Identifying Potential Sources of Phthalate Contamination in the Leaves of Stevia Rebaudiana (Bertoni) and the Development of Removal Technology.

Steviosides extracted from the leaves of the plant Stevia rebaudiana are increasingly used in the food industry as natural low-calorie sweeteners. Phthalates in food are often assumed to arise from food containers or packaging materials. Here, experiments were carried out to identify the potential sources of DMP, DBP, DIBP, and DEHP in the leaves of stevioside through investigation of their content in native stevioside tissues, soils, and associated agronomic materials. The results show that phthalate contamination was present in all the samples tested, and the influence of regional factors at the provincial level on the content of plasticizers in stevia leaves was not significant. Phthalates in stevia leaves can be absorbed into the plant body through leaves and roots. Using resin removal, the phthalate content in stevioside glycosides was reduced to less than 0.05 ppm, and some indicators were far lower than the limit standard in EU food.

Sucralose (C12H19Cl3O8) impact on microbial activity in estuarine and freshwater marsh soils.

As the general population's diet has shifted to reflect current weight-loss trends, there has been an increase in zero-calorie artificial sweetener usage. Sucralose (C12H19Cl3O8), commonly known as Splenda® in the USA, is a primary example of these sweeteners. In recent years, sucralose has been identified as an environmental contaminant that cannot easily be broken down via bacterial decomposition. This study focuses on the impact of sucralose presence on microbial communities in brackish and freshwater systems. Microbial respiration and fluorescence were measured as indicators of microbial activity in sucralose-dosed samples taken from both freshwater and estuarine marsh environments. Results showed a significant difference between microbial concentration and respiration when dosed with varying levels of sucralose. Diatom respiration implied a negative correlation of community abundance with sucralose concentration. The freshwater cyanobacterial respiration increased in the presence of sucralose, implying a positive correlation of community abundance with sucralose concentration. This was in direct contrast to its brackish water counterpart. However, further investigation is necessary to confirm any potential utility of these communities in the breakdown of sucralose in the marsh environment.

Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

BACKGROUND: Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. METHODS: The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. RESULTS: In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. CONCLUSION: SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.

Exploring the Impact of Saccharin on Neovascular Age-Related Macular Degeneration: A Comprehensive Study in Patients and Mice.

Purpose: We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. Methods: In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Results: Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Conclusions: Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.

The Molecular Theory of Sweet Taste: Revisit, Update, and Beyond.

The molecular origin of the sweet taste is still elusive. Herein, the canonical AH-B-X theory of sweet taste is extended by resurveying various sweeteners, which provides deeper insights into an analogous intramolecular connectivity pattern of both glucophores in sweeteners and their interaction counterparts in sweet taste receptor TAS1R2/TAS1R3: electrostatic complementarity and topochemical compatibility. Furthermore, their complementary interaction is elaborately illustrated, accounting for the common molecular feature of eliciting sweetness. Moreover, it highlights that multiple glucophores in a topological system synergistically mediate the elicitation and performance of sweetness. This perspective presents a meaningful framework for the structure-activity relationship-based molecular design and modification of sweeteners and sheds light on the mechanism of molecular evolution of TAS1R2s/TAS1R3s. The link between palatability of sweeteners and harmony relationships between their structural components via stereochemistry and network has significant implications to illuminate the underlying mechanisms by which nature designs chemical reactions to elicit the most important taste.

Development of a tamarind-based functional beverage with partially-hydrolyzed agave syrup and the health effects of its consumption in C57BL/6 mice.

Excess consumption of sweetened beverages is associated with a global rise in metabolic diseases. Tamarind and partially-hydrolyzed agave syrup have potential for developing healthier beverages. Our objective was to develop a functional beverage using these ingredients (PH-AS-B). We also evaluate shelf-life stability (physicochemical, microbiological, and antioxidant properties) and health effects in C57BL/6 mice compared with tamarind beverages sweetened with glucose or fructose. Optimal tamarind extraction conditions were a 1:10 ratio (g pulp/mL water) and boiling for 30 min, and the resulting beverage had a shelf life of two months at 4 °C. Non-volatile metabolites were identified using HPLC/MS. PH-AS-B was associated with decreased blood cholesterol (5%) and triglyceride (20-35%) concentrations in healthy mice as well as lower lipid (82%) concentrations and evidence of protein oxidation (42%) in the liver, compared with glucose- and fructose-sweetened tamarind beverages. In conclusion, PH-AS-B was stable and associated with beneficial metabolic properties in healthy mice.

