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Biological control of mycotoxins in food and feed grain commodities

MT Project 11/10. Biological control of mycotoxins in food and feed grain commodities. 1 Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa

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Biological control of mycotoxins in food and feed grain commodities

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  1. MT Project 11/10 Biological control of mycotoxins in food and feed grain commodities 1Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa 2Programme on Mycotoxins and Experimental Carcinogenesis (PROMEC Unit), Medical Research Council (MRC), South Africa J.F. Alberts1, W.H. Van Zyl1 and W.C.A. Gelderblom2

  2. Biotransformation of mycotoxins • Degradation/detoxification of mycotoxins by bacteria/fungi/yeasts or enzymes into non-toxic metabolites or compounds less toxic than the parent toxin molecule • Biotransformation – especially suitable for mycotoxins which hardly adsorb to minerals and organic polymers • Enzymatic degradation/detoxification: reactions resulting in cleavage of chemical bonds, oxidation, reduction, addition or deletion of a chemical moiety

  3. This project: Aim Develop practical, affordable and environmentally sound methods to biologically detoxify aflatoxin B1 and fumonisin B1mycotoxins Approach Eliminate mycotoxin contamination in food/feeds by treatment with recombinant microbial cultures and enzyme preparations • Cloning of fungal genes encoding enzymes capable of degrading mycotoxinsand expression in food-grade microorganisms

  4. BIOLOGICAL DEGRADATION OF AFLATOXIN B1 (AFB1)

  5. Degradation of AFB1 by Rhodococcus erythropolis extracellular extracts 1.6 a a a a 1.4 ab b 1.2 1 c AFB1Conc. (µg/ml) 0.8 d 0.6 e 0.4 0.2 0 0.5 1 2 4 24 48 72 C Treatment (h) • Significant (P<0.001) degradation of AFB1 • 33.2% AFB1 remaining after 72 h

  6. SDS-PAGE PROTEIN PROFILES Extracellular fractions of E. coli BL21 (DE3) liquid cultures containing recombinant 2,3-DHBD (Rhodococcuserythropolis) M 1 2 • Protein species greater than 35 kDa which correspond to the predicted molecular weight (36 kDa) of the bphC1 protein 170 kDa 130 kDa 100 kDa 70 kDa 55 kDa 40 kDa 36 kDa 35 kDa Extracellular 2,3-DHBD activity: • 34 mU/L 25 kDa

  7. SDS-PAGE PROTEIN PROFILES Extracellular fractions of E. coli BL21 (DE3) liquid cultures containing recombinant DHD (R. erythropolis) M 1 2 170 kDa 130 kDa • Protein species greater than 30 kDa which correspond to the predicted molecular weight (30 kDa) of the bphB protein 95 kDa 72 kDa 55 kDa 43 kDa 34 kDa 30 kDa 26 kDa

  8. DEGRADATION OF AFB1 BY EXTRACELLULAR FRACTIONS (RECOMBINANT 2,3-DHBD) Degradation of AFB1 by extracellular fractions (Recombinant 2,3-DHBD) • 50.68% AFB1 remaining after 72 h Baseline revertant count Salmonella typhimuriummutagenicity assay (Recombinant 2,3-DHBD) • 42.47% loss of mutagenicity

  9. Saccharomyces cerevisiae Y294- lcc2-fur1::LEU2 Y294 TV fur1:: LEU2 Y294 TV fur1::LEU2 Y294 Neg Control Y294 Neg Control M Y294-TV-lcc2 fur1::LEU2 Y294 Neg. Control Y294-TV-lcc2 170 Neg. Control Pos. Control 130 100 bp M 70 2568 55 Enzyme activity: ABTS plate assay 40 558 Laccase activity: 52.9 U/L 35 FUR1 disruption 25

  10. BIOLOGICAL DEGRADATION OF FUMONISIN B1(FB1)

  11. Fumonisin B1 • Degradation: • Deesterification (carboxylesterase) • Deamination (aminotransferases) Hartinger et al., 2011. Appl. Microbiol. Biotechnol.

