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.

Bio-Control of <i>Alternaria alternata</i> during Banana Storage by Purified AFP Using Isoelectric Focusing Technique

“…The established purification protocol of AFP could be efficient and valuable in AFP purification and yield. Comparing to mentioned purification protocol by [2] [5]- [8] [34], the isoelectric focusing could be 5 days shorter in time and cheaper in cost with producing higher amount of active AFP. Therefore, it recommended to be used for scaling up the AFP production.…”

“…Identification of A. alternate was carried out by comparing the individually DNA fragment to the DNA marker GeneRuler TM DNA Ladder Mix 100 -10,000 bp [25]. The relative fungal DNA amounts were calculated by measuring the PCR-Fragment intensity for all PCR bands using Kodak ID program v., 3.6 and mean of triplicate ± SE was calculated and performed by Excel program [8].…”

“…giganteus was cultivated on Olson medium as mentioned previously by [8] and the supernatant was obtained by filtration, centrifuged to get clear supernatant. The ammonium sulfate was added to the supernatant gradually to reach 75% of saturation at 4˚C for protein precipitation with constant stirring overnight.…”

“…Theis [7] found that 17 mg purified AFP•l −1 were obtained with CMC-52 and Sephadex G-50 column. Most recently 16.22 mg•AFP•l −1 were purified using Cellulose phosphate (Cph) as cation exchange chromatography and Sephadex G-50 with incubation the culture at 28˚C for about 100 hrs followed by an additional incubation period for 17 -20 hrs at 37˚C [8]. A few studies were established for purification different antifungal peptides applying the isoelectric focusing procedure [9]- [11], where no one used this technique for purification of AFP from A. gigenteus so far.…”

Target-Site-Specific Screening System for Antifungal Compounds

“…Therefore, investigation of the target site and the mode of action of an antifungal protein should reveal whether the protein is suitable for an application (Theis et al, 2005). The antifungal protein (AFP) are abundantly secreted by the filamentous fungus Aspergillus giganteus, this cysteine-rich protein have ability to disturb the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other eukaryotic systems (Barakat et al, 2010;Meyer, 2008).…”