Improving organoleptic and antioxidant properties by inhibition of novel miRstv_7 to target key genes of steviol glycosides biosynthetic pathway in Stevia rebaudiana Bertoni.

Stevioside (5-10%) and rebaudioside-A (2-4%) are well-characterized diterpene glycosides found in leaves of Stevia rebaudiana known to have natural sweetening properties with zero glycaemic index. Stevioside has after-taste bitterness, whereas rebaudioside-A is sweet in taste. The ratio of rebaudioside-A to stevioside needs to be changed in order to increase the effectiveness and palatability of this natural sweetener. Plant-specific miRNAs play a significant role in the regulation of metabolic pathways for the biosynthesis of economically important secondary metabolites. In this study inhibition of miRNA through antisense technology was employed to antagonize the repressive action of miRstv_7 on its target mRNAs involved in the steviol glycosides (SGs) biosynthesis pathway. In transgenic plants expressing anti-miRstv_7, reduced expression level of endogenous miRstv_7 was observed than the non-transformed plants. As a result, enhanced expression of target genes, viz. KO (Kaurene oxidase), KAH (Kaurenoic acid-13-hydroxylase), and UGT76G1 (UDP-glycosyltransferase 76G1) led to a significant increase in the rebaudioside-A to stevioside ratio. Furthermore, metabolome analysis revealed a significant increase in total steviol glycosides content as well as total flavonoids content. Thus, our study can be utilized to generate more palatable varieties of Stevia with improved nutraceutical values including better organoleptic and antioxidant properties.

High-quality haplotype-resolved chromosome assembly provides evolutionary insights and targeted steviol glycosides (SGs) biosynthesis in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is popular source of plant-derived low/no-calorie natural sweeteners (LNCSs), collectively known as steviol glycosides (SGs). Nevertheless, genetic predisposition for targeted biosynthesis of SGs is complex due to multi-substrate functionality of key uridine diphosphate glycosyltransferases (UGTs). Here, we created a high-quality monoploid assembly of 1.34 Gb with N50 value of 110 Mb, 55 551 predicted protein-coding genes, and ~80% repetitive regions in Rebaudioside-A (Reb-A) enriched cultivar of S. rebaudiana. Additionally, a haplotype-based chromosome assembly consisting of haplotype A and haplotype B with an overall genome size of 2.33Gb was resolved, harbouring 639 634 variants including single nucleotide polymorphisms (SNPs), indels and structural variants (SVs). Furthermore, a lineage-specific whole genome duplication analysis revealed that gene families encoding UGTs and Cytochrome-P450 (CYPs) were tandemly duplicated. Additionally, expression analysis revealed five tandemly duplicated gene copies of UGT76G1 having significant correlations with Reb-A content, and identified key residue (leu200val) in the glycosylation of Reb-A. Furthermore, missense variations identified in the acceptor region of UGT76G1 in haplotype resolve genome, transcriptional and molecular docking analysis were confirmed with resequencing of 10 diverse stevia genotypes (~25X). Gene regulatory network analysis identified key transcription factors (MYB, bHLH, bZIP and AP2-ERF) as potential regulators of SG biosynthesis. Overall, this study provides haplotype-resolved chromosome-level genome assembly for genome editing and enhancing breeding efforts for targeted biosynthesis of SGs in S. rebaudiana.

Transcriptomic Analyses Reveal Insights into the Shared Regulatory Network of Phenolic Compounds and Steviol Glycosides in Stevia rebaudiana.