Sweeteners in Orodispersible Films: How Much is too Much?

Four natural sweeteners (sucrose, xylitol, fructose, and isomalt) were selected to examine the influence of their qualities and amounts on the characteristics of orodispersible films. Sodium carboxymethylcellulose (2% w/w) was utilized as the film-forming polymer and 1% w/w glycerol as a plasticizer. Films were produced through the solvent casting method, rendering them suitable for convenient application in community or hospital pharmacy settings. The physicochemical and optical properties of the films were analyzed, and Fourier-transform infrared analysis was carried out. All films exhibited acceptable disintegration time, uniformity of mass, thickness, and optical characteristics, with significant dependence (p<0.05) on both sweetener type and quantity. Disintegration time varied based on the employed method, as well as the characteristics and amount of sweetener. Additionally, all films maintained pH values within the oral cavity range, suggesting no potential irritancy upon administration. Fourier-transform infrared analysis confirmed the formation of the film and demonstrated compatibility between its components.

Acute and two-week effects of neotame, stevia rebaudioside M and sucrose-sweetened biscuits on postprandial appetite and endocrine response in adults with overweight/obesity-a randomised crossover trial from the SWEET consortium.

BACKGROUND: Sweeteners and sweetness enhancers (S&SE) are used to replace energy yielding sugars and maintain sweet taste in a wide range of products, but controversy exists about their effects on appetite and endocrine responses in reduced or no added sugar solid foods. The aim of the current study was to evaluate the acute (1 day) and repeated (two-week daily) ingestive effects of 2 S&SE vs. sucrose formulations of biscuit with fruit filling on appetite and endocrine responses in adults with overweight and obesity. METHODS: In a randomised crossover trial, 53 healthy adults (33 female, 20 male) with overweight/obesity in England and France consumed biscuits with fruit filling containing 1) sucrose, or reformulated with either 2) Stevia Rebaudioside M (StRebM) or 3) Neotame daily during three, two-week intervention periods with a two-week washout. The primary outcome was composite appetite score defined as [desire to eat + hunger + (100 - fullness) + prospective consumption]/4. FINDINGS: Each formulation elicited a similar reduction in appetite sensations (3-h postprandial net iAUC). Postprandial insulin (2-h iAUC) was lower after Neotame (95% CI (0.093, 0.166); p < 0.001; d = -0.71) and StRebM (95% CI (0.133, 0.205); p < 0.001; d = -1.01) compared to sucrose, and glucose was lower after StRebM (95% CI (0.023, 0.171); p < 0.05; d = -0.39) but not after Neotame (95% CI (-0.007, 0.145); p = 0.074; d = -0.25) compared to sucrose. There were no differences between S&SE or sucrose formulations on ghrelin, glucagon-like peptide 1 or pancreatic polypeptide iAUCs. No clinically meaningful differences between acute vs. two-weeks of daily consumption were found. INTERPRETATION: In conclusion, biscuits reformulated to replace sugar using StRebM or Neotame showed no differences in appetite or endocrine responses, acutely or after a two-week exposure, but can reduce postprandial insulin and glucose response in adults with overweight or obesity. FUNDING: The present study was funded by the Horizon 2020 program: Sweeteners and sweetness enhancers: Impact on health, obesity, safety and sustainability (acronym: SWEET, grant no: 774293).

[Sweet protein brazzein as a promising sweetener].

