Aspartame carcinogenic potential revealed through network toxicology and molecular docking insights.

The research employed network toxicology and molecular docking techniques to systematically examine the potential carcinogenic effects and mechanisms of aspartame (L-α-aspartyl-L-phenylalanine methyl ester). Aspartame, a commonly used synthetic sweetener, is widely applied in foods and beverages globally. In recent years, its safety issues, particularly the potential carcinogenic risk, have garnered widespread attention. The study first constructed an interaction network map of aspartame with gastric cancer targets using network toxicology methods and identified key targets and pathways. Preliminary validation was conducted through microarray data analysis and survival analysis, and molecular docking techniques were employed to further examine the binding affinity and modes of action of aspartame with key proteins. The findings suggest that aspartame has the potential to impact various cancer-related proteins, potentially raising the likelihood of cellular carcinogenesis by interfering with biomolecular function. Furthermore, the study found that the action patterns and pathways of aspartame-related targets are like the mechanisms of known carcinogenic pathways, further supporting the scientific hypothesis of its potential carcinogenicity. However, given the complexity of the in vivo environment, we also emphasize the necessity of validating these molecular-level findings in actual biological systems. The study introduces a fresh scientific method for evaluating the safety of food enhancers and provides a theoretical foundation for shaping public health regulations.

Aspartame Intake Delayed Puberty Onset in Female Offspring Rats and Girls.

SCOPE: The disturbance of the hypothalamic-pituitary-gonadal (HPG) axis, gut microbiota (GM) community, and short-chain fatty acids (SCFAs) is a triggering factor for pubertal onset. The study investigates the effects of the long-term intake of aspartame on puberty and GM in animals and humans. METHODS AND RESULTS: Aspartame-fed female offspring rats result in vaginal opening time prolongation, serum estrogen reduction, and serum luteinizing hormone elevation. , 60 mg kg-1 aspartame treatment decreases the mRNA levels of gonadotropin-releasing hormone (GnRH), Kiss1, and G protein-coupled receptor 54 (GPR54), increases the mRNA level of RFamide-related peptide-3 (RFRP-3), and decreases the expression of GnRH neurons in the hypothalamus. Significant differences in relative bacterial abundance at the genus levels and decreased fecal SCFA levels are noted by 60 mg kg-1 aspartame treatment. Among which, Escherichia-Shigella is negatively correlated with several SCFAs. In girls, high-dose aspartame consumption decreases the risk of precocious puberty. CONCLUSIONS: Aspartame reduces the chance of puberty occurring earlier than usual in female offspring and girls. Particularly, 60 mg kg-1 aspartame-fed female offspring delays pubertal onset through the dysregulation of HPG axis and GM composition by inhibiting the Kiss1/GPR54 system and inducing the RFRP-3. An acceptable dose of aspartame should be recommended during childhood.

The effect of aspartame on accelerating caspase-dependent apoptosis of pancreatic islet via ZIPK/STAT3/caspase 3 signaling pathway.

Aspartame (ASP) as an important sugar substitute is widely used in pharmaceutical and food processing. Here, we compared the effects of ASP and sucrose on mice pancreatic islet cells in vivo and observed that ASP with the condition of high concentration and long-term exposure (HASP) could cause insulin secretion (500 mg/kg for 1 month). Next, we conducted iTRAQ mass spectrometry to profile the global phosphoproteome and found that phosphorylation of zipper-interacting protein kinase (ZIPK) in murine pancreatic islet tissues were induced at Thr197, Thr242, Thr282, and Ser328 by high-sucrose (HS) treatment, but only induced at Thr197 and Ser328 by HASP treatment. Simultaneously, phosphorylation of STAT3 could be induced at Tyr705 and Ser727 by HS but not by HASP. Furthermore, presence of activated STAT3 accompanied with autophagy was observed in HS treatment. In turn, the inactivation of STAT3 as well as enhanced expression of caspase 3 was observed in HASP treatment. We generated Thr242APro and Thr282Pro on ZIPK using CRISPR-Cas9 in ß-TC3 cells and found the weakened interaction with STAT3 as well as the reduced phosphorylation of STAT3 even under HS stimulation. Finally, we observed that ankyrin repeat domain containing 11 (ANKRD11) could interact with ZIPK and play an inhibitory role in the phosphorylation of Thr242APro and Thr282Pro of ZIPK. However, HASP can induce the retention of ANKRD11 in the cytoplasm by phenylpyruvic acid (the metabolite of ASP). Taken together, this study determined that ASP with high concentration and long-term exposure could lead to caspase-dependent apoptosis of pancreatic islet cells through ANKRD11/ZIPK/STAT3 inhibition. Our results give evidence of adverse effects of aspartame on islet cells in some extreme conditions, which might help people to reconsider the biosafety of non-nutritive sweeteners.

