Table 2.

Summary of changes to the gut microbiome in response to NNS.

Sweetener and ADI	Reference	Amount used and length of exposure	Study type/model	Reported effects
Acesulfame – K (Ace K) 15 mg/kg BW/day	(96)	3% Ace K	In vitro	• No significant effects
	(97)	1.7–33.2 mg/kg BW/day	Human	• No significant effects
	(93)	15 mg/kg BW/day 8 weeks	Male mice	• No significant effects
	(92)	37.5 mg/kg BW/day 4 weeks	Mice	• Increased Bacteroides, Anaerostipes, and Sutterella within male rats • Decreased Lactobacillus and Clostridium within female rats
	(88)		In vitro E. coli K-12	• Inhibit Escherichia coli HB101 and K-12
	(98)	ADI1x: 0.25 mg AceK + Sucralose (dams only) ADI2x: 0.5 mg AceK + Sucralose (dams only) 6 weeks	Pregnant dams and offspring	• Doubled Firmicutes • Diminished Akkermansia muciniphila
	(85)	0–6 mg/ml 5 h incubation	In vitro E. coli K-12	• Stimulated growth of E. coli
	(99)	150mg/kg BW/day 8 weeks	Male mice	• Decreased Clostridiaceae, Lachnospiraceae, and Ruminococcaceae
Aspartame 50 mg/kg BW/day	(26)	Concurrent with high fat/sucrose diet 5–7 mg/kg BW/day 8 weeks	Rat	• Increase Enterobacteriaceae and Clostridium leptum within normal chow diet • Increase Roseburia spp. with high fat diet
	(100)	135 or 400 mg Single dose	Humans (diabetic)	• No significant effects
	(101)	Concurrent with high fat/sucrose diet 5–7 mg/kg BW/day 18 weeks	Pregnant dams and offspring	• Increase Porphyromonadaceae
	(85)	0–6 mg/ml 5 h incubation	In vitro E. coli K-12	• Inhibit growth of E. coli K-12
	(102)	40mg/kg BW/day (dams only) 6 weeks	Obese pregnant dams and offspring	• Reduced Limosilactobacillus reuteri and Ligilactobacillus murinus
Saccharin 15 mg/kg BW/day	(91)	5 mg/kg BW/day 6 months	Male mice	• At 3 months: Increase Sporasarcina, Jeotgalicoccus, Akkermansia, Oscillopspira, Corynebacterium; Decrease Anaerostipes, Ruminococcus • At 6 months: Increase Corynebacterium, Roseburia, Turicibacter; Decrease Ruminococcus, Adlercreutzia, Dorea
Commercial saccharin was used, containing glucose (95%)	(25)	Mice: 5 mg/kg BW/day 5 weeks	Mice	• Mice: Increase Bacteroides, Clostridiales; Decrease Lactobacillus reuteri; Overrepresented Bacteroides vulgatis and Underrepresented Akkermansia muciniphila • Human: Increase Bacteroides fragilis and Weissella cibaria; Decrease Candidatus Arthromitus
		Human: 5mg/kg BW/day 1 week	Human
	(88)	Concurrent with high fat diet 5 mg/kg BW/day 10 weeks	Mice	• Decrease Tenericutes • Increase Proteobacteria and Actinobacteria • Increase Firmicute/Bacteroides ratio • Increase Akkermansia
	(103)	0.1 mg/ml 5 weeks	In vitro /Mice	• Inhibited Staphylococcus aureus (Firmicute), Klebsiella pneumonia and Pseudomonas aeruginosa (both Proteobacteria)
	(104)	250 mg/kg BW/day (mice) 10 weeks 400 mg/day 2 weeks	Mice Human (Randomized, double-blind, placebo controlled trial)	• No significant effects
	(105)	1.5 mM 4 weeks	Female guinea pig	• Increased Firmicutes and Lactobacillaceae-Lactobacillus
