Artificial sweeteners have become increasingly controversial due to their questionable influence on consumers’ health. They are introduced in most foods and many consume this added ingredient without their knowledge. Currently, there is still no consensus regarding the health consequences of artificial sweeteners intake as they have not been fully investigated. Consumption of artificial sweeteners has been linked with adverse effects such as cancer, weight gain, metabolic disorders, type-2 diabetes and alteration of gut microbiota activity. Moreover, artificial sweeteners have been identified as emerging environmental pollutants, and can be found in receiving waters, i.e., surface waters, groundwater aquifers and drinking waters. In this study, the relative toxicity of six FDA-approved artificial sweeteners (aspartame, sucralose, saccharine, neotame, advantame and acesulfame potassium-k (ace-k)) and that of ten sport supplements containing these artificial sweeteners, were tested using genetically modified bioluminescent bacteria from E. coli. The bioluminescent bacteria, which luminesce when they detect toxicants, act as a sensing model representative of the complex microbial system. Both induced luminescent signals and bacterial growth were measured. Toxic effects were found when the bacteria were exposed to certain concentrations of the artificial sweeteners. In the bioluminescence activity assay, two toxicity response patterns were observed, namely, the induction and inhibition of the bioluminescent signal. An inhibition response pattern may be observed in the response of sucralose in all the tested strains: TV1061 (MLIC = 1 mg/mL), DPD2544 (MLIC = 50 mg/mL) and DPD2794 (MLIC = 100 mg/mL). It is also observed in neotame in the DPD2544 (MLIC = 2 mg/mL) strain. On the other hand, the induction response pattern may be observed in its response in saccharin in TV1061 (MLIndC = 5 mg/mL) and DPD2794 (MLIndC = 5 mg/mL) strains, aspartame in DPD2794 (MLIndC = 4 mg/mL) strain, and ace-k in DPD2794 (MLIndC = 10 mg/mL) strain. The results of this study may help in understanding the relative toxicity of artificial sweeteners on E. coli, a sensing model representative of the gut bacteria. Furthermore, the tested bioluminescent bacterial panel can potentially be used for detecting artificial sweeteners in the environment, using a specific mode-of-action pattern.

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Figure 1. Artificial sweeteners toxicity. The toxicity index of different artificial sweeteners on the three tested bioluminescent bacteria strains: (A) TV1061; (B) DPD2544; (C) DPD2794. A strong induction response pattern may be observed in the response of the TV1061 strain to saccharin and DPD2794 strain to aspartame and saccharin. In addition, a strong inhibition response pattern may be observed in the response of the TV1061 strain to sucralose.

Figure 2. Sport supplements’ toxicity. Toxicity index of different sport supplements on the three tested bioluminescent bacteria strains: (A) TV1061; (B) DPD2544; (C) DPD2794.

Figure 3. Experimental process. (A) each bacteria strain tested was striked on an agar plate containing Kanamycin, and incubated overnight at 37 °C; (B) a starter was grown from a single colony from the striked plate, and incubated overnight at 37 °C in a shaking incubator; (C) the starter was refreshed by adding 200 μL of the overnight culture into 10 mL of fresh LB, and then grown for 3–4 h at 30 °C in a non-shaking incubator; (D) the bacteria strains were then exposed to the different samples of different concentrations in a high-throughput measurement using a 96-well plate; (E,F) the toxicity (Relative Light Unit (RLU)) and growth (O.D. 600 nm) signals were measured continuously during the 16 h incubation at 26 °C, in the Luminometer and TECAN reader, respectively.

Table 1. Artificial sweeteners toxicity and viability effect (mg/mL).

	Strain	MLIC	MLIndC	MGIC	MGIndC
Aspartame	TV1061	N.E.	N.E.	N.E.	N.E.
	DPD2544	N.E.	N.E.	N.E.	N.E.
	DPD2794	N.E.	4	N.E.	N.E.
Sucralose	TV1061	1	N.E.	50	N.E.
	DPD2544	50	N.E.	50	N.E.
	DPD2794	100	N.E.	50	N.E.
Saccharin	TV1061	N.E.	5	5	N.E.
	DPD2544	N.E.	N.E.	N.E.	N.E.
	DPD2794	N.E.	5	N.E.	N.E.
Advantame	TV1061	N.E.	N.E.	N.E.	2
	DPD2544	N.E.	N.E.	N.E.	N.E.
	DPD2794	N.E.	N.E.	N.E.	N.E.
Neotame	TV1061	N.E.	2	N.E.	N.E.
	DPD2544	2	N.E.	N.E.	N.E.
	DPD2794	N.E.	N.E.	N.E.	N.E.
Ace-K	TV1061	N.E.	N.E.	N.E.	N.E.
	DPD2544	N.E.	N.E.	N.E.	N.E.
	DPD2794	N.E.	10	N.E.	N.E.

MLIC—Minimum Luminescent Inhibition Concentration; MLIndC—Minimum Luminescent Induction Concentration; MGIC—Minimum Growth Inhibition Concentration; MGIndC—Minimum Growth Induction Concentration; N.E.—No Effect.