Beta-caryophyllene mitigates the cognitive impairment caused by repeated exposure to aspartame in rats: Putative role of BDNF-TrKB signaling pathway and acetylcholinesterase activity.

Aspartame (ASP) is a common sweetener, but studies show it can harm the nervous system, causing learning and memory deficits. ß-caryophyllene (BCP), a natural compound found in foods, including bread, coffee, alcoholic beverages, and spices, has already described as a neuroprotector agent. Remarkably, ASP and BCP are commonly consumed, including in the same meal. Therefore, considering that (a) the BCP displays plenty of beneficial effects; (b) the ASP toxicity; and (c) that they can be consumed in the same meal, this study sought to investigate if the BCP would mitigate the memory impairment induced by ASP in rats and investigate the involvement of the brain-derived neurotrophic factor (BDNF)/ tropomyosin receptor kinase B (TrKB) signaling pathway and acetylcholinesterase (AChE) activity. Young male Wistar rats received ASP (75 mg/kg; i.g.) and/or BCP (100 mg/kg; i.p.) once daily, for 14 days. At the end of the treatment, the animals were evaluated in the open field and object recognition tests. The cerebral cortex and hippocampus samples were collected for biochemical and molecular analyses. Results showed that the BCP effectively protected against the cognitive damage caused by ASP in short and long-term memories. In addition, BCP mitigated the increase in AChE activity caused by ASP. Molecular insights revealed augmented BDNF and TrKB levels in the hippocampus of rats treated with BCP, indicating greater activation of this pathway. In conclusion, BCP protected against ASP-induced memory impairment. AChE activity and the BDNF/TrkB signaling pathway seem to be potential targets of BCP modulatory role in this study.

Aspartame and Its Metabolites Cause Oxidative Stress and Mitochondrial and Lipid Alterations in SH-SY5Y Cells.

Due to a worldwide increase in obesity and metabolic disorders such as type 2 diabetes, synthetic sweeteners such as aspartame are frequently used to substitute sugar in the diet. Possible uncertainties regarding aspartame's ability to induce oxidative stress, amongst others, has led to the recommendation of a daily maximum dose of 40 to 50 mg per kg. To date, little is known about the effects of this non-nutritive sweetener on cellular lipid homeostasis, which, besides elevated oxidative stress, plays an important role in the pathogenesis of various diseases, including neurodegenerative diseases such as Alzheimer's disease. In the present study, treatment of the human neuroblastoma cell line SH-SY5Y with aspartame (271.7 µM) or its three metabolites (aspartic acid, phenylalanine, and methanol (271.7 µM)), generated after digestion of aspartame in the human intestinal tract, resulted in significantly elevated oxidative stress associated with mitochondrial damage, which was illustrated with reduced cardiolipin levels, increased gene expression of SOD1/2, PINK1, and FIS1, and an increase in APF fluorescence. In addition, treatment of SH-SY5Y cells with aspartame or aspartame metabolites led to a significant increase in triacylglycerides and phospholipids, especially phosphatidylcholines and phosphatidylethanolamines, accompanied by an accumulation of lipid droplets inside neuronal cells. Due to these lipid-mediating properties, the use of aspartame as a sugar substitute should be reconsidered and the effects of aspartame on the brain metabolism should be addressed in vivo.

Long-term intake of aspartame-induced cardiovascular toxicity is reflected in altered histochemical parameters, evokes oxidative stress, and trigger P53-dependent apoptosis in a mouse model.