	(106)	2.5% sodium saccharin Incorporated in feed	Rat	• Inhibited 3 strains of Lactobacillus and E. coli
	(107)	0.066% (w/v), with or without ethanol (10%) 4 weeks	Mice	• Increased Eubacteria in the pregnant group that received ethanol and saccharin • Reduced Clostridium population
	(88)		In vitro	• Inhibit E. coli HB101 and K-12
Sucralose 5 mg/kg BW/day	(93)	1.5 mg/kg BW/day 8 weeks	Mice	• Decreased of Clostridium cluster XIVa
Commercial sucralose (1.10%), glucose (1.08%), moisture (4.23%), and maltodextrin (93.59%)	(108)	Dosing range (100–1000 mg/kg BW/day) 12 weeks	Rat	• Decreased total anaerobes and aerobic bacteria • Decreased Lactobacilli, Bifidobacteria, Clostridia, and Bacteroirdes
	(27)	5 mg/kg BW/day 6 months	Male mice	• Increased Ruminococcus; Decrease Lachnospiraceae, Dehalobacteriaceae, Anaerostipes, Staphylococcus, Peptostreptococcaceae, Bacilles at 3 months • Increase Akkermansia, Turicibacter, Roseburia, Clostridiaceae, Christensenellaceae; Decrease Streptococcus, Lachnospiraceae, Dehalobacteriaceae, Erysipelotrichaceae at 6 months
	(109)	3.3 mg/kg BW/day (normal chow) 1.5 mg/kg BW/day (high fat diet) 8 weeks	Mice	• Increase Firmicutes (normal and high fat diet) • Increase Bifidobacterium (normal diet)
	(88)		In vitro	• Inhibit E. coli HB101
	(110)	3.5 mg/ml 6 weeks	Mice (induced Crohn's Disease model)	• Increased Proteobacteria
	(98)	ADI1x: 0.1 mg + Ace K (dams only) ADI2x: 0.2 mg + Ace K (dams only) 6 weeks	Pregnant dams and offspring (mouse)	• Increased Firmicutes
	(85)	0–6 mg/ml 5 h incubation	In vitro E. coli K-12	• No significant effects
	(111)	Concurrent with high fat diet 1.5% water solution 4 months	Male Rat	• increase in three Bacteroides species, B. fragilis
	(112)	0.1 mg/ml (dams only) 3 weeks	Pregnant dams and offspring	• Increased Akkermansia, Blautia, Corynebacterium, and Robinsoneilla • Diminished Alistipes, Barnesiella, Paraprevotella, Saccharibacteria incertae sedis, and Streptococcus
Steviol glycosides 4 mg/kg BW/day	(101)	2–3 mg/kg BW/day; 9 weeks	Rats	• Decrease Bifidobacteriaceae • Increase Bacteroides goldsteinii and Bacteroides thetaiotaomicron
	(113)	2–3 mg/kg BW/day 18 weeks	Obese dams and offspring	• Decrease Bifidobacteriaceae • Increase Bacteroides goldsteinii and Bacteroides thetaiotaomicron
	(95)	5 mg/kg BW/day Concurrent with high fat diet 10 weeks	Mice	• Increase Firmicutes/Bacteroides ratio • Increase Proteobacteria and Actinobacteria
	(114)	24 h	In vitro (human fecal samples)	• Bacteroides hydrolyze to steviol and rebaudioside A most efficiently
	(90)	95% (w/w) stevioside 97% rebaudioside A 24 h	In vitro Limosilactobacillus reuteri	• Inhibit L. reuteri growth
Neotame 0.3 mg/kg BW/day	(115)	0.75 mg/kg BW/day 4 weeks	Mice	• Decreased Firmicutes • Increased Bacteroidetes

BW, body weight; ADI1x, the recommended ADI; ADI2x, twice the recommended ADI; w/v, weight per volume; w/w, weight per weight; N/A, not applicable.