New insights into gastrointestinal stability and toxicity of bacteriocins for potential applications in the food, medical and veterinary sectors
Samira Soltani 1*, Séverine Zirah 2, Sylvie Rebuffat 2 , Eric Biron 3, Yvan Boutin 4 Frédéric Couture 4,  Muriel Subirade 1,5, Ismail Fliss 1,5

1 Food science department, Food and agriculture faculty, Laval University, Quebec, Quebec, Canada; 2 Muséum National d’Histoire Naturelle, Centre National de la Recherche Scientifique, Laboratory  Molecules of Communication and Adaptation of Microorganisms (MCAM), UMR 7245 CNRS-MNHN, CP 54, 57 rue Cuvier 75005, Paris, France; Faculty of Pharmacy, Laval University, Quebec, Quebec, Canada ; 4  TransBIOTech, 201 Rue Mgr Bourget, Lévis, Quebec, Canada G6V 6Z9; 5 Institute of Nutrition and Functional Foods, Laval University, Quebec, Quebec, Canada

Introduction: Bacteriocins have received substantial attention for potential applications in the food, veterinary and clinical settings. In order to be used for different applications, bacteriocins have to pass through strict toxicity evaluation to be legally approved. Although bacteriocins have been widely studied for their inhibitory activities, there is not sufficient data available on their gastrointestinal behaviour and toxicity for their intended use 1.

Objective: In this study, different in vitro models were used to provide scientific data and a complete portrait of gastrointestinal stability and oral toxicity as well as dermal toxicity (cytotoxicity, skin sensitization, and skin irritability) of selected bacteriocins, namely, microcin J25, pediocin PA-1, nisin Z, bactofencin A.

Method: All bacteriocins have been produced at a high level of purity. Their activity has been monitored using both agar diffusion and microtitration assays.Gastrointestinal stability of selected bacteriocins has been evaluated using in vitro simulated digestion according to INFOGEST protocol2. Also, hemolytic activity on rat erythrocyte was assessed and by LDH release assay, bacteriocins’ interaction with epithelial cells (Caco-2 cells) was studied.Dermal cytotoxicity was assessed using the neutral red and LDH release assays on normal human epidermal keratinocytes (NHEK) cells. Skin sensitization and skin irritability were studied according to OECD guideline using human cell line activation test (h-CLAT assay)3 and EpiDerm™ culture (EPI-200)4 as a human skin equivalent, respectively. 

Results: Pediocin PA-1, bactofencin A, and nisin were observed to lose their stability passing through the gastrointestinal tract, while microcin J25 is only partially degraded. Besides, selected bacteriocins were not toxic to Caco-2 cells, and the integrity of the cell membrane was observed to remain unaffected in the presence of these bacteriocins at concentrations up to 400 μg/mL. In the hemolysis study, pediocin PA-1, bactofencin A, and nisin were observed to lyse rat erythrocytes at concentrations higher than 50 μg/mL, while microcin J25 showed no effect on these cells. NHEK cells' viability and membrane integrity remained unaltered after exposure to bacteriocins at concentrations up to 400 µg/mL. Furthermore, microcin J25 showed no skin sensitization at concentrations up to 100 µg/mL, while pediocin PA-1, bactofencin A, and nisin Z caused sensitization at concentrations higher than 100 µg/mL. Tissue viability was unaffected in the presence of bacteriocins at concentrations up to 200 µg/mL.

Conclusion: The current study provides scientific evidence that pediocin PA-1, bactofencin A, and microcin J25, could be safely used in the food, medical and veterinary applications orally and topically.

1.         Soltani, S.;  Hammami, R.;  Cotter, P. D.;  et al., Bacteriocins as a new generation of antimicrobials: Toxicity aspects and regulations. FEMS Microbiol Rev 2020.
2.         Brodkorb, A.;  Egger, L.;  Alminger, M.;  et al., INFOGEST static in vitro simulation of gastrointestinal food digestion. Nat Protoc 2019,14 (4), 991-1014.
3.         OECD, Test No. 442E:In Vitro Skin Sensitisation: Human Cell Line Activation Test (h-CLAT). OECD Publishing: 2016.
4.         OECD, T. N., 439: In vitro skin irritation: Reconstructed human epidermis test method. OECD Guidel Test Chem Sect 2019,4.