Fundamental study of natural and synthetic antimicrobial peptides as novel preservatives against fungal food spoilage

Abstract

A large part of food loss is associated with the development of fungal contamination at early or late stages of production. This spoilage, via the production of mycotoxins, has potentially disastrous consequences on consumers’ health. The overuse of chemical antimicrobials in foods (e.g., sorbates) and pesticides (e.g., azoles) in crops has also led to the development of resistant strains, including human pathogens. This thesis aims to consider the potential of antimicrobial peptides as novel preservatives against spoilage fungi of food products. Antimicrobial peptides are short (<100 amino acids) components of the innate immune system widely spread amongst eukaryotic and prokaryotic kingdoms. A literature review about the diversity of antimicrobial peptides and proteins (AMPs) and their efficacy against fungal food spoilage is presented in Chapter 2. The studies presented in Chapters 3 and 4 highlight the activity of synthetic analogues of a human defensin, HBD-3, and a cowpea defensin-like peptide, Cp-thionin II, against selected species representing food contaminants, Fusarium culmorum, Penicillium expansum and Aspergillus niger. The peptides inhibit conidial germination, potentially with morphogenic changes and killing activity against F. culmorum hyphae, but suffer salt sensitivity. Mechanistic studies reveal membrane permeabilization and overgeneration of reactive oxygen species. The potential of the peptides for food applications has been exploited either with a recombinant yeast, able to express a recombinant HBD-3, or with the addition of synthetic Cp-thionin II in bakery products. However, the cost of synthesis of natural-based peptides is a major drawback to economically viable applications. To overcome this obstacle, a 4-residue peptide has been successfully tested against mould and yeast species. After conjugation with a lauric acid, synergistic antifungal activity is observed with strong improvements of peptide resistance. However, the price to pay for such improvement is a strong increase of toxicity. Based on rational design with key determinants of antifungal activity, a novel 14-residue peptide, made of the sequence KKFFRAWWAPRFLK-NH2, has been constructed in order to find a balance between activity and toxicity. Three analogues, created to improve the peptide’s properties, consist in residue substitution or enantiomerization. The different modifications hardly alter the antifungal potency but significant improvements in resistance are obtained with the enantiomerization or a Pro to Arg substitution. However, those modifications are associated with an increase of toxicity. In spite of continuous improvements and cost reduction, the launch of new synthetic compounds faces regulations and consumers’ demand for natural products. The natural approach consists in the isolation of an AMP from broccoli, a healthy raw material that belongs to the Brassicacea family, an AMP-rich plant family. Extracts of broccoli seeds show activity against the three fungal species studied. An antifungal napin-like protein has been isolated and purified via chromatographic methods. In addition, it shows no cytotoxicity and reduces fungal development on contaminated barley grains. Due its extraction and purification via “non-chemical”, food-grade, processes, this antifungal compound could represent a fully natural novel preservative. The world is facing a major crisis, with a boom of population worldwide, associated with a rise of undernutrition while food production is sufficient. The results of this work offer novel perspectives in the research of large spectrum food preservatives in a clean-label approach.