Aspartame (ASP) is probably the best known artificial sugar substitute that is used widely in food. Many experimental studies have reported the toxicity of long-term administration of ASP in various organ tissues. However, there is little evidence available about the nature and mechanisms of the adverse effects of long-term consumption of ASP on the cardiovascular system. This study was conducted to evaluate the possible effects of ASP on heart tissue. For this study 36 mature male mice were divided into one control group and three groups which received respectively 40 mg/kg, 80 mg/kg and 160 mg/kg ASP orally, for 90 days. ASP at the doses of 80 and 160 mg/kg increased the serum content of malondialdehyde (MDA), but decreased serum nitric oxide (NO), creatine kinase (CK) and CK-MB, as well as blood superoxide dismutase (SOD) levels. Serum level of total anti-oxidant capacity (TAC) in blood was also reduced in serum at the dose of 80 mg/kg. Histochemical staining, including Periodic acid-Schiff, Masson's trichrome and Verhoeff-van Gieson staining, indicated that ASP at doses of 80 and 160 mg/kg reduced glycogen deposition and decreased the number of collagen and elastic fibres in the cardiac tissue. The cardiac expression of pro-apoptotic genes, including P53, Bax, Bcl-2 and Caspase-3, was modulated at the dose of 160 mg/kg. Moreover, transcription of Caspase-3 was up-regulated at the dose of 80 mg/kg. In conclusion, long-term consumption of ASP any higher than the acceptable daily intake (40 mg/kg) appears to act by promoting oxidative stress, has the potential to alter both histopathological and biochemical parameters, and induces P53-dependent apoptosis in cardiac tissue.

Screening of α-amino acid ester acyl transferase variant with improved activity by combining rational and random mutagenesis.

Random and rational mutagenesis of an α-amino acid ester acyl transferase from Sphingobacterium siyangensis AJ2458 (SAET) was conducted to examine the production of aspartame, an α-l-aspartyl-l-phenylalanine methyl ester. We previously reported aspartame production via combination of enzymatic and chemical methods. However, the productivity of the aspartame intermediate by SAET was approximately one-fifth that of l-alanyl-l-glutamine (Ala-Gln), whose production method has already been established. Here, to improve the enzymatic activity of SAET, we performed random mutagenesis in the gene encoding SAET and obtained 10 mutations that elevated the enzymatic activity (1.2- to 1.7-fold increase) relative to that of wild-type SAET. To further improve the activity, we performed mutagenesis to optimize the combination of the obtained mutations and finally selected one SAET variant with 10 amino acid substitutions (M35-4 SAET). An Escherichia coli strain overexpressing M35-4 SAET displayed a 5.7-fold higher activity than that of the wild-type SAET, which was almost equal to that of Ala-Gln by an E. coli strain overexpressing wild-type SAET. The Vmax value of M35-4 SAET was 2.0-fold greater, and its thermostability was higher than those of wild-type SAET. These results suggest that the obtained SAET variants contribute to improvement in aspartame production.

Metformin induces mitochondrial remodeling and differentiation of pancreatic progenitor cells into beta-cells by a potential mechanism including suppression of the T1R3, PLCß2, cytoplasmic Ca+2, and AKT.

The main goal of this study was to investigate the molecular changes in pancreatic progenitor cells subject to high glucose, aspartame, and metformin in vitro. This scope of work glucose, aspartame, and metformin were exposed to pancreatic islet derived progenitor cells (PID-PCs) for 10 days. GLUT1's role in beta-cell differentiation was examined by using GLUT1 inhibitor WZB117. Insulin+ cell ratio was measured by flow cytometry; the expression of beta-cell differentiation related genes was shown by RT-PCR; mitochondrial mass, mitochondrial ROS level, cytoplasmic Ca2+, glucose uptake, and metabolite analysis were made fluorometrically and spectrophotometrically; and proteins involved in related molecular pathways were determined by western blotting. Findings showed that glucose or aspartame exposed cells had similar metabolic and gene expression profile to control PID-PCs. Furthermore, relatively few insulin+ cells in aspartame treated cells were determined. Aspartame signal is transmitted through PLCß2, CAMKK2 and LKB1 in PID-PCs. The most obvious finding of this study is that metformin significantly increased beta-cell differentiation. The mechanism involves suppression of the sweet taste signal's molecules T1R3, PLCß2, cytoplasmic Ca+2, and AKT in addition to the direct effect of metformin on mitochondria and AMPK, and the energy metabolism of PID-PCs is remodelled in the direction of oxidative phosphorylation. These findings are very important in terms of determining that metformin stimulates the mitochondrial remodeling and the differentiation of PID-PCs to beta-cells and thus it may contribute to the compensation step, which is the first stage of diabetes development.

