Antimicrobiens naturels d'origine bactérienne : contribution à l'étude de leur activité biologique, leur stabilité gastrointestinale et leur toxicité
|Advisor:||Fliss, Ismaïl; Biron, Éric; Subirade, Muriel|
|Abstract:||In recent decades, natural antimicrobials produced by bacteria, such as bacteriocins and reuterin, have received considerable attention as food preservatives or as alternatives to antibiotics for the human and animal sectors. Approval of bacteriocins and reuterin as bio-preservatives or sanitizers by regulatory agencies is based not only on evidence of efficacy but also on sufficient data on cytotoxicity and behavior in the gastrointestinal conditions. Although the antimicrobial activities of bacteriocins and reuterin have been widely studied, very little data is available on their behavior in biological matrices and their toxicity for different applications. Therefore, the general objective of this thesis was to evaluate the gastrointestinal stability, the biological activity, and the toxicity of bacteriocins and reuterin using different in vitro models for their intended use in food, medical, and veterinary applications. The first specific objective was to produce, purify and evaluate the antimicrobial activity of bacteriocins and reuterin alone and in combination against a wide range of pathogenic and spoilage organisms of interest to different sectors. Five different synergistic consortia were identified, of which three of them (microcin J25 + citric acid + lactic acid, microcin J25 + reuterin + citric acid, and microcine J25 + reuterin + lactic acid) showed significant activity against Salmonella enterica subsp. enterica serovar Newport ATCC 6962 (FIC index = 0.5). Two other combinations (pediocin PA-1 + citric acid + lactic acid, and reuterin + citric acid + lactic acid) showed partially synergistic effect against Listeria ivanovii HPB28 (FIC index = 0.75). The second specific objective was to evaluate the gastrointestinal stability of reuterin using an in vitro simulated digestion conditions in humans. Furthermore, the hemolytic activity, and in vitro cytotoxicity of reuterin using neutral red assay and lactate dehydrogenase (LDH) release assays were determined on assay on the Caco-2 cell line. Reuterin was observed to be very stable biologically during gastrointestinal transit. Moreover, exposure of Caco-2 cells to different concentrations of reuterin showed no negative effect on cell viability or membrane integrity at concentrations up to 1080 mM. In addition, no hemolysis was observed on erythrocytes exposed to concentrations up to 270 mM of reuterin. As a result, this study demonstrated that reuterin can be safely used at concentrations up to 270 mM, in various applications. The third objective was to assess the gastrointestinal stability and activity of microcin J25, pediocin PA-1, bactofencin A and nisin using in vitro models. In addition, the cytotoxicity and hemolytic activity of these bacteriocins were studied on Caco-2 cells and rat erythrocytes, respectively. According to the results, pediocin PA-1, bactofencin A and nisin were degraded at different levels during gastrointestinal transit, while microcin J25 was only partially degraded. Moreover, bacteriocins did not show cytotoxic effects on Caco-2 cells at concentrations up to 400 µg/mL. Finally, pediocin PA-1, bactofencin A, and nisin at concentrations higher than 50 µg/mL showed hemolytic potential, while microcin J25 did not cause hemolysis. In the fourth objective, the dermal toxicity of reuterin, microcin J25, pediocin PA-1, bactofencin A and nisin Z was studied in vitro using neutral red and LDH release assays on NHEK cells. The skin sensitization and irritation potentials of these bacteriocins and reuterin were also determined using the h-CLAT test and the human epidermal model EpiDerm ™, respectively. The results demonstrated that exposure of NHEK cells to these bacteriocins and reuterin at concentrations up to 400 µg/mL and 80 mg/mL, respectively, did not induce any cytotoxic effect. Inaddition, microcin J25 and reuterin showed no skin sensitization at concentrations up to 100 µg/mL and 40 mg/mL, respectively, while pediocin PA-1, bactofencin A and nisin Z caused sensitization at concentrations higher than 100 µg/mL. Tissue viability was not affected in the presence of bacteriocins and reuterin at concentrations up to 200 µg/mL and 40 mg/mL, respectively. Taken together, these results showed that certain bacteriocins and reuterin could be safely incorporated as an antimicrobial ingredient in various topical preparations intended for human and animal applications. Finally, the last chapter of the thesis was devoted to the development and characterization of different antimicrobial hydrogels based on bacteriocins or reuterin. Reuterin, pediocin PA-1, microcin J25 were incorporated into hydrogel formulations developed from biopolymers; chitosan (1.5%, 2.5%) or carboxymethyl cellulose (CMC, 3, 5%). The developed hydrogels were characterized in terms of general appearance, viscosity, antimicrobial activity, and stability during storage at room temperature. The results showed that the 1.5% and 2.5% chitosan containing microcin J25 or pediocin PA-1 have significant inhibitory activity throughout the storage period. The inhibitory effect of microcin J25 and reuterin in the 3% and 5% CMC hydrogels remained unchanged while the pediocin PA-1 added to the 3% and 5% CMC hydrogels totally lost its activity after one week. In terms of viscosity, the addition of antimicrobials to the hydrogels did not affect their viscosity and all formulations remained stable during the four weeks of storage. The results suggested that the formulas developed have great potential for application as sanitizer in the food, medical and veterinary sectors. In conclusion, this thesis has generated unique results on the gastrointestinal behavior, biological activity, stability, and toxicity of several bacterial drived antimicrobials including different bacteriocins and reuterin. Different in vitro models were adopted for this study. The results obtained are very relevant and are essential for the approval process of these natural compounds by the various regulatory agencies for different applications in food, medical or veterinary sector.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||20 December 2021|
|Collection:||Thèses et mémoires|
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