A Study On Plant-derived Small Proteins And Peptides With Antiyeast Activity And Preliminary Investigations In Food Materials
Authors:   ,   Munster Technological University, Mignone Giulia 
Published: September 2024

The discovery of novel antimicrobial molecules would benefit considerably different biotechnological fields, including medicine, agriculture and the food industry, on which this thesis work focuses. Specifically, here are presented fundamental studies on plant-derived antimicrobial proteins for the reduction of food spoilage caused by yeast microorganisms. Small proteins and peptides from the plant innate immune system possess several characteristics which could make them ideal candidates for a novel and natural antispoilage agent. The first Chapter reviews proteins families associated with the plant innate immune system that exhibit antiyeast activity and low molecular weight (<30 kDa), focusing on their structure and antiyeast mode of action. Chapter 2 investigates two peptides, Pn-AMP1 and Pn-AMP2 belonging to the hevein-type peptides family; peptides were synthetically generated using their native amino acid sequence. Synthetic Pn-AMP1 showed fast action activity against common food spoilage yeasts, it resulted potentially safe for consumption, and it was successfully incorporated in UHT milk and Fanta Orange, where it contained the growth of Kluyveromyces lactis and Zygosaccharomyces bailii, respectively. A different family of antimicrobial plant proteins (2S albumins) was studied in Chapters 3, 4, 5 and 6. Chapter 3 describes the selective extraction of an antiyeast protein (labelled WMS1 in this thesis work) of ~14 kDa from white mustard (Brassica hirta) seeds. The protein was purified in a relatively high yield, using a simple protocol that included only one chromatographic method step. In Chapter 4, bioinformatics tools and wet-lab techniques are used to study the structure and classify the antiyeast protein. The WMS1 antiyeast protein was identified as an isoform of the Napin protein Allergen Sin a 1 belonging to the 2S albumins family, characterised by high structural stability which donates to the protein resistance heat treatment and proteolytic degradation. Chapter 5 describes the antimicrobial spectrum of WMS1 , its antiyeast mechanism of action and its validity as preservative agent. Although WMS1 resulted potentially safe for consumption and inhibited the growth of Z. bailii in several beverage systems, its biotechnological potential is limited by its allergic properties. In Chapter 6, the protein WMS1 was cloned in a pET28a-MBP vector and expressed in an E. coli BL21 system. Interestedly, recombinant WMS1 and native WMS1 showed similar activity against the yeast Z. bailii. Thesis abstract 2 Chapter 7 includes a protocol for the rational design of ultra-short antimicrobial peptides. Since ultra-short antimicrobial sequences can be found within the sequence of any larger protein, the protocol was applied using WMS1 primary sequence, and seven ultra-short SinA-AMPs were chemically generated. Among the various SinA-AMPs, the peptide labelled SinA-pepIII had in vitro activity against several common yeast spoilage strains via membrane permeabilisation and it could be employed as a food preservative as it was pH- and heat- stable, potentially safe in terms of human consumption and it prevented the growth of Z. bailii in cranberry juice. The results obtained in this work offer new perspectives in the research for novel and clean-label food preservatives to fight the food waste phenomenon.