Hepatic susceptibility to oxidative damage after repeated concomitant exposure to aspartame and aflatoxin B1 in rats.

The potential interactions among food additives/contaminants and the consequences to biological systems is a topic that is rarely addressed in scientific literature. Thus, the current study investigated if the combined administration of ASP and AFB1 would impair hepatic and renal oxidative status. Male Wistar rats received during 14 days once a day ASP (75 mg/Kg) and/or AFB1 (250 µg/Kg) through intragastric route. At the end of experimental protocol, samples of liver and kidneys were collected for assessing biochemical markers of oxidative status. In the hepatic tissue, the treatment with a single substance (ASP or AFB1) caused an increase in TBARS levels, and a reduction in non-enzymatic antioxidant defenses (Vit C and NPSH levels and FRAP test). In the kidneys, TBARS levels were increased only in the group that received ASP + AFB1. The association reduced NPSH content, while the treatment with AFB1 reduced the FRAP levels. GST and CAT activities were increased in all treatments. Overall, ASP and AFB1 association presented higher toxic effects to the tissues. To the best of our knowledge, this is the first study demonstrating that the associated use of both ASP and AFB1 induces more extensive injuries in comparison to the effects caused by each one alone. Therefore, these data demonstrated that concomitant exposure to ASP and AFB1 potentiated their oxidative damage in hepatic tissue, suggesting that this organ is particularly sensitive to the toxic action induced by these substances.

Aspartame-True or False? Narrative Review of Safety Analysis of General Use in Products.

Aspartame is a sweetener introduced to replace the commonly used sucrose. It was discovered by James M. Schlatter in 1965. Being 180-200 times sweeter than sucrose, its intake was expected to reduce obesity rates in developing countries and help those struggling with diabetes. It is mainly used as a sweetener for soft drinks, confectionery, and medicines. Despite its widespread use, its safety remains controversial. This narrative review investigates the existing literature on the use of aspartame and its possible effects on the human body to refine current knowledge. Taking to account that aspartame is a widely used artificial sweetener, it seems appropriate to continue research on safety. Studies mentioned in this article have produced very interesting results overall, the current review highlights the social problem of providing visible and detailed information about the presence of aspartame in products. The studies involving the impact of aspartame on obesity, diabetes mellitus, children and fetus, autism, neurodegeneration, phenylketonuria, allergies and skin problems, its cancer properties and its genotoxicity were analyzed. Further research should be conducted to ensure clear information about the impact of aspartame on health.

Spectroscopic studies on the interaction of aspartame with human serum albumin.

In this work the binding of artificial sweetener aspartame with human serum albumin (HSA) was studied at physiological pH. Binding studies of aspartame (APM) with HSA are useful to understand APM -HSA interaction, mechanism and providing guidance for the application and design of new and more efficient artificial sweeteners. The interaction was investigated by spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD) techniques. The results indicated that the binding of APM to HSA caused fluorescence quenching of HSA through static quenching mechanism with binding constant 1.42 × 10+4 M-1 at 298 K and the number of binding sites is approximately one. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be -41.20 kJ mol-1 and -58.19 J mol-1 K-1, respectively, according to van't Hoff equation, which indicated that reaction is enthalpically driven. Quenching of the fluorescence of HSA was found to be a static quenching process. The binding constants and number of binding sites were obtained at three different temperatures (298, 308 and 318 K). Combining above results and those of spectrofluorometric competition experiment and circular dichroism (CD), indicated that APM binds to HSA via Sudlow's site I. Furthermore, the study of molecular docking on HSA binding also indicated that APM can strongly bind to the site I (subdomain IIA) of HSA mainly by hydrophobic interaction and hydrogen bond interactions exist between APM and HSA.

