Ambrosio, Rosa Luisa (2021) Innovative food safety and hygiene management systems in the agri- food industry. [Tesi di dottorato]

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Food sector contributes significantly to the financial statements of the European Union (EU), despite the economic losses caused by the contamination of the foods, which are exposed to various hazards along the food chain. The microbiological contamination plays an important role in the food spoilage and foodborne diseases, causing public health and economic damage. Indeed, in the EU, around 90 million tons of food are wasted annually, with associated burden of microbial spoilage estimated to affect more than a quarter of the total weight. Furthermore, the European Food Safety Authority has attributed to pathogenic bacteria the role of mail culprit agents of human diseases linked to the ingestion of contaminated food. These microorganisms are also partly responsible of the spread of the antimicrobial resistance phenomenon, which has become one of the major global public health concerns. In this context, the general aim of the thesis was to search for molecules capable of replacing the chemical additives and conventional antibiotics, and develop an “end-product” with antimicrobial activity ready to be put on the food market. Ideally, the novel preservatives should have broad spectrum of action, be non-toxic, be active a low concentration, no affect organoleptic properties of food, and have a good ratio cost-effectiveness. Plants and their extracts, and antimicrobial peptides (AMPs) are gaining attention due to their naturalness, high bioavailability, and innumerable antimicrobial properties. In Chapter 1, Loranthus europaeus, well‐known medicinal plant, was chosen to evaluate its potential antimicrobial action. To this aim, different protocols were performed to selectively extract protein compounds, from L. europaeus yellow fruits, and evaluate the antimicrobial activity against four phytopathogenic fungi (Aspergillus niger, Alternaria spp., Penicillium spp., Botrytis cinerea) and a number of foodborne bacterial pathogens (Listeria monocytogenes, Staphylococcus aureus strains, Salmonella Typhimurium and Escherichia coli) by using serial dilutions and colony formation assays. Results evidenced no antifungal activity but a notable bactericidal efficiency of a crude protein extract against two foodborne pathogens, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values between 0.2 and 0.5 mg/mL, being S. aureus strains the most susceptible bacteria. Moreover, a strong bactericidal activity against S. aureus M7 was observed by two partially purified protein fractions of about 600 and 60 kDa molecular mass in native conditions. Therefore, these plant protein extracts could be used as natural alternative preventives to control food poisoning diseases and preserve foodstuff avoiding health hazards of chemically antimicrobial applications. Chapter 2 aimed at evaluating the antimicrobial and antibiofilm activity of the innate defense peptide, named 1018-K6, against Salmonella enterica. A total of 42 strains, belonging to three different subspecies and 32 serotypes, were included in this study. The antibiotic resistance profile of all the strains and the cytotoxic effects of 1018-K6 on mammalian fibroblast cells were also investigated. Results revealed that MIC (minimum inhibitory concentrations) and MBC (minimum bactericidal concentrations) values were in the ranges of 8–64 μg/mL and 16–128 μg/mL, respectively, although most strains (97%) showed MICs between 16 and 32 μg/mL. Moreover, sub- inhibitory concentrations of 1018-K6 strongly reduced the biofilm formation in several S. enterica strains, whatever the initial inoculum size. The results showed that 1018-K6 was able to control and manage S. enterica growth with a large potential for applications in the fields of active packaging and disinfectants. To this purpose, the goal of Chapter 3 was to investigate the effects of a food contact surface of polyethylene terephthalate (PET) functionalized with a previously characterized antimicrobial peptide mitochondrial-targeted peptide 1 (MTP1), in reducing the microbial population related to spoilage and in extending the shelf- life of different types of fresh foods such as ricotta cheese and buffalo meat. Modified polymers were characterized concerning the procedure of plasma-activation by water contact angle measurements and Fourier transform infrared spectroscopy measurements in attenuated total reflection mode (ATR-FTIR). Results showed that the MTP1-PETs provided a strong antimicrobial effect for spoilage microorganisms with no cytotoxicity on human cell line. Finally, the activated polymers revealed high storage stability and good reusability. Chapter 4 aimed to evaluate the efficacy of packaging in polyethylene terephthalate (PET) functionalized with the antimicrobial peptide 1018-K6 to extend the shelf life of fish burgers of dolphinfish (Coryphaena hippurus). At 1, 3, 5 and 7 days, microbiological analyses, pH, aw (activity water) and the sensory analyses were carried out. Sensory results showed that samples packed with functionalized packaging were considered acceptable two days longer than the control group. Microbiological data underlined an evident antimicrobial activity of the active packaging at 5 days against coliforms, Enterobacteriaceae, Pseudomonas spp., E. coli and fecal streptococci, with a reduction of about 1 Log (CFU/g) compared to the control group. The advantage to use natural antimicrobial compounds is also due to their poor link with the common bacterial resistance than conventional drugs. To this purpose, a better comprehension of the mechanisms of action of the antimicrobial peptide is essential for promoting their use in the food industry. Although antimicrobial activity of 1018-K6 towards both Gram-positive and Gram-negative bacteria responsible for foodborne diseases has been extensively demonstrated, the mechanism of action at the molecular level is still unclear. In Chapter 5, mechanistic studies (binding saturation assay, and fluorescence methodologies) were carried out using model membranes and bacterial membranes extracted from pathogenic bacteria isolated in various food matrices. Results reveal that the antimicrobial activity of 1018-K6 is connectable to its affinity for bacterial membranes. The study highlights differences in peptide-lipid interaction and partitioning between mimetic membranes of bacteria, suggesting that peptides have to establish more deeper interactions for binding to Gram- positive membrane that could be favored with arginine (Arg) residues. Moreover, the peptide ability to interact with bacterial mimetic liposomes was confirmed by the formation of vesicular aggregates at ratios of lipid to peptide lower than 80. In conclusion, it is worth pointing out the higher affinity of 1018-K6 for bacterial membranes than those of the respective multilaminar vesicles (MLVs). Thus far, the knowledge gathered on the mechanism of action of this peptide shows a high specificity against anionic liposomes and provides valuable information on the time of action. By calculating that the peptide needs only about 30 minutes or 2 hours of contact to enter in the membranes of Gram- negative and Gram-positive, respectively, the prospect of using 1018-K6 in the formulation of active packaging becomes more concrete. The antimicrobial packaging is offering a viable solution to tackle the economic and safety issues above mentioned, by extending the shelf-life and improving the quality and safety of fresh products. Overall, this thesis provided valuable information to develop alternative antimicrobial packaging for enhancing and extending the microbial quality and safety of perishable foods during storage by using natural compounds.

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