Table 2. Sport supplements’ toxicity and viability effect (µg/mL).

	Strain	MLIC	MLIndC	MGIC	MGIndC
SS1	TV1061	N.E.	2000	N.E.	N.E.
	DPD2544	2 × 10−3	2 × 10−6	N.E.	N.E.
	DPD2794	N.E.	N.E.	2000	N.E.
SS2	TV1061	N.E.	N.E.	N.E.	N.E.
	DPD2544	1 × 10−3	1 × 10−6	N.E.	N.E.
	DPD2794	N.E.	N.E.	1000	N.E.
SS3	TV1061	N.E.	N.E.	N.E.	4000
	DPD2544	4 × 10−3	4 × 10−6	N.E.	N.E.
	DPD2794	4000	N.E.	N.E.	4000
SS4	TV1061	N.E.	N.E.	N.E.	5000
	DPD2544	5 × 10−3	5 × 10−6	N.E.	N.E.
	DPD2794	N.E.	N.E.	N.E.	5000
SS5	TV1061	N.E.	5000	N.E.	N.E.
	DPD2544	5 × 10−3	5 × 10−6	N.E.	N.E.
	DPD2794	N.E.	5000	5000	N.E.
SS6	TV1061	N.E.	N.E.	N.E.	3000
	DPD2544	3 × 10−3	3 × 10−6	N.E.	N.E.
	DPD2794	N.E.	N.E.	N.E.	N.E.
SS7	TV1061	5000	500	5000	N.E.
	DPD2544	5 × 10−3	5 × 10−6	N.E.	N.E.
	DPD2794	5000	N.E.	5000	N.E.
SS8	TV1061	N.E.	N.E.	N.E.	2000
	DPD2544	2 × 10−3	2 × 10−6	N.E.	N.E.
	DPD2794	N.E.	N.E.	N.E.	2000
SS9	TV1061	N.E.	3000	N.E.	N.E.
	DPD2544	3 × 10−3	3 × 10−6	N.E.	N.E.
	DPD2794	N.E.	N.E.	N.E.	N.E.
SS10	TV1061	N.E.	N.E.	N.E.	3000
	DPD2544	3 × 10−3	3 × 10−6	N.E.	N.E.
	DPD2794	N.E.	N.E.	N.E.	3000

MLIC—Minimum Luminescent Inhibition Concentration; MLIndC—Minimum Luminescent Induction Concentration; MGIC—Minimum Growth Inhibition Concentration; MGIndC—Minimum Growth Induction Concentration; N.E.—No Effect.

Table 3. Sport supplement profile.

	Artificial Sweeteners Content	Recommended Amount for Consumption (1 oz = 30 mL)	Ingredients
SS1	Sucralose	2 tablets (5 g), recommended to drink a lot of water	Creatine Hydrochloride, Cellulose, Dicalcium phosphate, Enteric Coating (Cellulose, Sodium Alginate, Medium Chain Triglycerides, Oleic and Stearic Acid), Natural Mint Flavor, Sucralose, Titanium Dioxide
SS2	Acesulfame Potassium-K and Sucralose	2 (7 g) to 8 (28 g) scoops in 8–10 oz per serving (2 scoops)	Black Tea Extract, Green Tea Extract, Green Coffee Extract, Micronized Taurine, Micronized l-Glutamine, Micronized l-Arginine, Micronized l-Leucine, Beta-Alanine (as CarnoSyn®), Micronized Citrulline, Micronized l-Isoleucine, Micronized l-Valine, Micronized l-Tyrosine, Micronized l-Histidine, Micronized l-Lysine, Micronized l-Phenylalanine, Micronized l-Threonine, Micronized l-Methionine Other Ingredients: Inulin, Acesulfame Potassium, Citric Acid, FD&C Red #40, Malic Acid, Natural and Artificial Flavors, Sucralose, Silion Dioxide
SS3	Acesulfame Potassium-K and Sucralose	1 (31 g) to 2 (62 g) scoops in 6–8 oz per scoop	Calcium, Cholesterol, Dietary Fibers, Potassium, Protein, Saturated Fat, Sodium, Sugars, Trans Fat Other Ingredients: Acesulfame Potassium, Cocoa (Processed with Alkali), Enzyme Blend (Aminogen®, Lactase), Lecithin, Natural and Artificial Flavors, Salt, Sucralose, Whey Protein Blend (Whey Protein Isolate, Whey Protein Concentrate, Whey Protein Hydrolysate), Xanthan Gum
SS4	Sucralose	1 (31 g) to 2 (62 g) scoops in 4–10 oz per scoop	Calcium, Cholesterol, proteins, Sodium, Saturated Fat, sugars, Trans Fat Other Ingredients: Citric Acid, FD&C Red #40 Lake, Lactase, Sucralose, Natural and Artificial Flavors, Soy Lecithin, Whey Protein Isolate, Whey Protein Concentrate, Whey Peptides
SS5	Sucralose	2 (9 g) to 6 (27 g) scoops in 10–12 oz per serving (2 scoops)	Caffeine, Green Tea Extract, Green Coffee Extract, Micronized Taurine, Micronized l-Glutamine, Micronized l-Arginine, Micronized l-Leucine, Beta-Alanine (as CarnoSyn®), Micronized Citrulline, Micronized l-Isoleucine, Micronized l-Valine, Micronized l-Tyrosine, Micronized l-Histidine, Micronized l-Lysine HCI, Micronized l-Phenylalanine, Micronized l-Threonine, Micronized l-Methionine Other Ingredients: Calcium Citrate, Calcium Silicate, Citric Acid, Gum Blend (Cellulose Gum, Xanthan Gum, Carrageenan), FD&C Blue #2, FD&C Red #40, Inulin, Lecithin, Malic Acid, Natural and Artificial Flavors, Silicon Dioxide, Sucralose, Tartaric Acid
SS6	Acesulfame Potassium-K	1 (29.4 g) rounded scoop in 4–10 oz	Calcium, Protein, Saturated Fat, Sodium, Sugars, Trans Fat Other Ingredients: Acesulfame Potassium, Aminogen®, Lactase, Lecithin, Natural and Artificial Flavor, Whey Protein Isolate, Whey Protein Concentrate, Whey Peptides
SS7	Acesulfame Potassium-K and Sucralose	1 (49 g) to 2 (98 g) scoops in 6 oz per scoop	Alpha lipoic acid, Calcium, Citric Acid, Creatine Monohydrate, Creatine HCI, Dicalcium Phosphate, Dextrose, l-alanine, l-Isoleucine, l-Leucine, l-Valine, Magnesium Oxide, Potassium, Sodium, Sugar, Taurine, Vitamin B6, Vitamin C, Vitamin B12 Other Ingredients: Acesulfame-Potassium, Dextrose, Ethyl-Cellulose, Glucose Polymers, Modcarb™ [Oat Bran, Amaranth, Quinoa, Buckwheat, Millet, Chia], Natural Flavors, Calcium Silicate, Salt, Sucralose, FD&C Yellow No. 6, Soy Lecithin, FD&C Yellow No. 5, Waxy Maize (Corn Starch), (Cluster Dextrin)
SS8	Acesulfame Potassium-K and Sucralose	1 (34 g) scoop in 6 oz water or skim milk	Calcium, Cholesterol, Dietary Fiber, Iron, Protein, Saturated Fat, Sodium, Sugar Other Ingredients: Acesulfame-Potassium, Alkalized Cocoa Powder, Calcium Carbonate, Gum Blend (Cellulose Gum, Xanthan Gum, Carrageenan), Natural and Artificial Flavors, Salt, Soy Lecithin, Sucralose, Sunflower-based Creamer (Sunflower oil, Corn syrup solids, Sodium Caseinate, Mono-Diglycerides, Dipotassium Phosphate, Tocopherols), Tricalcium Phosphate, Whey Protein Isolate, Whey Peptides, whey Protein Concentrate
SS9	Acesulfame Potassium-K and Sucralose	1 (32.4 g) to 2 (64.8 g) scoops in 8–12 oz	Calcium, Cholesterol, Dietary Fiber, Iron, Potassium, Protein, Saturated Fat, Sodium, Sugar, Trans Fat, Vitamin A, Vitamin C Other Ingredients: Acesulfame-Potassium, Amino Matrix (l-Glycine, l-Taurine, BCAAs (Leucine, Iso-Leucine, Valine), l-Glutamine), Flax Seed Oil, Glucose Polymers, Lactase, Natural and Artificial Flavors, Sucralose, Sea Salt, Suspension Matrix (Xanthan Gum, Cellulose Gum, Guar Gum), Whey Protein Concentrate, Whey Protein Isolate, Whey Protein Hydrolysate
SS10	Acesulfame Potassium-K and Sucralose	1 (34.9 g) to 2 (69.8 g) scoops in 8–12 oz	Calcium, Cholesterol, Dietary Fiber, Iron, Multi-level Amino Acid Growth Matrix, Potassium, Protein, Saturated Fat, Sodium, Trans Fat Other Ingredients: Alanine, Arginine, Aspartic Acid, BCAAs (l-Leucine, l-isoleucine, l-Glutamine, l-valine), Cystine, Digestive Enzyme Blend, Egg Albumen, Glycine, Histidine, Lactase, Lysine, Methionine, Micellar Casein, Partially-hydrolyzed Whey Concentrate, Phenylalanine, Proline, Protease, Serine, Tyrosine, Threonine, Tryptophan, Whey Protein Isolate, Whey Protein Concentrate

Table 4. Bioluminescent bacterial strains.

Strain	E. coli Host Strain	Promoter	Plasmid	Stress Sensitivity	Reference
TV1061	RFM443	grp E	pGrpELux5	Heat Shock (Cytotoxic)	[86]
DPD2544	W3110	fab A	pFabALux6	Fatty Acid Availability (Cytotoxic)	[53]
DPD2794	RFM443	rec A	pRecALux3	SOS—DNA Damage (Genotoxicity)	[87]

• Sample Availability: Not available.

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