High Concentrations of Aspartame Induce Pro-Angiogenic Effects in Ovo and Cytotoxic Effects in HT-29 Human Colorectal Carcinoma Cells.

Aspartame (ASP), an artificial sweetener abundantly consumed in recent years in an array of dietary products, has raised some concerns in terms of toxicity, and it was even suggested a link with the risk of carcinogenesis (colorectal cancer), though the present scientific data are rather inconclusive. This study aims at investigating the potential role of aspartame in colorectal cancer by suggesting two experimental approaches: (i) an in vitro cytotoxicity screening in HT-29 human colorectal carcinoma cells based on cell viability (Alamar blue assay), cell morphology and cell migration (scratch assay) assessment and (ii) an in ovo evaluation in terms of angiogenic and irritant potential by means of the chorioallantoic membrane method (CAM). The in vitro results showed a dose-dependent cytotoxic effect, with a significant decrease of viable cells at the highest concentrations tested (15, 30 and 50 mM) and morphological cellular changes. In ovo, aspartame (15 and 30 mM) proved to have a pro-angiogenic effect and a weak irritant potential at the vascular level. These data suggest new directions of research regarding aspartame's role in colorectal cancer.

Taste compound - Nanocellulose interaction assessment by fluorescence indicator displacement assay.

Interactions between taste compounds and nanofibrillar cellulose were studied. For this, a new fluorescent indicator displacement method was developed. Two fluorescent indicators, namely, Calcofluor white and Congo red, were chosen because of their specific binding to cellulose and intrinsic fluorescence. Seven taste compounds with different structures were successfully measured together with nanofibrillar cellulose (NFC) and ranked according to their binding constants. The most pronounced interactions were found between quinine and NFC (1.4 × 104 M-1), whereas sucrose, aspartame and glutamic acid did not bind at all. Naringin showed moderate binding while stevioside and caffeine exhibited low binding. The comparison with microcrystalline cellulose indicates that the larger surface area of nanofibrillated cellulose enables stronger binding between the binder and macromolecules. The developed method can be further utilized to study interactions of different compound classes with nanocellulose materials in food, pharmaceutical and dye applications, using a conventional plate reader in a high-throughput manner.

Maternal low-dose aspartame and stevia consumption with an obesogenic diet alters metabolism, gut microbiota and mesolimbic reward system in rat dams and their offspring.

OBJECTIVE: We examined the impact of maternal low-dose aspartame and stevia consumption on adiposity, glucose tolerance, gut microbiota and mesolimbic pathway in obese dams and their offspring. DESIGN: Following obesity induction, female Sprague-Dawley rats were allocated during pregnancy and lactation to: (1) high fat/sucrose diet (HFS) +water (obese-WTR); (2) HFS +aspartame (obese-APM; 5-7 mg/kg/day); (3) HFS +stevia (obese-STV; 2-3 mg/kg/day). Offspring were weaned onto control diet and water and followed until 18 weeks. Gut microbiota and metabolic outcomes were measured in dams and offspring. Cecal matter from offspring at weaning was used for faecal microbiota transplant (FMT) into germ-free (GF) mice. RESULTS: Maternal APM and STV intake with a HFS diet increased body fat in offspring at weaning and body weight long-term with APM. Maternal APM/HFS consumption impaired glucose tolerance in male offspring at age 8 weeks and both APM and STV altered faecal microbiota in dams and offspring. Maternal obesity/HFS diet affected offspring adiposity and glucose tolerance more so than maternal LCS consumption at age 12 and 18 weeks. APM and STV altered expression of genes in the mesolimbic reward system that may promote consumption of a palatable diet. GF mice receiving an FMT from obese-APM and obese-STV offspring had greater weight gain and body fat and impaired glucose tolerance compared with obese-WTR. CONCLUSION: Maternal low-calorie sweetener consumption alongside HFS may disrupt weight regulation, glucose control and gut microbiota in dams and their offspring most notably in early life despite no direct low-calorie sweetener consumption by offspring.

Commercially available non-nutritive sweeteners modulate the antioxidant status of type 2 diabetic rats.

