrogen yields tetrahydroxyaflatoxin.

Food and feed contaminated with mycotoxins pose a severe health risk to animals and they may cause big economical losses due to the lower efficacy of animal husbandry and crop performances.

In addition, directly or indirectly (carry on through animal products) contaminated foods may also pose a health risk to humans. For this reason it is understandable that many re‐ search has been addressed in an attempt to salvage mycotoxin contaminated commodities and to avert health risks associated with the toxins.

Relevant basic criteria to be followed when a decontamination strategy is assessed have been suggested (Scott, 1990; Pomeranz, Bechtel, Sauer, Seitz, 1990):


**•** it must be economically feasible (the cost of decontamination should be less than the val‐ ue of contaminated commodity).

*pus oryzae, Rhizopus solonifer* and a protozoan *Tetrahymena pyriformis* (Doyle, Applebaum,

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 363

Recently, a growing interest can be observed concerning the use of *Rhodococci* for aflatoxins degradation: these microorganisms have a wide-range ability to degrade compounds like aflatoxins (Alberts, Engelbrecht, Steyn, Holzapfel, van Zyl, 2006; Teniola, Addo, Brost, Farb‐ er, Jany, Alberts, Van Zyl, Steyn, Holzapfel, 2005). Teniola et al. (Teniola, Addo, Brost, Farb‐ er, Jany, Alberts, Van Zyl, Steyn, Holzapfel, 2005) reported the degradation of aflatoxin B1 using liquid cultures of *Rhodococcus erythropolis* and the analysis of the intracellular extracts separated from *Rhodococcus erythropolis* liquid cultures suggested that a cascade of enzymat‐ ic reactions with loss of fluorescence (the intact aflatoxin is a fluorescent compound and degradation results in loss of fluorescence in time) occurred. Aflatoxin B1 is probably de‐ graded by the same enzymes (biphenyl-dioxygenases, dihydro-diol-dehydrogenases, and hydrolases) that are involved in catabolic pathways of polychlorinated biphenyls. Knowl‐ edge of gene coding for these enzymes may be helpful in development and production of new effective enzyme preparations for degradation of aflatoxins. The role of trace metal ions in microbial aflatoxin B1 degradation has been studied studied by Souza et al. (Souza, Brack‐ ett, 1998) who found that copper and zinc ions may inhibit the degradation of aflatoxin B1 by *Flavobacterium aurantiacum*. This effect is probably connected with an influence on the en‐ zyme system involved in the degradation process. Peltonen et al. (Peltonen, El-Nezami, Sal‐ minen, Ahokas, 2000) and El Nezami et al. (El-Nezami, Kankaanpaa, Salminen, Ahokas, 1998) studied the ability of dairy strains of lactic acid bacteria to bind Aflatoxin B1. It has been observed that *Lactobacillus rhamnosus* can significantly remove Aflatoxin B1 compared with other strains. Removal was observed as very rapid, with 80% of toxin removed within

Several bacterial species, such as *Bacillus*, *Lactobacilli*, *Pseudomonas*, *Ralstonia* and *Burkholderia* spp., have shown ability to inhibit fungal growth and production of aflatoxins by *Aspergillus* spp. in laboratory tests. Palumbo et al. (Palumbo, Baker, Mahoney, 2006) reported that a number of *Bacillus*, *Pseudomonas*, *Ralstonia* and *Burkholderia* strains could completely inhibit *A. flavus* growth. *B*.*subtilis* and *P*. *solanacearum* strains isolated from maize soil were also able to inhibit aflatoxin accumulation. In most cases, although these strains were highly effective against fungal growth and against the produced toxins in laboratory conditions, they did not give good efficacies on field. This could be attributed to the difficulty to bring the bacte‐ rial cells to the *Aspergillus* infection sites on commodities under field conditions. Saprophytic yeast species, such as *Candida krusei* and *Pichia anomala*, have revealed promising efficacy as biocontrol agents for aflatoxins decontamination (Yin, Yan, Jiang, Ma, 2008). In a similar way to bacterial agents, these yeast strains were able to significantly inhibit *Aspergillus* growth and resultant toxins in laboratory conditions. Shetty et al. (Shetty, Hald, Jespersen, 2007) observed that the ability of *S. cerevisiae* to bind aflatoxin B1 was strain specific with 7 strains binding 10-20%, 8 strains binding 20-40% and 3 strains binding more than 40% of the added aflatoxin B1. Though the yeasts are considered to be potential biocontrol agents for the aflatoxins management, further experiments conducted on field are necessary to test

their efficacies in reducing aflatoxin contamination in real on field situations.