Stevia rebaudiana (Bertoni) is a highly valuable crop for the steviol glycoside content in its leaves, which are no-calorie sweeteners hundreds of times more potent than sucrose. The presence of health-promoting phenolic compounds, particularly flavonoids, in the leaf of S. rebaudiana adds further nutritional value to this crop. Although all these secondary metabolites are highly desirable in S. rebaudiana leaves, the genes regulating the biosynthesis of phenolic compounds and the shared gene network between the regulation of biosynthesis of steviol glycosides and phenolic compounds still need to be investigated in this species. To identify putative candidate genes involved in the synergistic regulation of steviol glycosides and phenolic compounds, four genotypes with different contents of these compounds were selected for a pairwise comparison RNA-seq analysis, yielding 1136 differentially expressed genes. Genes that highly correlate with both steviol glycosides and phenolic compound accumulation in the four genotypes of S. rebaudiana were identified using the weighted gene co-expression network analysis. The presence of UDP-glycosyltransferases 76G1, 76H1, 85C1, and 91A1, and several genes associated with the phenylpropanoid pathway, including peroxidase, caffeoyl-CoA O-methyltransferase, and malonyl-coenzyme A:anthocyanin 3-O-glucoside-6″-O-malonyltransferase, along with 21 transcription factors like SCL3, WRK11, and MYB111, implied an extensive and synergistic regulatory network involved in enhancing the production of such compounds in S. rebaudiana leaves. In conclusion, this work identified a variety of putative candidate genes involved in the biosynthesis and regulation of particular steviol glycosides and phenolic compounds that will be useful in gene editing strategies for increasing and steering the production of such compounds in S. rebaudiana as well as in other species.

Efficient Bioconversion of Stevioside and Rebaudioside A to Glucosylated Steviol Glycosides Using an Alkalihalobacillus oshimesis-Derived Cyclodextrin Glucanotransferase.

The enzymatic transglycosylation of steviol glycosides can improve the edulcorant quality of steviol glycosides. Cyclodextrin glucanotransferase (CGTase) is one of the most popular glucanotransferases applied in this reaction. Herein, the CGTase-producing strain Alkalihalobacillus oshimensis CGMCC 23164 was isolated from Stevia planting soil. Using mass spectrometry-based secretome profiling, a high-efficiency CGTase that converted steviol glycosides to glucosylated steviol glycosides was identified and termed CGTase-13. CGTase-13 demonstrated optimal transglycosylation activity with 10 g/L steviol glycoside and 50 g/L soluble starch as substrates at <40 °C. Under the above conditions, the conversion rate of stevioside and rebaudioside A, two main components of steviol glycosides, reached 86.1% and 90.8%, respectively. To the best of our knowledge, this is the highest conversion rate reported to date. Compared with Toruzyme® 3.0 L, the commonly used commercial enzyme blends, glucosylated steviol glycosides produced using CGTase-13 exhibited weaker astringency and unpleasant taste, faster sweetness onset, and stronger sweetness intensity. Thus, CGTase provides a novel option for producing high-quality glucosylated steviol glycoside products and has great potential for industrial applications.

In vitro fermentation assay on the bifidogenic effect of steviol glycosides of Stevia rebaudiana plant for the development of dietetic novel products.

The relationship between excessive sugar consumption and many diseases such as dental caries, obesity, diabetes and coronary heart has been increasing in recent years. In this study, utilization of natural sugar replacer steviol glycosides and bifidogenic effect by Bifidobacterium animalis subsp. lactis was assayed in vitro model system. The basal medium (non-carbohydrate containing MRS, Man, Rogosa and Sharpe Agar) were supplemented with 0.025% and 1% stevia, 0.025% stevia + 1% inulin, %1 stevia + 1% inulin. The medium which contained no carbohydrate was designated as negative control, whereas the medium containing 1% glucose or inulin were evaluated as positive and evaluated on the 0, 12, 24, 36 and 48 h of fermentation. Steviol glycosides in both system significantly stimulated the growth of Bifidobacterium animalis subsp. lactis to varying degrees with highest prebiotic activity score, short chain fatty acid production and growth parameters as much as glucose and prebiotic inulin. The viability of the probiotic bacteria was determined within the bio-therapeutic level with potential prebiotic effects depending on the probiotic bacterial strain growing and the type of carbohydrate source utilized. In the study, stevia at lower concentration showed a higher growth rate of with inulin. In conclusion, stevia can be used as functional ingredients for the modulation of the gut microbiota and design of synbiotic systems as a prebiotic substrate and sugar substitute.

Steviol Glycosides from Stevia rebaudiana: An Updated Overview of Their Sweetening Activity, Pharmacological Properties, and Safety Aspects.

