Synthesis and characterization of new acid-functionalized porphyrins displaying antimicrobial activity against gram positive bacteria, yeasts and filamentous fungi with or without ultra-high irradiance.

The antimicrobial activity of new acid-functionalized porphyrins, with or without ultra-high irradiance, was investigated. Antibacterial efficacy was evaluated against Staphylococcus aureus (methicillin-resistant or methicillin-sensitive strains) and antifungal efficacy was evaluated against the yeast Candida albicans and the filamentous fungi Aspergillus fumigatus. Overall, the porphyrins tested are more effective against S. aureus. The best results were obtained with zinc diacid porphyrins 4 and 5 after only 3 min of ultra-high irradiation (500 mW/cm2, 405 nm), demonstrating that acid-functionalized porphyrins are promising as novel antimicrobial drugs for surface disinfection.

Ultra-high irradiance (UHI) blue light: highlighting the potential of a novel LED-based device for short antifungal treatments of food contact surfaces.

Microbial food spoilage is an important cause of health and economic issues and can occur via resilient contamination of food surfaces. Novel technologies, such as the use of visible light, have seen the light of day to overcome the drawbacks associated with surface disinfection treatments. However, most studies report that photo-inactivation of microorganisms with visible light requires long time treatments. In the present study, a novel light electroluminescent diode (LED)-based device was designed to generate irradiation at an ultra-high power density (901.1 mW/cm2). The efficacy of this technology was investigated with the inactivation of the yeast S. cerevisiae. Short-time treatments (below 10 min) at 405 nm induced a ~4.5 log reduction rate of the cultivable yeast population. The rate of inactivation was positively correlated to the overall energy received by the sample and, at a similar energy, to the power density dispatched by the lamp. A successful disinfection of several food contact surfaces (stainless steel, glass, polypropylene, polyethylene) was achieved as S. cerevisiae was completely inactivated within 5 min of treatments. The disinfection of stainless steel was particularly effective with a complete inactivation of the yeast after 2 min of treatment. This ultra-high irradiance technology could represent a novel cost- and time-effective candidate for microbial inactivation of food surfaces. These treatments could see applications beyond the food industry, in segments such as healthcare or public transport. KEY POINTS : • A novel LED-based device was designed to emit ultra-high irradiance blue light • Short time treatments induced high rate of inhibition of S. cerevisiae • Multiple food contact surfaces were entirely disinfected with 5-min treatments.

Inhibitory effect of four novel synthetic peptides on food spoilage yeasts.

The spoilage of foods caused by the growth of undesirable yeast species is a problem in the food industry. Yeast species such as Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Kluyveromyces lactis and Saccharomyces cerevisiae have been encountered in foods such as high sugar products, fruit juices, wine, mayonnaise, chocolate and soft drinks. The demand for new methods of preservations has increased because of the negative association attached to chemical preservatives. The sequence of a novel short peptide (KKFFRAWWAPRFLK-NH2) was modified to generate three versions of this original peptide. These peptides were tested for the inhibition of the yeasts mentioned above, allowing for the better understanding of their residue modifications. The range of the minimum inhibitory concentration was between 25 and 200 µg/mL. Zygosaccharomyces bailii was the most sensitive strain to the peptides, while Zygosaccharomyces rouxii was the most resistant. Membrane permeabilisation was found to be responsible for yeast inhibition at a level which was a two-fold increase of the MIC (400 µg/mL). The possibility of the production of reactive oxygen species was also assessed but was not recognised as a factor involved for the peptides' mode of action. Their stability in different environments was also tested, focusing on high salt, pH and thermal stability. The newly designed peptides showed good antifungal activity against some common food spoilage yeasts and has been proven effective in the application in Fanta Orange. These efficient novel peptides represent a new source of food preservation that can be used as an alternative for current controversial preservatives used in the food industry.

Isolation and characterisation of the antifungal activity of the cowpea defensin Cp-thionin II.

As a result of the rapidly growing human population, reducing post-harvest crop losses of cereals due to microbial pests has major importance. Plant defensins have the potential to fulfil these demands, being highly specific and efficient antimicrobial agents. Hence, this study aimed to extract and characterise a peptide from cowpea seeds and investigate its antifungal performance. After extraction and partial purification, N-terminal sequencing was used to identify the primary peptide in the extract as cowpea-thionin II. Antifungal activity in vitro was found against Fusarium culmorum (MIC = 50 µg/mL), but Aspergillus niger and Penecillium expansum showed an MIC > 500 µg/mL. The extract was resistant against heat treatment (100 °C, 15 min) but lost its antifungal activity in presence of cations (Na+, K+, Ca2+ and Mg2+, respectively). Membrane permeabilization of fungal hyphae was evident at 25 µg/mL, while induction of oxidative stress only had minor contribution to the antifungal performance. The extract did not induce haemolysis at all concentrations tested (up to 200 µg/mL). Finally, it was successfully used to protect stored wheat grains from fungal spoilage (determined via ergosterol content) when applied at 100 µg/mL. In conclusion, the defensin Cp-thionin II showed the potential for future application as food bio-preservative.

Natural Antifungal Peptides/Proteins as Model for Novel Food Preservatives.