  12. Objectives • 1. Bio-prospecting for fumonisin degrading fungi • Culture media: growth of fungi capable of utilizing molecules containing groups related to the 2-amino-3-hydroxy butyl moiety of fumonisin as nitrogen source • US and the PROMEC Unit Culture Collections: • Ability to utilize FB1 for growth • Aspergillusand Pleurotusspp. (FB1 as nitrogen source)

  13. Table 1. Fungal strains from culture collections of SU and the PROMEC Unit evaluated for the ability to utilize FB1 for growth

  14. Objectives • 1. Bio-prospecting for fumonisin degrading fungi • US and the PROMEC Unit Culture Collections: • Ability to utilize FB1 for growth • Aspergillusand Pleurotusspp. (FB1 as nitrogen source) • Media: growth of fungi capable of utilizing molecules containing groups related to the 2-amino-3-hydroxy butyl moiety of fumonisin as nitrogen source • Enrichment from compost-rich soil: • Select fungi capable of utilizing 1,4 diaminobutane (putrescine) as nitrogen source • Identificaton of positive fungal isolates: Traditional microscopic examination, sequencing of the ITS regions and BLAST sequence comparison

  15. Develop recombinant microbial culture- and enzyme preparations capable of effectively degrading fumonisins • Cloning of fungal genes encoding carboxylesterase and aminotransferase enzymes and expression through genomic integration in S. cerevisiaeMH1000 or Aspergillus niger • Enzymatic activity • Degradation of FB1 • The effect of the recombinant mycotoxin degrading enzyme- as well as recombinant bacterial, fungal and yeast treatments on the cytotoxic and carcinogenic properties of the fumonisins • Degradation of FB1 in contaminated grain, including samples obtained from subsistence farmers in rural populations

  16. Results • 1. Screening of fungal isolates from culture collections • Several fungal strains could initially maintain growth in FB1 minimal medium, possibly due to oligotrophic growth • However, after several times sub-culturing in FB1 minimal medium and eventually in MCD medium, only a few strains continue to grow • Strains capable of growing in the selective media include • A. nigerstrains • PleurotussanguineusSCC108 • Aspergillusfischeri MRC 1148

  17. Table 1. Fungal strains from culture collections of SU and the PROMEC Unit evaluated for the ability to utilize FB1 for growth

  18. Results 1. Screening of fungal isolates from culture collections Several fungal strains could initially maintain growth in FB1 minimal medium, possibly due to oligotrophic growth However, after several times sub-culturing in FB1 minimal medium and eventually in MCD medium, only a few strains continue to grow Strains capable of growing in the selective media include A. niger SU 10864, Pleurotussanguineus SCC108, Aspergillusfischeri MRC 1148 • Positive A. niger strains: able to exist in the FB1 minimal medium in MCD medium • Traditional microscopic examination, sequencing of the ITS regions and BLAST sequence comparison resulted in 100% homology with A. niger CBS 513.88 / A. nigerATCC 1015

  19. Enrichment from compost-rich soil: fungi capable of utilizing 1,4 diaminobutane as nitrogen source • One fungal isolate capable of growing in a medium with 1,4 diaminobutane as nitrogen source • Isolate was also capable to grow in FB1 minimal medium • Species identification: Fusariumsolani

  20. Enrichment from compost-rich soil:fungi capable of utilizing FB1 as nitrogen source • One fungal isolate capable of utilizing FB1 as nitrogen source. • The isolate was capable of existing in the FB1 minimal medium for four months as well as in MCD medium with FB1 as sole nitrogen source. • Species identification: A. nigerATCC 1015

  21. Subsequent experiments: aminotransferase encoding genes of • A. niger CBS 513.88 targeted for expression in S. cerevisiaeor A. niger

  22. A. niger (as source and host organism) • World Health Organisation(WHO): A. niger acceptable for daily intake • US Food and Drug Administration (FDA) under the Federal Food, Drug and Cosmetic Act: A.niger fermentation is "generally recognized as safe" (GRAS) • Widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid and gluconic acid • A. nigerglucoamylaseis used in the production of high fructose corn syrup • Pectinases are used in cider and wine clarification • α-Galactosidase - an enzyme that breaks down certain complex sugars

  23. S. cerevisiae as host organism • GRAS status: long association with the food and beverage industries • Easy to manipulate genetically • Ferments naturally • Natural robustness in industrial processes

  24. Biological degradation: Application • Transformation of genes encoding degradative enzymes into maize plants • Application of microbial and enzyme preparations pre-harvest • Treatment of contaminated grain during processing • Ruminal organisms engineered and added to feed in the form of • an inoculum/probiotic inoculant • Incorporation of enzymes into feed to act in the intestinal tract of animals

  25. Application: Treatment of contaminated grain • Enzymes: introduced during processing in appropriate manner (wash, spray, dried, lyophilized form or powdered form) depending upon the nature of the milling process and/or the stage of processing  

