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The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley

Abstract

Secondary growth is a common post-harvest problem when pre-infected crops are attacked by filamentous fungi during storage or processing. Several antifungal approaches are thus pursued based on chemical, physical, or bio-control treatments; however, many of these methods are inefficient, affect product quality, or cause severe side effects on the environment. A protein that can potentially overcome these limitations is the antifungal protein AFP, an abundantly secreted peptide of the filamentous fungus Aspergillus giganteus. This protein specifically and at low concentrations disturbs the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other pro- and eukaryotic systems. We thus studied in this work the applicability of AFP to efficiently prevent secondary growth of filamentous fungi on food stuff and chose, as a case study, the malting process where naturally infested raw barley is often to be used as starting material. Malting was performed under lab scale conditions as well as in a pilot plant, and AFP was applied at different steps during the process. AFP appeared to be very efficient against the main fungal contaminants, mainly belonging to the genus Fusarium. Fungal growth was completely blocked after the addition of AFP, a result that was not observed for traditional disinfectants such as ozone, hydrogen peroxide, and chlorine dioxide. We furthermore detected reduced levels of the mycotoxin deoxynivalenol after AFP treatment, further supporting the fungicidal activity of the protein. As AFP treatments did not compromise any properties and qualities of the final products malt and wort, we consider the protein as an excellent biological alternative to combat secondary growth of filamentous fungi on food stuff.

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References

  • Andrews S, Pardoel D, Harun A, Treloar T (1997) Chlorine inactivation of fungal spores on cereal grains. Int J Food Microbiol 35:153–162

    Article  CAS  Google Scholar 

  • Bengtson P, Bastviken D, de Boer W, Oberg G (2009) Possible role of reactive chlorine in microbial antagonism and organic matter chlorination in terrestrial environments. Environ Microbiol 11:1330–1339

    Article  CAS  Google Scholar 

  • Bullerman LB, Bianchini A (2007) Stability of mycotoxins during food processing. Int J Food Microbiol 119:140–146

    Article  CAS  Google Scholar 

  • Chaidez C, Lopez J, Vidales J, Campo NC (2007) Efficacy of chlorinated and ozonated water in reducing Salmonella typhimurium attached to tomato surfaces. Int J Environ Health Res 17:311–318

    Article  CAS  Google Scholar 

  • Coca M, Bortolotti C, Rufat M, Penas G, Eritja R, Tharreau D, del Pozo AM, Messeguer J, San Segundo B (2004) Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast fungus Magnaporthe grisea. Plant Mol Biol 54:245–259

    Article  CAS  Google Scholar 

  • Demeke T, Clear RM, Patrick SK, Gaba D (2005) Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis. Int J Food Microbiol 103:271–284

    Article  Google Scholar 

  • Demirkol O, Cagri-Mehmetoglu A, Qiang Z, Ercal N, Adams C (2008) Impact of food disinfection on beneficial biothiol contents in strawberry. J Agric Food Chem 56:10414–10421

    Article  CAS  Google Scholar 

  • EBC-Analytica (1998) Analytica EBC. Section 3 (barley analysis), 4 (malt analysis) and 8 (wort analysis), Hans Carl, Nürnberg-ISBN 3-418-00759-7

  • Fredlund E, Gidlund A, Olsen M, Borjesson T, Spliid NH, Simonsson M (2008) Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels. J Microbiol Methods 73:33–40

    Article  CAS  Google Scholar 

  • Fukuzaki S (2006) Mechanisms of actions of sodium hypochlorite in cleaning and disinfection processes. Biocontrol Sci 11:147–157

    CAS  Google Scholar 

  • Girgi M, Breese WA, Lorz H, Oldach KH (2006) Rust and downy mildew resistance in pearl millet (Pennisetum glaucum) mediated by heterologous expression of the afp gene from Aspergillus giganteus. Transgenic Res 15:313–324

    Article  CAS  Google Scholar 

  • Hagen S, Marx F, Ram AF, Meyer V (2007) The antifungal protein AFP from Aspergillus giganteus inhibits chitin synthesis in sensitive fungi. Appl Environ Microbiol 73:2128–2134

    Article  CAS  Google Scholar 

  • Hasan HA (2001) Phytotoxicity of pathogenic fungi and their mycotoxins to cereal seedling viability. Acta Microbiol Immunol Hung 48:27–37

    Article  CAS  Google Scholar 

  • Hazel CM, Patel S (2004) Influence of processing on trichothecene levels. Toxicol Lett 153:51–59

    Article  CAS  Google Scholar 

  • Jones BL, Marinac L (2002) The effect of mashing on malt endoproteolytic activities. J Agric Food Chem 50:858–864

    Article  CAS  Google Scholar 

  • Lacadena J, Martinez del Pozo A, Gasset M, Patino B, Campos-Olivas R, Vazquez C, Martinez-Ruiz A, Mancheno JM, Onaderra M, Gavilanes JG (1995) Characterization of the antifungal protein secreted by the mould Aspergillus giganteus. Arch Biochem Biophys 324:273–281