Brackett, Marth, 1982; Karlovsky, 1999).

the first 60 min of treatment.

The main three possibilities to avoid any possible harmful effects of contamination of food and feed caused by mycotoxins habe been described by Halàsz et al. (Halasz, Lasztity, Abo‐ nyi, Bata, 2009):


Although the different methods used at present are to some extent successful, they have big disadvantages with limited efficacy and possible losses of important nutrients and normally with high costs. It is a common opinion that the best solution for decontamination should be detoxification by biodegradation, giving a possibility for removal of mycotoxins under mild conditions without using harmful chemicals without significant losses in nutritive value and palatability of decontaminated food and feed. One of the most frequently used strategies for biodegradation of mycotoxins includes isolation of microorganisms able to degrade the giv‐ en mycotoxin and treatment of food or feed in an appropriate fermentation process.

Thousand of microorganisms habe been screened for their ability to degrade aflatoxins from solutions (Lillehoj, Ciegler, Hall, 1967; Ciegler, Lillehoj, Peterson, Hall, 1996). As a result it was found that only one bacterium, the *Flavobacterium aurantiacum* B-184, was able to elimi‐ nate aflatoxin from solutions and uptake of the mycotoxin by the cells was influenced by pH and temperature.

Another interesting result was that an high concentration populations of the cells, more than 1011 per mL, is more useful to remove the aflatoxin from solutions than lower cell concentra‐ tions. Large populations of heat inactivated cells were also shown to bind some aflatoxin from solution, which was easily recovered by washing with water (Line, Brackett, 1967). The ability of *Flavobacterium aurantiacum* B-184 to remove aflatoxins from foods was demonstrat‐ ed in milk, vegetable oil, corn, peanut, peanut butter and peanut milk (Hao, Brackett, 1988; Hao, Brackett, 1989; Line, Brackett, 1995). To assess the exact fate of the aflatoxin B1 treated with *Flavobacterium aurantiacum*, Line et al. used radio-labeled carbon (C14) aflatoxin B1 and detected the formed radioactive carbon dioxide confirming this way the biodegradation pathway of aflatoxin (Line, Brackett, Wilkinson, 1994).

It should be noted that the interest of the biological approach to degrade aflatoxin is increas‐ ing since the consumers prefer this tool to chemical treatments used on food and feed to eliminate aflatoxins.

Nevertheless, one of the big obstacle to the developing of biological approaches is the bright pigmentation associated with the bacterium treatment, that hampers the applicability for food and feed. Microorganisms that are able to degrade aflatoxin B1 include *Corynebacterium rubrum*, *Aspergillus niger, Trichoderma viride, Mucor ambiguus, Dactylium dendroides, Mucor gri‐ seocyanus, Absidia repens, Helminthosporium sativum, Mucor alternans, Rhizopus archisus, Rhizo‐* *pus oryzae, Rhizopus solonifer* and a protozoan *Tetrahymena pyriformis* (Doyle, Applebaum, Brackett, Marth, 1982; Karlovsky, 1999).

**•** it must be economically feasible (the cost of decontamination should be less than the val‐

The main three possibilities to avoid any possible harmful effects of contamination of food and feed caused by mycotoxins habe been described by Halàsz et al. (Halasz, Lasztity, Abo‐

Although the different methods used at present are to some extent successful, they have big disadvantages with limited efficacy and possible losses of important nutrients and normally with high costs. It is a common opinion that the best solution for decontamination should be detoxification by biodegradation, giving a possibility for removal of mycotoxins under mild conditions without using harmful chemicals without significant losses in nutritive value and palatability of decontaminated food and feed. One of the most frequently used strategies for biodegradation of mycotoxins includes isolation of microorganisms able to degrade the giv‐

Thousand of microorganisms habe been screened for their ability to degrade aflatoxins from solutions (Lillehoj, Ciegler, Hall, 1967; Ciegler, Lillehoj, Peterson, Hall, 1996). As a result it was found that only one bacterium, the *Flavobacterium aurantiacum* B-184, was able to elimi‐ nate aflatoxin from solutions and uptake of the mycotoxin by the cells was influenced by pH