  26. Application of this study Degradation of FB1in contaminated grain including samples obtained from subsistence farmers in impoverished rural populations Treatment method Method in line with a previous study (van der Westhuizen et al., 2010. Food Addit. Contam. 27:11, 1582-1588)

  27. The method involves.. • A (culturally acceptable intervention) method to reduce fumonisin exposure in a community where high rates of oesophageal cancer have been associated with home-grown maize contaminated with fumonisins • The method involves sorting and washing procedures that were customised under laboratory-controlled conditions • The washing procedure involves a 10-min maize-washing procedure consisting of a 5-min hand agitation followed by a 1-min agitation immediately prior to a 10-min end point • Recombinant enzyme preparations exhibiting degradation properties will be incorporated

  28. Food Safety Assessment Guidelines for foods derived from GM microorganisms • [The European Network on the Safety Assessment of Genetically Modified Food Crops • (ENTRANSFOOD); Codex Alimentarius Commission and FAO/WHO] • Detailed descriptions of Donor organisms • Detailed descriptions of Host organisms and its use in food • Description of the Genetic modifications • Characterization of the genetic modifications • Potential horizontal gene transfer • Compositional analyses of key components: Qualitative and quantitative composition of nutrients, micronutrients and predictable secondary metabolites • Assessment of possible toxicity (animal feeding trials with GM foods/feed) • Assessment of possible allergenicity • Potential environmental impact • Long-term nutritional impact • Influence of food/feed processing • Potential dietary intake and change in dietary pattern

  29. Examples of approved GM food/feed crops • Maize with nutritionally improved amino acid profile (lysine) and/or vitamin content • Rice with ß-carotene, enhanced iron content or lower levels of allergens • Soybeans with improved amino acid composition (methionine, cysteine) or higher levels of oleic acid or isoflavones • Tomatoes with higher levels of lycopene, ß-carotene or flavonoids

  30. Acknowledgements • South African Maize Trust • National Research Foundation • PROMEC Unit, MRC • Prof. W.H. van Zyl, Stellenbosch University • Prof. A. Botha, Stellenbosch University

  31. Fumonisin B1 degradation • Duvick et al. (1998): Exophiala spinifera, Rhinocladiellaatrovirens, Sphingomonas and Xanthomonas • Bacterium ATCC 55552 • Benedetti et al. (2006): Delftia/Comomonas group • Täubel, 2005: Sphingomonas sp. MTA144 • Heinl et al. (2010): • The source of the genes,Sphingopyxis sp. MTA144 is associated with mild infections in humans • Will enzyme preparations originating from this organism be considered as safe • for treatment of food intended for human and animal consumption?

  32. Evaluating the effectiveness of detoxifying feed additives • 1. Effects of specific mycotoxins and detoxifying agents differ for each animal species. The efficacy of detoxifying agents against the different mycotoxins is undertaken separately for poultry, swine, ruminants, rodents and other species • 2. Assessment of performance parameters of the animals. Other parameters: relative weight of target organs such as liver, kidneys, heart, spleen, etc. Haematological and biochemical serum parameters: total proteins, albumins, globulins, enzymatic activities as γ-glutamyl-transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT) - indicators of the effects of mycotoxins and the preventive action of detoxifying agents

  33. GM crop-derived foods/feed category: improved nutritional characteristics • Enhanced levels of essential amino acids or improved nutritional value of proteins • Altered starch composition • Improved fiber/lignancontent • Altered oil/lipids composition • Enhanced levels of vitamins or minerals • Lower levels of anti-nutrients, toxins or allergens • Enhanced taste characteristics, or • Improved processing properties

  34. Examples • Detoxification of deoxynivalenol (Avad et al., 2010. Food Addit. Contam. 27:510-20) • Eubacterium sp. BBSH 797: Isolated from bovine rumen fluid; deactivate deoxynivalenol by anaerobic reduction of the epoxidering • Mode of action proven in vitro (demonstrated in the presence of pieces of gut) and in vivo: demonstrated in chickens (as a feed additive)

  35. Detoxification of AFB1 by Rhodococcus sp. as a feed additive: 1 day old chicks (Tejada-Castaneda et al., 2008. Poult. Sci. 87:1569-76) • Aspergillus parasiticus cultivated on maize; autoclaved • Contaminated maize inoculated with Rhodococcus sp. and incubated; autoclaved • The maize lots dried, milled and incorporated into chick feed • Several parameters monitored: body weight, food intake, histopathology on several organs, lipid content and residual AFB1 in the liver was monitored • Conclusion: Rhodococcus sp. significantly reduced the AFB1 associated lesion severity in liver, duodenum and kidneys

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