    Article  CAS  Google Scholar 

  • Laitila A, Sarlin T, Kotaviita E, Huttunen T, Home S, Wilhelmson A (2007) Yeasts isolated from industrial maltings can suppress Fusarium growth and formation of gushing factors. J Ind Microbiol Biotechnol 34:701–713

    Article  CAS  Google Scholar 

  • Lancova K, Hajslova J, Poustka J, Krplova A, Zachariasova M, Dostalek P, Sachambula L (2008) Transfer of Fusarium mycotoxins and 'masked' deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 25:732–744

    CAS  Google Scholar 

  • Linko M, Haikara A, Ritala A, Penttilä M (1998) Recent advances in the malting and brewing industry. J Biotechnol 65:85–98

    Article  CAS  Google Scholar 

  • Lowe DP, Arendt EK (2004) The use and effects of lactic acid bacteria in malting and brewing with their relationships to antifungal activity, mycotoxins and gushing: a review. J Inst Brew 110:163–180

    CAS  Google Scholar 

  • Magan N, Hope R, Colleate A, Baxter ES (2002) Relationship between growth and mycotoxin production by Fusarium species, biocides and environment. Eur J Plant Pathol 108:685–690

    Article  CAS  Google Scholar 

  • Meyer V (2008) A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value. Appl Microbiol Biotechnol 78:17–28

    Article  CAS  Google Scholar 

  • Moreno AB, Penas G, Rufat M, Bravo JM, Estopa M, Messeguer J, San Segundo B (2005) Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice. Mol Plant Microb Interact 18:960–972

    Article  CAS  Google Scholar 

  • Nicholson P, Simpson DR, Weston G, Rezanoor HN, Lees AK, Parry DW, Joyce D (1998) Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol Mol Plant Pathol 53:17–37

    Article  CAS  Google Scholar 

  • Oldach KH, Becker D, Lorz H (2001) Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat. Mol Plant Microbe Interact 14:832–838

    Article  CAS  Google Scholar 

  • Parry DW, Nicholson P (1996) Development of a PCR assay to detect Fusarium poae in wheat. Plant Pathology 45:383–391

    Article  CAS  Google Scholar 

  • Pekkarinen AI, Longstaff C, Jones BL (2007) Kinetics of the inhibition of Fusarium serine proteinases by barley (Hordeum vulgare L.) inhibitors. J Agric Food Chem 55:2736–2742

    Article  CAS  Google Scholar 

  • Rabie CJ, Lubben A, Marais GJ, Jansen van Vuuren H (1997) Enumeration of fungi in barley. Int J Food Microbiol 35:117–127

    Article  CAS  Google Scholar 

  • Reverberi M, Fabbri AA, Zjalic S, Ricelli A, Punelli F, Fanelli C (2005) Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes inhibits aflatoxin production. Appl Microbiol Biotechnol 69:207–215

    Article  CAS  Google Scholar 

  • Sambrook J, Russel DW (2002) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, NY

    Google Scholar 

  • Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    Article  Google Scholar 

  • Szappanos H, Szigeti GP, Pal B, Rusznak Z, Szucs G, Rajnavolgyi E, Balla J, Balla G, Nagy E, Leiter E, Pocsi I, Hagen S, Meyer V, Csernoch L (2006) The antifungal protein AFP secreted by Aspergillus giganteus does not cause detrimental effects on certain mammalian cells. Peptides 27:1717–1725

    Article  CAS  Google Scholar 

  • Theis T, Wedde M, Meyer V, Stahl U (2003) The antifungal protein from Aspergillus giganteus causes membrane permeabilization. Antimicrob Agents Chemother 47:588–593

    Article  CAS  Google Scholar 

  • Theis T, Marx F, Salvenmoser W, Stahl U, Meyer V (2005) New insights into the target site and mode of action of the antifungal protein of Aspergillus giganteus. Res Microbiol 156:47–56

    Article  CAS  Google Scholar 

  • Vila L, Lacadena V, Fontanet P, Martinez del Pozo A, San Segundo B (2001) A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens. Mol Plant Microbe Interact 14:1327–1331

    Article  CAS  Google Scholar 

  • Wolf-Hall CE (2007) Mold and mycotoxin problems encountered during malting and brewing. Int J Food Microbiol 119:89–94

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Iris Fechter and Jean-Paul Ouedraogo for excellent technical assistance. This study was partially supported by a grant to HB from the Ministry of High Education, Mission Department, Egypt.

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Correspondence to Ulf Stahl.

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Hassan Barakat and Anja Spielvogel equally contributed to this work.

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Barakat, H., Spielvogel, A., Hassan, M. et al. The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley. Appl Microbiol Biotechnol 87, 617–624 (2010). https://doi.org/10.1007/s00253-010-2508-4

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  • DOI: https://doi.org/10.1007/s00253-010-2508-4

Keywords

  • Antifungal protein AFP
  • Barley
  • Malting
  • Fusarium
  • Mycotoxin
  • Bio-control