This literature-based review synthesizes the available scientific information about steviol glycosides as natural sweeteners and molecules with therapeutic potential. In addition, it discusses the safety concerns regarding human consumption. Steviol glycosides exhibit a superior sweetener proficiency to that of sucrose and are noncaloric, noncariogenic, and nonfermentative. Scientific evidence encourages stevioside and rebaudioside A as sweetener alternatives to sucrose and supports their use based on their absences of harmful effects on human health. Moreover, these active compounds isolated from Stevia rebaudiana possess interesting medicinal activities, including antidiabetic, antihypertensive, anti-inflammatory, antioxidant, anticancer, and antidiarrheal activity. The described bioactivities of steviol glycosides deserve special attention based on their dose dependence and specific pathological situations. Further clinical research is needed to understand underlying mechanisms of action, therapeutic indexes, and pharmacological applications.

Environmental life cycle assessment of production of the high intensity sweetener steviol glycosides from Stevia rebaudiana leaf grown in Europe: The SWEET project.

Purpose: There is an increasing interest in the use of non-nutritive sweeteners to replace added sugar in food and beverage products for reasons of improving consumer health. Much work has been done to understand safety of sweeteners, but very little on sustainability. To address that gap, this study presents the results of a life cycle assessment (LCA) of production of rebaudioside A 60%, 95% pure (RA60) steviol glycoside mix from Stevia rebaudiana leaf grown in Europe. Methods: An attributional cradle-to-factory-gate life cycle assessment was conducted on growing of stevia leaves and extraction of steviol glycosides in Europe. Primary data were used from a case study supply chain. Results are reported in impact categories from the ReCiPe 2016 (H) method, with focus given to global warming potential, freshwater eutrophication, water consumption, and land use. Impacts are expressed both in terms of production mass and sweetness equivalence, a common metric for understanding high intensity sweetener potency. Sweetness equivalence of RA60 is typically 200 to 300 times that of sugar. Comparison of environmental impact is made to sugar (sucrose) produced from both cane and beets. The research is part of the EU project SWEET (sweeteners and sweetness enhancers: impact on health, obesity, safety, and sustainability). Results and discussion: Global warming potential for production of RA60 was found to be 20.25 kgCO2-eq/kgRA60 on a mass basis and 0.081 kgCO2-eq/kgSE on a sweetness equivalence basis. Field production of stevia leaves was found to be the main source of impact for most impact categories, and for all four focus categories. Extraction of the RA60 was the main source of impact for the others. Leaf processing and seedling propagation were minor contributors to life cycle impact. Removal of international transport from the supply chain reduced global warming potential by 18.8%. Compared with sugar on a sweetness equivalence basis, RA60 has approximately 5.7% to 10.2% the impact for global warming potential, 5.6% to 7.2% the impact for land use, and is lower across most other impact categories. Conclusion: This is the first LCA of steviol glycoside mix RA60 produced from leaf in Europe. The results indicate that RA60 can be used to reduce environmental impact of providing a sweet taste by replacing sugar across all impact categories. However, it is important to note that specific formulations in which RA60 is used will have a bearing on the final environmental impact of any food or beverage products. For solid foods, this requires further research. Supplementary Information: The online version contains supplementary material available at 10.1007/s11367-022-02127-9.

Stevia (Stevia rebaudiana) as a common sugar substitute and its application in food matrices: an updated review.

Stevia (Stevia rebaudiana) has been employee for developing food products as a substitute for sucrose, low caloric, and natural sweetener. Different studies have evaluated the effect of this ingredient on the physicochemical, nutritional and technological properties of products; their application in dairy products affects some sensory characteristics such as taste; in the case of bread, cookies, and cakes modify the properties of the dough, altering attributes of the manufactured product, including color, texture, and flavor; also the use of stevia reduces gelling and affects the optical properties of the final product; and in beverages cases, the total substitution of sugar has led to bitter and unpleasant flavor. This review presents stevia general information and its employees in dairy, bakery, beverages, and jelly products.

Steviol glycosides affect functional properties and macromolecular expression of breast cancer cells.

