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-NH 2 , 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 β-diphenylalanine or a full Denantiomerization 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.
In the food industry, food spoilage is a real issue that can lead to a significant amount of waste. Although current preservation techniques are being applied to reduce the occurrence of spoilage microorganisms, the problem persists. Food spoilage yeast are part of this dilemma, with common spoilers such as Zygosaccharomyces, Kluyveromyces, Debaryomyces and Saccharomyces frequently encountered. Antimicrobial peptides derived from plants have risen in popularity due to their ability to reduce spoilage. This study examines the potential application of a synthetic defensin peptide derived from barley endosperm. Its inhibitory effect against common spoilage yeasts, its mechanisms of action (membrane permeabilisation and overproduction of reactive oxygen species), and its stability in different conditions were characterised. The safety of the peptide was evaluated through a haemolysis and cytotoxicity assay, and no adverse effects were found. Both assays were performed to understand the effect of the peptide if it were to be consumed. Its ability to be degraded by a digestive enzyme was also examined for its safety. Finally, the peptide was successfully applied to different beverages and maintained the same inhibitory effects in apple juice as was observed in the antiyeast assays, providing further support for its application in food preservation.
Proteins and peptides belonging to the plant immune system can possess natural antibacterial, antifungal and antiviral properties. Due to their broad range of activity and stability, they represent promising novel alternatives to commonly used antifungal agents to fight the emergence of resistant strains. An isolation protocol was optimised to target proteins found in plants’ defence system, and it was applied to white mustard (
Brassica hirta
) seeds. Firstly, a ∼14 kDa protein with activity against
S. cerevisiae
was extracted and purified; secondly, the protein was identified as the mustard Napin protein named
Allergen Sin a 1
. Napin is the name given to seed storage (2S) albumin proteins belonging to the
Brassicaceae
family. While several Napins have been described for their antimicrobial potential,
Sin a
1 has been mainly studied for its allergenic properties. The antimicrobial activity of
Sin a 1
is described and characterised for the first time in this study; it possesses antifungal and antiyeast
in vitro
activity, but no antibacterial activity was recorded. The yeasts
Zygosaccharomyces bailii
Sa 1403 and
Saccharomyces cerevisiae
DSM 70449 along with the filamentous fungi
Fusarium culmorum
FST 4.05 were amongst the most senstitive strains to
Sin a 1
(MICs range 3–6 μM). The antimicrobial mechanism of membrane permeabilisation was detected, and in general, the antifungal activity of
Sin a 1
seemed to be expressed in a dose-dependent manner. Data collected confirmed
Sin a 1
to be a stable and compact protein, as it displayed resistance to α-chymotrypsin digestion, heat denaturation and insensitivity to pH variations and the presence of salts. In addition, the protein did not show cytotoxicity towards mammalian cells.
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