A large range of ingredients for food and food products are subject to fungal contamination, which is a major cause of destruction of crops, exposure of animals and humans to invasive mycotoxins, and food spoilage. The resistance of fungal species to common preservation methods highlights the necessity of new ways to increase the shelf life of raw material for food and food products. Antimicrobial peptides and proteins (AMPs) are essential members of the immune system of most living organisms. Due to their broad range of activity and their stability to commonly used food processes, they represent promising alternatives to traditional preservatives. However, despite the growing number of reports of potential food applications of these AMPs, the number of approved peptides is low. Poor solubility, toxicity, and a time-consuming extraction are hurdles that limit their application in food products. Thanks to a deep understanding of the key determinants of their activity, the development of optimized synthetic peptides has reduced these drawbacks. This review presents natural and synthetic antifungal peptides/proteins (AFPs), effective against food-related fungi, with particular emphasis on AFPs from plant sources. The design of novel antifungal peptides via key elements of antifungal activity is also reviewed. The potential applications of natural and synthetic AFPs as novel antifungal food preservatives are finally discussed.

Antifungal activity of a de novo synthetic peptide and derivatives against fungal food contaminants.

The development of novel solutions to fight microbial food contaminants rests upon two pillars, which are the development of resistant strains and consumers' desire for a reduced consumption of synthetic drugs. Natural antimicrobial peptides possess the qualities to overcome these issues. De novo synthesis of novel antifungal compounds is a major progress that has been facilitated by the identification of parameters involved in the antimicrobial activity. A 14-residue peptide named KK14, with the sequence KKFFRAWWAPRFLK-NH2 , was designed and inhibited conidial germination and fungal growth of food contaminants within the range 6.25 to 50 µg/ml and 6.25 to 100 µg/ml, respectively. The study of three analogues of the peptide highlighted the role of some residues in the structural conformation of the peptide and its antifungal activity. The substitution of a Pro residue with Arg increased the helical content of the peptide not only its antifungal activity but also its cytotoxicity. The insertion of an unnatural bulky residue ß-diphenylalanine or a full d-enantiomerization overall increased the antifungal potency. The four peptides showed similar behaviour towards salt increase, heat treatment, and pH decrease. Interestingly, the denantiomer remained the most active at high pH and after proteolytic digestion. The four peptides did not present haemolytic activity up to 200 µg/ml but had different behaviours of cytotoxicity. These differences could be crucial for potential application as pharmaceutical or food preservatives.

Antifungal activity of synthetic cowpea defensin Cp-thionin II and its application in dough.

Plant defensins are small, cysteine-rich antimicrobial peptides of the immune system found in several organs during plant development. A synthetic peptide, KT43C, a linear analogue of the native Cp-thionin II found in cowpea seeds, was evaluated for its antifungal potential. It was found that KT43C displayed antifungal activity against Fusarium culmorum, Penicillium expansum and Aspergillus niger. Like native plant defensins, KT43C showed thermostability up to 100 °C and cation sensitivity. The synthetic peptide decreased the fungal growth without inducing morphogenic changes in the fungal hyphae. Non-inhibitory concentrations of the peptide induced permeabilization of the fungal membrane. In addition, high concentrations of KT43C induced the production of reactive oxygen species in the granulated cytoplasm. To investigate potential applications, the peptide was used as an additive in the preparation of dough which did not contain yeast. This peptide delayed the development of fungal growth in the dough by 2 days. Furthermore, KT43C did not induce red blood cell lysis up to a concentration of 200 µg.ml-1. These results highlight the potential for the use of synthetic antimicrobial defensins for shelf-life extension of food products.

Optimisation of the antifungal potency of the amidated peptide H-Orn-Orn-Trp-Trp-NH2 against food contaminants.

The design of novel efficient antimicrobial peptides (AMPs) faces several issues, such as cost of synthesis, proteolytic stability or cytotoxicity. The identification of key determinants involved in the activity of AMPs, such as cationicity and amphipathicity, allowed the synthesis of short peptides with optimized properties. An ultrashort peptide made of the sequence H-Orn-Orn-Trp-Trp-NH2 (O3TR) showed antifungal activity against several contaminants from food products. This peptide inhibited the growth of the filamentous fungi Fusarium culmorum, Penicillium expansum and Aspergillus niger within a range of concentration of 12.5-50µg/ml. In addition, O3TR inhibited the growth of the yeast Saccharomyces cerevisiae, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii and Kluyveromyces lactis within the range 12.5-50µg/ml. A derivative peptide, called C12O3TR, made by the addition of lauric acid at the N-terminus of O3TR was 2- to 8-fold more active than O3TR against every species. In addition to the inhibition of conidial germination, O3TR and C12O3TR killed F. culmorum hyphae at 100 and 50µg/ml respectively. The MIC of the two peptides against F. culmorum and Z. bailii after heat treatment at 100°C for 60 min and within the pH range 3-10, were not changed. However, the activity of O3TR against F.culmorum and Z. bailii was strongly reduced in salt solutions, whereas the lauric acid peptide kept its antifungal activity and resistance to proteolytic digestion. The conjugation with lauric acid reduced the random coiled structure and increased the α-helical content of O3TR. After conjugation with the dye tetramethylrhodamine (TMR), both peptides entered F. culmorum spores. They also both induced permeabilization of F. culmorum hyphae but only C12O3TR permeabilized Z. bailii membrane. In contrast to the lipopeptide, O3TR did not show haemolytic or cytotoxic activity when applied at the concentrations that exhibited antifungal potency. The two peptides were challenged against a yeast cocktail of S. cerevisiae and Z. bailii, and A. niger in different commercial beverages. After 7 days, O3TR was able to inhibit the yeast cocktail in a commercial lager and carbonated drink. Due to its antifungal potency, high stability and low cytotoxicity, the tetrapeptide could represent a promising starting point of a novel food preservative.