Table 3.

Common criteria generally considered as essential for the safety of NF/probiotic products (required for both novel food and health claim regulations)

	What		How	Why	Comments and Propositions for improvement
Survival in GI tract conditions	Resistance to intestinal stress		In vitro Growth curves/Detection in feces after consumption	Resistance to GI tract conditions may favor the beneficial effects	Not valuable for all beneficial effects
					Development of new protectors/encapsulators
	Bile salt deconjugation		High-performance liquid chromatography	Large amounts of deconjugated bile salts may have undesirable effects on the human host	Evaluation of property in vitro has poor relevance; assessment of bile salt deconjugation in vivo
			Mass spectrometry
Preservation of the homeostasis of gut barrier components	Microbiota	Perturbation of commensal consortium	In vitro production of bacteriocins or antibiotics (AB)	Bacteriocins and AB may perturb microbiota.	Development of growth inhibitory references with major commensal bacteria
				AB may interact with a patient’s treatment
		Antibiotics (AB) resistance	In silico *prediction and in vitro antibiogram	AB resistance may be transmitted between bacteria	Development of in vivo assessment (animal model) indicating the microbiota homeostasis (composition and activities) after probiotic consumption
			Minimal inhibitory concentration test (MIC)
			Minimal bactericidal concentration test (CMB)		If plasmids are detected: the presence/absence of genes encoding the most common resistance determinants should be characterized
		Presence of plasmids	In silico *prediction or DNA extraction followed by analysis by gel electrophoresis	Plasmids favor the transmission of antibiotic resistance	Requirement to up-date the antibiotic list
	Mucus	Mucus degradation	Mucin degradation test (agarose gel or liquid culture)	Excessive mucus degradation may lead to intestinal barrier weakening	The capacity to degrade mucus seems to be a poor criterion to estimate the protective or deleterious effect of bacteria on the intestinal barrier
Adhesion and translocation risk	Intestinal/Mucosal adhesion		Test bacterial strain adhesion to epithelial cell line	Mucosal adhesion may interfere with pathogenic microorganisms, stimulate beneficial cellular processes, or favor bacterial translocation	The intestinal/mucosal adhesion capacity can be either a beneficial or a deleterious criterion
	Intestinal mucosa degradation		Gelatinase activity assay	Mucosal degradation may weaken the intestinal barrier	It could be useful to evaluate in vivo translocation capacities
	Hemolytic activity		Blood agar culture	Hemolysis damages red blood cells
Metabolic activities	D-Lactate production		Colorimetric assay	D-Lactate accumulation in blood leads to acidosis	The production of D-lactate should be compared with the amount produced by usual strains (like in yoghurt)
	Toxin production		Protocol recommended by the European scientific committee of animal nutrition.	Toxic molecules	Establishment of threshold values relevant in humans
	Biogenic amine production		Colorimetric assay	Immune responses such as allergic responses
Remote effects	Platelet aggregation		Aggregation test	Risk of thrombosis	Development of ex vivo protocols (explants, organoids)
	Genotoxicity		Toxicity testing on animals models (chronic and subchronic tests)	Risk of cancer
	Allergenicity			Allergic response

(*: if the complete genome is available).