Steviol glycosides, the active sweet components of stevia plant, have been recently found to possess a number of therapeutic properties, including some recorded anticancer ones against various cancer cell types (breast, ovarian, cervical, pancreatic, and colon cancer). Our aim was to investigate this anticancer potential on the two most commonly used breast cancer cell lines which differ in the phenotype and estrogen receptor (ER) status: the low metastatic, ERα+ MCF-7 and the highly metastatic, ERα-/ERß+ MDA-MB-231. Specifically, glycosides' effect was studied on cancer cells': (a) viability, (b) functionality (proliferation, migration, and adhesion), and (c) gene expression (mRNA level) of crucial molecules implicated in cancer's pathophysiology. Results showed that steviol glycosides induced cell death in both cell lines, in the first 24 hr, which was in line with the antiapoptotic BCL2 decrease. However, cells that managed to survive showcased diametrically opposite behavior. The low metastatic ERα+ MCF-7 cells acquired an aggressive phenotype, depicted by the upregulation of all receptors and co-receptors (ESR, PGR, AR, GPER1, EGFR, IGF1R, CD44, SDC2, and SDC4), as well as VIM and MMP14. On the contrary, the highly metastatic ERα-/ERß+ MDA-MB-231 cells became less aggressive as pointed out by the respective downregulation of EGFR, IGF1R, CD44, and SDC2. Changes observed in gene expression were compatible with altered cell functions. Glycosides increased MCF-7 cells migration and adhesion, but reduced MDA-MB-231 cells migratory and metastatic potential. In conclusion, the above data clearly demonstrate that steviol glycosides have different effects on breast cancer cells according to their ER status, suggesting that steviol glycosides might be examined for their potential anticancer activity against breast cancer, especially triple negative breast cancer (TNBC).

Influence of calcium and magnesium elimination on plant biomass and secondary metabolites of Stevia rebaudiana Bertoni.

This study reports the increment in the secondary metabolites in Stevia rebaudiana plant after exposure to the elimination of Ca and Mg from Murashige and Skoog culture medium. The effect of nutrient stress on regenerants of S. rebaudiana is measured, which reveals significantly enhanced growth parameters, steviol glycosides (SGs) content, and nonenzymatic antioxidants; total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power, and DPPH-free radical scavenging activity as compared with the control treatment. However, significantly highest amounts are obtained in a medium with only Ca deficiency. The amount of rebaudioside A (Reb A) and stevioside (ST) obtained in the case of Ca-deficient medium is 4.08 and 0.69%, respectively. It is followed by the results obtained from both Ca- and Mg-deprived medium [Reb A (3.23%) and ST (0.52%)] and the lowest values are obtained from medium lacking Mg only [Reb A (2.60%) and ST (0.40%)]. The most probable adaptation mechanism might be the production of reactive oxygen species by nutrients' stress, which results in secondary metabolites production as defensive moieties to overcome stress situation. This effective protocol needs to be refined to apply on an industrial scale in bioreactors for increasing quantities of commercially important pharmaceutical compounds.

Do steviol glycosides affect the oxidative and genotoxicity parameters in BALB/c mice?

This study was performed with 40 (20 males, 20 females) BALB/c mice divided into 4 experimental groups and a control group, each consisting of 8 mice (4 males, 4 females). Experimental groups were administered 470, 620, 940, and 1880 mg/kg doses of steviol glycosides, orally, for 4 weeks. The total antioxidants and the oxidant status, paraoxonase-1 enzyme activity, high density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol levels were analyzed from blood samples. The chromosomal aberrations and cell cycle activities were examined from bone marrow samples. Plasma lipid parameters were not affected by the dose of steviol glycosides, however, the total antioxidants, oxidant status, and paraoxonase-1 enzyme activity were found to be negatively correlated with the doses. A positive correlation was found between the total oxidant status and the dose (r = 0.65) and between the mitotic index and the dose (r = 0.74). The dose of steviol glycosides also increased the percentage of the abnormal cells and the CA/cell dose in a dependent manner (r = 0.74 and 0.76, respectively). The study findings concluded that steviol glycosides slightly increased the oxidative damage, cell cycle activity, and chromosomal aberration frequency. However, we did not evaluate the potential of steviol glycosides as genotoxic and mitogenic agents, and, therefore, further investigations are required. CAS number: 58543-16-1.

LC-MS metabolomics analysis of Stevia rebaudiana Bertoni leaves cultivated in Malaysia in relation to different developmental stages.