Non-nutritive sweeteners (NNS) are increasingly being used by diabetics, but little is known about their effects on antioxidant status. We investigated the effects of ad libitum consumption of commercially available NNS (aspartame, saccharin, sucralose, and cyclamate-based sweeteners) on antioxidative markers in a rat model of type 2 diabetes (T2D). NNS consumption reduced (p < 0.05) T2D-induced lipid peroxidation and boosted serum, hepatic, renal, cardiac, and pancreatic glutathione (GSH) levels. Catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase activity was increased in the serum and most organs upon diabetes induction, perhaps due to adaptative antioxidant response to the diabetes-induced lipid peroxidation. NNS showed varying effects on serum and tissue antioxidant enzymes of animals. An antioxidant capacity scores sheet of NNS, suggest that aspartame-based NNS may not exert antioxidant effects in diabetics, while saccharin-based NNS may be a potent antioxidative sweetener as seen in the animal model of T2D. PRACTICAL APPLICATIONS: The use of NNS is becoming more popular, especially for diabetic individuals. While there are several commercial NNS available in the market, little is known about how they affect the antioxidant status of consumers. We therefore investigated how some commercially available NNS affect the antioxidant status of diabetic rats. Observed data revealed varying effects of NNS on serum and different organs, which suggest that some NNS may be better than others for diabetic oxidative stress and thus may be recommended for consumers. However, this finding is subject to additional corroborative clinical studies.

Introducing a New Model of Sweet Taste Receptor, a Class C G-protein Coupled Receptor (C GPCR).

The structure of sweet taste receptor (STR), a heterodimer of class C G-protein coupled receptors comprising T1R2 and T1R3 molecules, is still undetermined. In this study, a new enhanced model of the receptor is introduced based on the most recent templates. The improvement, stability, and reliability of the model are discussed in details. Each domain of the protein, i.e., VFTM, CR, and TMD, were separately constructed by hybrid-model construction methods and then assembled to build whole monomers. Overall, 680 ns molecular dynamics simulation was performed for the individual domains, the whole monomers and the heterodimer form of the VFTM orthosteric binding site. The latter's structure obtained from 200 ns simulation was docked with aspartame; among various binding sites suggested by FTMAP server, the experimentally suggested binding domain in T1R2 was retrieved. Local three-dimensional structures and helices spans were evaluated and showed acceptable accordance with the template structures and secondary structure predictions. Individual domains and whole monomer structures were found stable and reliable to be used. In conclusion, several validations have shown reliability of the new and enhanced models for further molecular modeling studies on structure and function of STR and C GPCRs.

The debate over neurotransmitter interaction in aspartame usage.

Aspartame (NutraSweet®, Equal®) is a widely used artificial sweetener, has been reported to be accountable for neurological and behavioural disturbances in people. Upon ingestion, aspartame is hydrolyzed in gut and provides its metabolite; such as essential amino acid phenylalanine (Phy) (50%), aspartic acid (40%), and methanol (10%). Altered brain neurochemical compositions [such as dopamine (DA), norepinephrine (NE), and serotonin (5-HT)] have long been a concern and being involved in observed neurophysiological symptom (such as headaches, memory loss, mood changes, as well as depression) in aspartame consumers. Aspartames might act as chemical stressor through increasing plasma cortisol level. Aspartame consumption similarly altered gut microbiota. Taken together all this factors, we reviewed to search for convincing evidence, in what manner aspartame metabolites, stress hormones (cortisol), and gut dysbiosisis involved in altering brain neurochemical composition. We concluded that aspartame metabolite; mainly Phy and its interaction with neurotransmitter and aspartic acid by acting as excitatory neurotransmitter causes this pattern of impairments. Along with elevated cortisol and gut dysbiosis via interactions with different biogenic amine may also have additional impact to modulate neuronal signaling lead to neurobiological impairments. Hence ongoing research is instantly needed to understand the specific roles of aspartame metabolite, elevated cortisol, and gut dysbiosis with emerging neurophysiological symptom in aspartame consumers to improve healthy life in its consumers.

Impact of Food Ingredients (Aspartame, Stevia, Prebiotic Oligofructose) on Fertility and Reproductive Outcomes in Obese Rats.

