Degradation of aflatoxin B 1 by fungal laccase enzymes J.F. Alberts a , W.C.A. Gelderblom b,c , A. Botha a , W.H. van Zyl a, ⁎ a Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa b Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa c PROMEC Unit, Medical Research Council, P.O. Box 19070, Tygerberg 7505, South Africa abstract article info Article history: Received 3 March 2009 Received in revised form 18 July 2009 Accepted 22 July 2009 Keywords: Aflatoxin B 1 AFB 1 Detoxification Fungi Laccase The enzymatic degradation of aflatoxin B 1 (AFB 1 ) by white rot fungi through laccase production was investigated in different liquid media. A significant (P b 0.0001) correlation was observed between laccase activity and AFB 1 degradation exhibited by representatives of Peniophora and Pleurotus ostreatus cultivated in minimal salts (MSM) (r = 0.93) and mineral salts — malt extract (MSB–MEB) (r = 0.77) liquid media. Peniophora sp. SCC0152 cultured in MSB–MEB liquid medium supplemented with veratryl alcohol and sugarcane bagasse showed high laccase activity (496 U/L), as well as 40.45% AFB 1 degradation as monitored using high performance liquid chromatography. P. ostreatus St2-3 cultivated in MSM liquid medium supplemented with veratryl alcohol resulted in laccase activity of 416.39 U/L and 35.90% degradation of AFB 1 . Aflatoxin B 1 was significantly (P b 0.0001) degraded when treated with pure laccase enzyme from Trametes versicolor (1 U/ml, 87.34%) and recombinant laccase produced by Aspergillus niger D15-Lcc2#3 (118 U/L, 55%). Aflatoxin B 1 degradation by laccase enzyme from T. versicolor and recombinant laccase enzyme produced by A. niger D15-Lcc2#3 coincided with significant (P b 0.001) loss of mutagenicity of AFB 1 , as evaluated in the Salmonella typhimurium mutagenicity assay. The degradation of AFB 1 by white rot fungi could be an important bio-control measure to reduce the level of this mycotoxin in food commodities. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Aflatoxins are difuranocoumarin derivates (Eaton and Gallagher, 1994; Payne and Brown, 1998) predominantly produced as secondary metabolites by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus (Pitt, 2000). Other Aspergillus spp. producing aflatoxin include Aspergillus nomius, Aspergillus tamarii (Goto et al., 1997) and Aspergillus pseudotamarii (Ito et al., 2001). Aflatoxin B 1 , the most abundantly produced aflatoxin, is highly mutagenic, toxic, carcino- genic and teratogenic to humans and animals (Eaton and Gallagher, 1994; Mishra and Das, 2003). Aflatoxin contamination of feed and foodstuffs is responsible for significant economic losses due to loss of crops and animals and in some years estimated losses ranged between $85 and $100 millions in certain states of the United States of America (Yabe and Nakajima, 2004). In parts of Africa, China and South East Asia aflatoxin contamination is correlated with the incidence of liver cancer and classified as a Group I human carcinogen by the International Agency for Research on Cancer (Wogan, 2000). Reduction of AFB 1 in food sources by various decontamination procedures has been studied extensively. Inactivation of aflatoxin by physical and chemical methods has not yet proved to be effective and economically feasible (Mishra and Das, 2003). However, biological detoxification offers an attractive alternative for eliminating toxins and safe-guarding the quality of food and feed. In recent years it became clear that fungi play a major role in the degradation of AFB 1 . The biosynthesis of AFB 1 by cultures of A. flavus and A. parasiticus reaches a maximum, where after it is degraded, presumably under nitrogen limiting conditions (Hamid and Smith, 1987; Shih and Marth, 1975). Other fungi that have been implicated in AFB 1 degradation include zygomycetous fungi (Rhizopus sp. and Mucor sp.), ascomy- cetous fungi (Aspergillus niger and Trichoderma sp.), plant pathogens (Phoma sp. and Alternaria sp.), as well as basidiomycetous fungi (Armillariella tabescens and other white rot fungi) (Leonowicz et al., 1999; Liu et al.,1998a; Nakazato et al., 1990; Shantha, 1999; Shantha et al., 1990; Yao et al., 1998). The current approach to the biological degradation of AFB 1 is based on the microbial processes involved in the degradation of complex organic aromatic compounds such as lignin. When considering polyphenolic compounds in nature, lignin is undoubtedly the most abundant and possibly also the most heterogeneous and recalcitrant compound to be degraded microbially (de Jongh et al., 1994). However, microbial communities have developed means that can effectively degrade this complex compound. Different microbial consortia are responsible for initially opening the lignin structure, depolymerisation of the complex compounds and finally mineralization of the more recalcitrant phenolic compounds. There are indications that Aspergillus International Journal of Food Microbiology 135 (2009) 47–52 ⁎ Corresponding author. Tel.: +27 21 8085854, fax: +27 21 8085846. E-mail address: whvz@sun.ac.za (W.H. van Zyl). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.07.022 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro