Aspartame

The Pan American Health Organization (PAHO) nutrient profile model (NPM) was used to classify foods according to their nutritional profile because it was developed to be used in various food and nutrition policies in Latin America, including labeling, and identifies unhealthy foods being aligned with the proposed FoP regulations under discussion in Brazil [30 ]. This NPM considers the level and degree of industrial processing, according to the NOVA classification, as an eligibility criterion. It classifies food products as containing or not excessive amounts of five nutrients: free sugar, total fats, saturated fats, trans fat, and sodium. In addition, the presence of nonnutritive sweeteners in the list of ingredients is also considered in the model. The thresholds determined in the model are applied on the ratio between the content of critical nutrients and the content of energy, and is based on the World Health Organization (WHO) recommendations to prevent obesity and chronic diseases [30 ]. In our study, we included a modification to the original PAHO NPM, in which a food or beverage was eligible to be regulated and therefore receive FOP warning signs based on the Chilean nutritional labeling law eligibility criteria (Law 20.606/2015). In the Chilean law, only foods and beverages with added salt, sugar, or saturated fat were eligible to receive FOP warning signs for “high in” critical nutrients [31 ]. Culinary ingredients, as sugar, salt, oils, butter, and milk creams were only included if the product had the addition of another critical nutrient in excessive amounts (for instance, butter made with milk cream and salt is eligible to be regulated and receives a warning sign for high content of sodium if this nutrient is in excess—however, it does not receive a warning sign related to the high content of fats). For this modified PAHO NPM, we used the same targeted five nutrients (free sugar, total fats, saturated fats, trans fat, and sodium) as well as nonnutritive sweeteners and applied the same threshold levels as the PAHO NPM, and the model behaved similarly to the originally proposed PAHO NPM in terms of identifying foods high in critical nutrients [32 ].
The PAHO NPM considers free sugars, information that is not available on food labels of products sold in Brazil. We thus estimated the amount of free sugars using the method proposed by PAHO that considers the information on the amount of total sugars declared on food labels [30 ]. In this method, foods are classified by the information available on the nutrition facts panel (total sugars or added sugars) and by food category. For instance, if information on total sugars is available and the product has no or a minimal amount of naturally occurring sugars, such as soda and sports drinks, then the total amount of added sugars is considered free sugars. For milk or yogurt with any type of sugar in the list of ingredients, 50% of the declared added sugars were considered free sugars, so lactose, galactose, and other types of naturally occurring sugars were less likely to be considered free sugars. In Brazil, the content of total sugar is not required to be present on nutrition facts panel in the country, and analyses that considered free sugars were conducted for 10% of the sample that provided this information.
In order to apply the PAHO nutrient profile model, products were classified as containing added sugar, sodium, fat, and nonnutritive sweeteners on the basis of keyword searches in the list of ingredients. Briefly, ingredients used as a proxy for added sugars included sugar, honey, syrups, molasses, maltodextrin, glucose, fructose, and concentrated fruit and vegetables juices, as well as chocolate and milk fondant. Ingredients for the addition of salt included salt, sodium chloride, cheeses, and processed meats. For fat, we considered oils, olives, butter, creams, and animal and vegetal fats. Nonnutritive sweeteners included aspartame, saccharin, sucralose, cyclamate, acesulfame k, stevia, polydextrose, maltitol, mannitol, isomaltose, neotame, xylitol, thaumatin, and advantame. All searches were made in Portuguese.

Approximately 1,600 journal articles previously manually curated for CTD were used as a baseline data set, or "gold standard," to evaluate the performance of our prototype text-mining applications. These documents were a subset of the approximately 25,000 documents reviewed by biocurators since CTD manual curation began in 2005. The 1,600 documents contained 6,664 curated actors, including chemicals, genes, and diseases and represented data for 10 different priority chemicals: urethane, aspartame, 2-acetylaminofluorene, cyclophosphamide, indomethacin, aniline, raloxifene, amsacrine, phenacetin, and doxorubicin.