The excessive consumption of sugar-containing foods contributes to the development of a number of diseases, including obesity, diabetes mellitus, etc. As a substitute for sugar, people with diabetes mellitus and obesity most often use sweeteners. Sweet proteins, in particular brazzein, are an alternative to synthetic sweeteners that have natural origin, are broken down in the intestines along with food proteins, and do not affect blood sugar and insulin levels. The purpose of the review was to analyze the available data on the sweet protein brazzein, its physical and chemical properties, existing biotechnological methods of production, and prospects for application in the food industry in order to further develop an optimized heterologous expression system. Material and methods. Google Scholar, Scopus, Web of Science, PubMed, RSCI and eLibrary.ru databases were used for collecting and analyzing literature. Search depth - 30 years. Results. Numerous studies of the physical and chemical properties of brazzein have demonstrated its high potential for use in the food industry. In particular, a short amino acid sequence, thermal stability, the ability to maintain its structure and sweet properties in a wide pH range, hypoallergenicity, lack of genotoxicity, and an extremely high level of sweetness compared to sucrose allow us to conclude that its use is promising. Mutant variants of brazzein have been generated, the sweetest of which (with three amino acid substitutions H31R/E36D/E41A) exceeds sucrose sweetness by 22 500 times. To date, various systems for the expression of recombinant brazzein have already been developed, in which bacteria (Escherichia coli, Lactococcus lactis, Bacillus licheniformis), yeast (Komagataella phaffii, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum, Daucus carota) and animals (Mus musculus) have been used. Conclusion. Due to its high sweetness, organoleptic properties and long history of human consumption, brazzein can be considered as a promising natural sweetener. Despite the short peptide sequence, the production of the recombinant protein faced a number of problems, including low protein yield (for example, it could only be detected in mouse milk by Western blot hybridization) and loss of sweetness. Thus, further optimization of the process is necessary for widespread brazzein use in the food industry, which includes the selection of an adequate producer and the use of extracellular expression systems to reduce the final cost of the product.

Rethinking Sweetener Discovering: Multiparameter Modeling of Molecular Docking Results between the T1R2-T1R3 Receptor and Compounds with Different Tastes.

Molecular docking has been widely applied in the discovery of new sweeteners, yet the interpretation of computational results sometimes remains difficult. Here, the interaction between the T1R2-T1R3 sweet taste receptor and 66 tasting compounds, including 26 sweet, 19 bitter, and 21 sour substances was investigated by batch molecular docking processes. Statistical analysis of the docking results generated two novel methods of interpreting taste properties. Quantitative correlation between relative sweetness (RS) and docking results created a multiparameter model to predict sweetness intensity, whose correlation coefficient r = 0.74 is much higher than r = 0.17 for the linear correlation model between sweetness and binding energy. The improved correlation indicated that docking results besides binding energy contain undiscovered information about the ligand-protein interaction. Qualitative discriminant analysis of different tasting molecules generated an uncorrelated linear discriminant analysis (UDLA) model, which achieved an overall 93.1% accuracy in discriminating the taste of molecules, with specific accuracy for verifying sweet, bitter, and sour compounds reaching 88.0%, 92.1%, and 100%. These unprecedented models provide a unique perspective for interpreting computational results and may inspire future research on sweetener discovery.

Study on the Thermal Stability of the Sweet-Tasting Protein Brazzein Based on Its Structure-Sweetness Relationship.

Brazzein (Brz) is a sweet-tasting protein composed of 54 amino acids and is considered as a potential sugar substitute. The current methods for obtaining brazzein are complicated, and limited information is available regarding its thermal stability. In this study, we successfully expressed recombinant brazzein, achieving a sweetness threshold of 15.2 µg/mL. Subsequently, we conducted heat treatments at temperatures of 80, 90, 95, and 100 °C for a duration of 2 h to investigate the structural changes in the protein. Furthermore, we employed hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to analyze the effect of heating on the protein structure-sweetness relationships. Our results indicated that the thermal inactivation process primarily affects residues 6-14 and 36-45 of brazzein, especially key residues Tyr8, Tyr11, Ser14, Glu36, and Arg43, which are closely associated with its sweetness. These findings have significant implications for improving the thermal stability of brazzein.

Sweetened Beverages, Genetic Susceptibility, and Incident Atrial Fibrillation: A Prospective Cohort Study.