Another interesting result was that an high concentration populations of the cells, more than 1011 per mL, is more useful to remove the aflatoxin from solutions than lower cell concentra‐ tions. Large populations of heat inactivated cells were also shown to bind some aflatoxin from solution, which was easily recovered by washing with water (Line, Brackett, 1967). The ability of *Flavobacterium aurantiacum* B-184 to remove aflatoxins from foods was demonstrat‐ ed in milk, vegetable oil, corn, peanut, peanut butter and peanut milk (Hao, Brackett, 1988; Hao, Brackett, 1989; Line, Brackett, 1995). To assess the exact fate of the aflatoxin B1 treated with *Flavobacterium aurantiacum*, Line et al. used radio-labeled carbon (C14) aflatoxin B1 and detected the formed radioactive carbon dioxide confirming this way the biodegradation

It should be noted that the interest of the biological approach to degrade aflatoxin is increas‐ ing since the consumers prefer this tool to chemical treatments used on food and feed to

Nevertheless, one of the big obstacle to the developing of biological approaches is the bright pigmentation associated with the bacterium treatment, that hampers the applicability for food and feed. Microorganisms that are able to degrade aflatoxin B1 include *Corynebacterium rubrum*, *Aspergillus niger, Trichoderma viride, Mucor ambiguus, Dactylium dendroides, Mucor gri‐ seocyanus, Absidia repens, Helminthosporium sativum, Mucor alternans, Rhizopus archisus, Rhizo‐*

**•** inhibition of absorption of mycotoxin in consumed food in the digestive tract.

en mycotoxin and treatment of food or feed in an appropriate fermentation process.

ue of contaminated commodity).

362 Aflatoxins - Recent Advances and Future Prospects

**•** prevention of contamination;

**•** decontamination of mycotoxin-containing food and feed;

pathway of aflatoxin (Line, Brackett, Wilkinson, 1994).

nyi, Bata, 2009):

and temperature.

eliminate aflatoxins.

Recently, a growing interest can be observed concerning the use of *Rhodococci* for aflatoxins degradation: these microorganisms have a wide-range ability to degrade compounds like aflatoxins (Alberts, Engelbrecht, Steyn, Holzapfel, van Zyl, 2006; Teniola, Addo, Brost, Farb‐ er, Jany, Alberts, Van Zyl, Steyn, Holzapfel, 2005). Teniola et al. (Teniola, Addo, Brost, Farb‐ er, Jany, Alberts, Van Zyl, Steyn, Holzapfel, 2005) reported the degradation of aflatoxin B1 using liquid cultures of *Rhodococcus erythropolis* and the analysis of the intracellular extracts separated from *Rhodococcus erythropolis* liquid cultures suggested that a cascade of enzymat‐ ic reactions with loss of fluorescence (the intact aflatoxin is a fluorescent compound and degradation results in loss of fluorescence in time) occurred. Aflatoxin B1 is probably de‐ graded by the same enzymes (biphenyl-dioxygenases, dihydro-diol-dehydrogenases, and hydrolases) that are involved in catabolic pathways of polychlorinated biphenyls. Knowl‐ edge of gene coding for these enzymes may be helpful in development and production of new effective enzyme preparations for degradation of aflatoxins. The role of trace metal ions in microbial aflatoxin B1 degradation has been studied studied by Souza et al. (Souza, Brack‐ ett, 1998) who found that copper and zinc ions may inhibit the degradation of aflatoxin B1 by *Flavobacterium aurantiacum*. This effect is probably connected with an influence on the en‐ zyme system involved in the degradation process. Peltonen et al. (Peltonen, El-Nezami, Sal‐ minen, Ahokas, 2000) and El Nezami et al. (El-Nezami, Kankaanpaa, Salminen, Ahokas, 1998) studied the ability of dairy strains of lactic acid bacteria to bind Aflatoxin B1. It has been observed that *Lactobacillus rhamnosus* can significantly remove Aflatoxin B1 compared with other strains. Removal was observed as very rapid, with 80% of toxin removed within the first 60 min of treatment.