The food additives tested in this study were purchased from Sigma-Aldrich (Castle Hill, Australia). Solutions of carboxymethylcellulose sodium salt (21904), ι-carrageenan (C1138), saccharin (109185, ≥ 99%), sucralose (69293, ≥98.0%), sodium sulfite (S0505, ≥98%), Tween (polysorbate) 80 (polysorbate 8074), aluminum silicate (520179), and aspartame (A5139) were prepared at 1% (w/v or v/v) concentration in milli-Q water and flushed with N₂ for an hour. The solutions were transferred to an anaerobic chamber (N₂:H₂, 95:5) and transferred to glass serum bottles sealed with a rubber stopper and crimp sealed. These solutions were then autoclaved and stored at room temperature prior to use.
For the growth studies with the F. prausnitzii strains, the food additive solutions were used to supplement the basal M2G medium by aseptically transferring 1 ml of each stock solution to individual tubes, to provide final concentrations of 0.1% (w/v or v/v), which is intended to approximate concentrations found within the gut milieu. The same approach was used with the Proteobacteria strains with the exception that Luria Bertani (LB) broth was used as the basal medium.

The growth experiments were repeated at least twice and with three technical replicates for each treatment group. For initial growth experiments, a single colony of each F. prausnitzii strains was transferred to M2G broth and incubated at 37°C overnight (14 − 16 hours). Aliquots (100 μl) of these cultures were used to inoculate Hungate tubes containing anaerobically prepared M2G broth medium supplemented with either the food additives, or additional sterile water, to provide a final volume of 10 ml. Microbial growth was assessed hourly by measuring the culture optical density at 600 nm (OD₆₀₀). Based on the results, a second series of experiments with all three F. prausnitzii strains were performed. All three strains were cultured with M2G broth and growth monitored as described above. Once the cultures had reached mid-exponential phase of growth, either the sodium sulfite or polysorbate 80 stock solutions were added to provide a final concentration of 0.1% and bacterial growth monitored longitudinally for another 8 hours, as described above. As controls, separate cultures received a similar volume of sterile anaerobic water or were left untreated.
The growth studies with Proteobacteria strains were conducted within a 96-well microtiter plate format. Here, the inoculating cultures were prepared by using a single colony of each strain transferred to 5 ml of LB broth within a 50 ml centrifuge tube (Corning, US) and incubated for 5 hours aerobically with shaking at 200 rpm at 37°C. Aliquots (0.135 ml) of either sterile LB broth, or LB broth prepared to contain 0.1% final concentration one of the food additives described above (with the exceptions of aluminum silicate and aspartame) were dispensed into individual wells, which were then inoculated with 0.015 ml of a 1:100 dilution (with LB) of the inoculating cultures described above. As controls, wells containing 0.15 ml of uninoculated broth (with and without food additives) were also prepared and positioned around the edge of the microtiter plates. The plates were covered with Breathe-Easy sealing membranes and placed within a Multiskan GO microplate reader (ThermoFisher, US) for measurements of aerobic growth. The plate reader was programmed to shake at 300 rpm and OD₆₀₀ measurements were taken every 30 minutes for 17 hours. A parallel series of growth studies were also done with the 7 Proteobacteria strains using a 96-well microtiter plate format under “microaerobic” conditions. Here, the LB medium was prepared with an additional step of gassing the solution with a steady stream of nitrogen gas for 60 minutes, and the manipulations described above were performed within a Coy Anaerobic chamber filled with a mixture of CO₂:H₂:N₂ (15:5:80). The “microaerobic” conditions resulted from the periodic introduction of surrounding atmosphere during interchange operations, which resulted in transient readings of ~300 ppm oxygen within the chamber atmosphere, before returning to zero. Bacterial growth was monitored by OD₆₀₀ measurements using a FLUOStar Omega (BMG Labtech, Germany) plate reader permanently housed within the chamber using the same operational parameters described above.