OBJECTIVE: This study aimed to investigate the interaction between obesity, low-calorie sweeteners, and prebiotic oligofructose on reproductive parameters in rats. METHODS: Data were derived from two separate studies of female Sprague-Dawley rats with (1) Lean (n = 24), (2) Obese (n = 27), (3) Obese+Aspartame (n = 14), (4) Obese+Stevia (n = 15), and (5) Obese+Prebiotic (n = 15) groups. Obesity was induced with a high-fat/high-sucrose diet prior to pregnancy. In one study, human-approved doses of aspartame (5-7 mg/kg/d) and stevia (2-3 mg/kg/d) in drinking water were examined, and in the second, 10% prebiotics (oligofructose) in the diet was examined. Reproductive parameters, including fertility, pregnancy, and delivery indexes, were analyzed. RESULTS: Obesity significantly reduced pregnancy index in Obese dams (60.7% successful pregnancies) compared with lean (100%). Obesity also reduced the number of pups born alive and pup survival percentage compared with those of Lean dams (P < 0.001). Only 53.3% of rats were able to conceive in the Obese+Stevia group, but if rats did become pregnant, they had 100% pregnancy and delivery index. While prebiotic administration rescued the pregnancy index, it could not remediate pup survival percentage (P = 0.025) in Obese dams. CONCLUSIONS: Both obesity status and dietary ingredients affect the ability to conceive. Future rigorously controlled studies designed to examine reproductive outcomes in depth are needed to confirm these findings.

Dynamic Proteome Alteration and Functional Modulation of Human Saliva Induced by Dietary Chemosensory Stimuli.

Saliva flow measurements and SDS-PAGE separation of human whole saliva freshly collected after oral stimulation with citric acid (sour), aspartame (sweet), iso-α-acids (bitter), mono sodium l-glutamate (umami), NaCl (salty), 6-gingerol (pungent), hydroxy-α-sanshool (tingling), and hydroxy-ß-sanshool (numbing), followed by tryptic digestion, nano-HPLC-MS/MS, and label-free protein quantitation demonstrated a stimulus- and time-dependent influence of the dietary chemosensates on salivation and the salivary proteome composition. Gene ontology enrichment analysis showed evidence for stimulus-induced alterations of the saliva proteome to boot an efficient molecular defense network of the oral cavity, e.g., 6-gingerol increased salivary lactoperoxidase activity, catalyzing the oxidation of thiocyanate to produce the antimicrobial and antifungal hypothiocyanate, from 0.37 ± 0.02 to 0.91 ± 0.05 mU/mL 45 s after stimulation. In comparison, oral citric acid stimulation induced an increase of myeloperoxidase activity, catalyzing the chloride oxidation to generate antimicrobial hypochloride in saliva, from 0.24 ± 0.04 to 0.70 ± 0.1 mU/mL as well as an increase of salivary levels of lysozyme, exhibiting antimicrobial activity on Gram-positive bacteria, from 6.0-10 to 100-150 µg/mL. Finally, microbial growth experiments clearly demonstrated for the first time that the increase of the salivary lysozyme abundance upon oral citric acid stimulation translates into an enhanced biological function, that is an almost complete growth inhibition of the two lysozyme-sensitive Gram-positive bacteria tested.

Enhancement of Z-aspartame synthesis by rational engineering of metalloprotease.

Metalloprotease PT121Y114S, an effective catalyst for Z-aspartame synthesis under the substrate (Z-Asp:l-Phe-OMe) molar ratio of 1:5, was obtained previously. Herein, a computational strategy combining molecular dynamics simulation of the enzyme-substrate complex with binding free energy (ΔG) calculations was established to guide the further engineering of PT121Y114S. One His224 residue proximal to the PT121Y114S active site was selected on the basis of the difference in ΔG decomposition of PT121Y114S toward l-Phe-NH2 and l-Phe-OMe. Site-saturation mutagenesis of His224 resulted in the mutants H224D, H224N, and H224S, which showed great improvement in Z-aspartame synthesis under an economical substrate molar ratio approaching 1:1. Furthermore, the kinetic constants of PT121Y114S and its mutants revealed that the affinity of mutants toward the l-Phe-OMe was significantly higher than that of PT121Y114S. Molecular dynamic simulation revealed that the enhanced synthetic activity may be attributed to the mutation stabilizing the transient state of the enzyme-l-Phe-OMe complex.