Several bacterial species, such as *Bacillus*, *Lactobacilli*, *Pseudomonas*, *Ralstonia* and *Burkholderia* spp., have shown ability to inhibit fungal growth and production of aflatoxins by *Aspergillus* spp. in laboratory tests. Palumbo et al. (Palumbo, Baker, Mahoney, 2006) reported that a number of *Bacillus*, *Pseudomonas*, *Ralstonia* and *Burkholderia* strains could completely inhibit *A. flavus* growth. *B*.*subtilis* and *P*. *solanacearum* strains isolated from maize soil were also able to inhibit aflatoxin accumulation. In most cases, although these strains were highly effective against fungal growth and against the produced toxins in laboratory conditions, they did not give good efficacies on field. This could be attributed to the difficulty to bring the bacte‐ rial cells to the *Aspergillus* infection sites on commodities under field conditions. Saprophytic yeast species, such as *Candida krusei* and *Pichia anomala*, have revealed promising efficacy as biocontrol agents for aflatoxins decontamination (Yin, Yan, Jiang, Ma, 2008). In a similar way to bacterial agents, these yeast strains were able to significantly inhibit *Aspergillus* growth and resultant toxins in laboratory conditions. Shetty et al. (Shetty, Hald, Jespersen, 2007) observed that the ability of *S. cerevisiae* to bind aflatoxin B1 was strain specific with 7 strains binding 10-20%, 8 strains binding 20-40% and 3 strains binding more than 40% of the added aflatoxin B1. Though the yeasts are considered to be potential biocontrol agents for the aflatoxins management, further experiments conducted on field are necessary to test their efficacies in reducing aflatoxin contamination in real on field situations.

Many reports exists on the use of physically separated yeast cell walls obtained from brew‐ ery as feed additive in poultry diet resulting in amelioration of aflatoxins toxic effects (Shet‐ ty, Jespersen, 2003; (Santin, Paulillo, Maiorka, Okada Nakaghi, Macari, Fischer da Silva, Alessi, 2003). When dried, yeast and yeast cell walls have been added to rat-ration along with aflatoxin B1, and a significant reduction in the toxicity has been observed (Baptista et al., 2004). In an *in vitro* study with the cell wall material, there was a dose dependent bind‐ ing of as much as 77% (w/w) and modified mannan-oligosaccharides derived from the *S. cerevisiae* cell resulted in as much as 95% (w/w) binding (Girish and Devegowda, 2006).

able to detoxify aflatoxins without causeing any unwanted side effect, e.g. changes in senso‐ rial and technological properties of foods. In addition, any new method should be economi‐ cally convenient if compared with any actually used procedure, especially for food industry

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 365

Safe food is a non-negotiable topic both for ethic reasons and for economic aspects. The so‐ cial costs linked to an increase of health conditions like liver diseases, or the problems con‐ nected to crop destruction, withdrawal of food from the shelves, etc., can be more expensive

1 Department of Food Science, University of Napoli "Federico II", Portici, Napoli, Italy

2 Department of Pharmaceutical and Toxicological Chemistry, University of Napoli "Federi‐

[1] Abbas, H. K., Cartwright, R. D., Xie, W., & Shier, W. T. (2006). Aflatoxin and fumoni‐ sin contamination of corn (maize, Zea mays) hybrids in Arkansas. Crop Prot. , 25(1),

[2] Abbas, H. K., Williams, W. P., Windham, G. L., Pringle, H. C., Xie, W., & Shier, W. T. (2002). Aflatoxin and Fumonisin Contamination of Commercial Corn (Zea mays) Hy‐

[3] Abdel-Wahhab, M. A., Ahmed, H. H., & Hagazi, M. M. (2006). Prevention of aflatox‐ in B1-initiated hepatotoxicity in rat by marine algae extracts. J. Appl. Toxicol. , 26,

[4] Alberts, J. F., Engelbrecht, Y., Steyn, P. S., Holzapfel, W. H., & van Zyl, W. H. (2006). Biological degradation of aflatoxin B1 by Rhodococcus erythropolis cultures.

[5] Arcand, Y., Mainville, I., & Farnworth, E. R. (2007). A dynamic model that simulates the human upper gastrointestinal tract for the study of probiotics. Int. J. Food Micro‐

brids in Mississippi. J. Agric. Food Chem. , 50, 5246-5254.

than a preventive actions to reduce aflatoxin presence in the food chain.

that may hamper for these economic reasons.

Antonello Santini1\* and Alberto Ritieni2

\*Address all correspondence to: asantini@unina.it

Int.J.Food Microbiol. , 109, 121-126.

**Author details**

co II", Napoli, Italy

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