BACKGROUND: An association between sweetened beverages and several cardiometabolic diseases has been reported, but their association with atrial fibrillation (AF) is unclear. We aimed to investigate the associations between consumption of sugar-sweetened beverages (SSB), artificially sweetened beverages (ASB), and pure fruit juice (PJ) and risk of consumption with AF risk and further evaluate whether genetic susceptibility modifies these associations. METHODS: A total of 201 856 participants who were free of baseline AF, had genetic data available, and completed a 24-hour diet questionnaire were included. Cox proportional hazard models were used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: During a median follow-up of 9.9 years, 9362 incident AF cases were documented. Compared with nonconsumers, individuals who consumed >2 L/wk of SSB or ASB had an increased risk of AF (HR, 1.10 [95% CI, 1.01-1.20] and HR, 1.20 [95% CI, 1.10-1.31]) in the multivariable-adjusted model. A negative association was observed between the consumption of ≤1 L/wk of PJ and the risk of AF (HR, 0.92 [95% CI, 0.87-0.97]). The highest HRs (95% CIs) of AF were observed for participants at high genetic risk who consumed >2 L/wk of ASB (HR, 3.51 [95% CI, 2.94-4.19]), and the lowest HR were observed for those at low genetic risk who consumed ≤1 L/wk of PJ (HR, 0.77 [95% CI, 0.65-0.92]). No significant interactions were observed between the consumption of SSB, ASB, or PJ and genetic predisposition to AF. CONCLUSIONS: Consumption of SSB and ASB at >2 L/wk was associated with an increased risk for AF. PJ consumption ≤1 L/wk was associated with a modestly lower risk for AF. The association between sweetened beverages and AF risk persisted after adjustment for genetic susceptibility to AF. This study does not demonstrate that consumption of SSB and ASB alters AF risk but rather that the consumption of SSB and ASB may predict AF risk beyond traditional risk factors.

Metabolic Effects of Selected Conventional and Alternative Sweeteners: A Narrative Review.

Sugar consumption is known to be associated with a whole range of adverse health effects, including overweight status and type II diabetes mellitus. In 2015, the World Health Organization issued a guideline recommending the reduction of sugar intake. In this context, alternative sweeteners have gained interest as sugar substitutes to achieve this goal without loss of the sweet taste. This review aims to provide an overview of the scientific literature and establish a reference tool for selected conventional sweeteners (sucrose, glucose, and fructose) and alternative sweeteners (sucralose, xylitol, erythritol, and D-allulose), specifically focusing on their important metabolic effects. The results show that alternative sweeteners constitute a diverse group, and each substance exhibits one or more metabolic effects. Therefore, no sweetener can be considered to be inert. Additionally, xylitol, erythritol, and D-allulose seem promising as alternative sweeteners due to favorable metabolic outcomes. These alternative sweeteners replicate the benefits of sugars (e.g., sweetness and gastrointestinal hormone release) while circumventing the detrimental effects of these substances on human health.

Lack of Biological Plausibility and Major Methodological Issues Cast Doubt on the Association between Aspartame and Autism. Comment on Fowler et al. Daily Early-Life Exposures to Diet Soda and Aspartame Are Associated with Autism in Males: A Case-Control Study. Nutrients 2023, 15, 3772.

The case-control study by Fowler et al [...].

Time-lapse confocal microscopy to study in vitro Streptococcus mutans surface colonization.

The cariogenicity of Streptococcus mutans relates to its ability to form biofilms on dental surfaces. The aim of this work was to develop a flowcell system compatible with time-lapse confocal microscopy to compare the adhesion and accumulation of S. mutans cells on surfaces in unsupplemented media against media containing sucrose or sucralose (a non-metabolized sweetener) over a short period of time. Fluorescent S. mutans 3209/pVMCherry was suspended in unsupplemented media or media supplemented with 1% sucrose or 1% sucralose and passed through a 3D-printed flowcell system. Flowcells were imaged over 60 minutes using a confocal microscope. Image analysis was performed, including a newly developed object-movement-based method to measure biomass adhesion. Streptococcus mutans 3209/pVMCherry grown in 1% sucrose-supplemented media formed small, dense, relatively immobile clumps in the flowcell system measured by biovolume, surface area, and median object centroid movement. Sucralose-supplemented and un-supplemented media yielded large, loose, mobile aggregates. Architectural metrics and per-object movement were significantly different (P < 0.05) when comparing sucrose-supplemented media to either unsupplemented or sucralose-supplemented media. These results demonstrate the utility of a flowcell system compatible with time-lapse confocal microscopy and image analysis when studying initial biofilm formation and adhesion under different nutritional conditions.

Natural Sweetener Glycyrrhetinic Acid Monoglucuronide Improves Glucose Homeostasis in Healthy Mice.

Noncaloric or low-caloric sweeteners have become popular worldwide, although debates persist regarding their impact on health. To investigate whether the sweeteners are favorable for glucose homeostasis, our study assessed the effects of glycyrrhetinic acid monoglucuronide (GAMG) and several commonly used sweeteners [glycyrrhetinic acid (GA), stevioside, erythritol, sucralose, and aspartame] on glycometabolism and elucidated the underlying mechanisms. The C57BL/6J male mice were exposed to different sweeteners for 10 weeks, and our results showed that GAMG significantly reduced fasting blood glucose (FBG) levels (FBG-control: 3.81 ± 0.42 mmol/L; FBG-GAMG: 3.37 ± 0.38 mmol/L; p < 0.05) and the blood glucose levels 15 and 30 min after sucrose or maltose loading (p < 0.05). Furthermore, it improved glucose tolerance (p = 0.028) and enhanced insulin sensitivity (p = 0.044), while the other sweeteners had negligible or adverse effects on glucose homeostasis. Subsequent experiments showed that GAMG inhibited α-glucosidases potently (IC50 = 0.879 mg·mL-1), increased three SCFA-producing bacteria and SCFAs levels (p < 0.05), and promoted the gene expression of SCFA receptor GPR43 (p = 0.018). These results suggest that GAMG may regulate blood glucose by inhibiting α-glucosidases and modulating gut microbial SCFAs. Our findings prove that GAMG, beneficial to blood glucose regulation, is a promising natural sweetener for future utilization.

Optimizing Trilobatin Production via Screening and Modification of Glycosyltransferases.

Trilobatin (TBL) is a key sweet compound from the traditional Chinese sweet tea plant (Rubus suavissimus S. Lee). Because of its intense sweetness, superior taste profile, and minimal caloric value, it serves as an exemplary natural dihydrochalcone sweetener. It also has various health benefits, including anti-inflammatory and glucose-lowering effects. It is primarily produced through botanical extraction, which impedes its scalability and cost-effectiveness. In a novel biotechnological approach, phloretin is used as a precursor that is transformed into TBL by the glycosyltransferase enzyme ph-4'-OGT. However, this enzyme's low catalytic efficiency and by-product formation limit the large-scale synthesis of TBL. In our study, the enzyme Mdph-4'-OGT was used to screen 17 sequences across species for TBL synthesis, of which seven exhibited catalytic activity. Notably, PT577 exhibited an unparalleled 97.3% conversion yield within 3 h. We then optimized the reaction conditions of PT577, attaining a peak TBL bioproduction of 163.3 mg/L. By employing virtual screening, we identified 25 mutation sites for PT577, thereby creating mutant strains that reduced by-products by up to 50%. This research enhances the enzymatic precision for TBL biosynthesis and offers a robust foundation for its industrial-scale production, with broader implications for the engineering and in silico analysis of glycosyltransferases.

Characterization and sweetness-enhancing effect of peptides from yeast extract based on sensory evaluation and molecular docking approaches.

Yeast extract (YE) is derived from the soluble component in yeast cells, which is rich in peptides and has been used as a sweet-enhancing agent. It has the potential to be utilized to produce natural sweet-flavored peptides or sweet-enhancing peptides. To study the synergistic effect and mechanism of sweetness-enhancing peptides derived from YE, ultrafiltration fraction with molecular weight less than 1 kDa was screened according to sensory analysis, which showed a synergistic sweetening effect in stevioside and mogroside solution. Twenty potential taste peptides were identified from the screened fractions, among which EV, AM, AVDNIPVGPN and VDNIPVGPN showed sweetness-enhancing effects on both stevioside and mogroside. The sweetener-receptor-peptide complex was constructed to investigate the interaction of stevioside and mogroside to taste receptor type 1 member 2 accompanied by these peptides. The results of the molecular docking indicated that new hydrophobic interactions (Leu 279, Pro 308, Val 309, etc.) and hydrogen bonds (Ser 40, Ala 43, Asp 278, etc.) were formed between sweeteners and active sites in the venus flytrap domain. In conclusion, the presence of sweetness-enhancing peptides from YE improved the binding stability of sweeteners and receptors by increasing the binding interaction, especially the hydrophobic interactions, which contribute to the synergistic effect of sweetness-enhancing peptides.