INTRODUCTION: Stevia is known for its sweet taste, attributed to the presence of steviol glycosides. Although reports on the dynamic changes of steviol glycosides during development of stevia are available, the data are mainly focused on stevioside and rebaudioside A. Information concerning the comprehensive metabolite profile of stevia in relation to different developmental stages is still lacking. OBJECTIVE: This study investigated the metabolite changes along the developmental stages of a local stevia cultivar. METHODOLOGY: Stevia leaves were harvested at 4 different developmental stages (early vegetative, late vegetative, budding, and flowering). Samples were then subjected to LC-MS metabolomics analysis to determine the metabolite variations. RESULTS: A total of 55 metabolites, comprising phenolic acids, flavonoids, and terpenoids were identified by MS/MS analysis of the stevia leaf extracts, revealing a metabolite profile which was comparatively similar with those of cultivars grown in other countries. PLS-DA differentiated the early vegetative stage stevia leaf samples from those of the later stages by higher content of phenolic acids. The leaf metabolomes of the later 3 stages (late vegetative, budding, and flowering) were collectively richer in flavonoids. Meanwhile, the content of steviol glycosides is highest during the late vegetative and budding stages. CONCLUSION: The present study provided, for the first time, a general overview of the metabolite variations with regard to the different developmental stages of stevia. The information may facilitate decision making of suitable harvesting times for higher yields of steviol glycosides or a more balanced metabolite profile in terms of pharmacologically useful metabolites.

Resilience of Stevia rebaudiana (Bertoni) Bertoni in the Underwater Biospheres of Nemo's Garden®: Adaptation to New Cultivation Systems.

The Nemo's Garden® project is an alternative production system for areas with scarce cultivable land but significant presence of water; thus, it is an interesting intervention to address the climate crisis. This work aimed to evaluate the micromorphological, biochemical, and phytochemical characteristics of Stevia rebaudiana (Bertoni) Bertoni grown underwater compared to the terrestrial specimens. The micromorphological analyses, performed on the leaves using light microscopy, fluorescence microscopy, and scanning electron microscopy, evidenced a general uniformity of the trichome morphotype and distribution pattern. The histochemical investigation indicated the simultaneous presence of terpenes and polyphenols in the trichome secreted material from the underwater samples and a prevailing polyphenolic content in the terrestrial specimens; this was also confirmed by biochemical analyses (26.6 mg GAE/g DW). The characterization of non-volatile components, performed using HPLC-MS, showed similar chemical profiles in all the samples, which were characterized by phenolic compounds and steviol glycosides. The volatile compounds, evaluated using HS-SPME coupled with GC-MS, showed sesquiterpene hydrocarbons as the main class in all the analyzed samples (80.1-93.9%). However, the control plants were characterized by a higher content of monoterpene hydrocarbons (12.1%). The underwater biosphere environment did not alter S. rebaudiana micro-morphological characters, although slight qualitative changes were evidenced for the compounds produced as a response to the growth conditions.

Infusions prepared with Stevia rebaudiana: application of a simplex centroid mixture design for the study of natural sweeteners and phenolic compounds.

Three mixture designs were used to characterize herbal formulations for infusions prepared with cedron, boldo, and yerba mate in addition to stevia. This study aimed to investigate if the inclusion of stevia in infusions could affect the recovery of phenolic compounds with antioxidant activity. Infusions with higher phenolic content and higher antioxidant activity were obtained when yerba mate or boldo predominated in the infusion. The highest tannin content was found in mixtures containing yerba mate, boldo, and stevia, while the minimum tannin contents were found in some cedron infusions. The content of steviol glycosides increased as the proportion of stevia increased in the infusions. In general, the recovery of natural sweeteners or phenolic compounds with antioxidant activity exhibited different patterns, depending on the components of the infusions. The presence of stevia and steviol glycosides did not influence the recovery of phenolic compounds with antioxidant activity. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s13197-021-04979-9).

Membrane separation processes for the extraction and purification of steviol glycosides: an overview.

Steviol glycosides (SGs), as natural sweeteners from Stevia rebaudiana, are currently employed for replacing sugar and its derivatives in several food products and formulations. Such compounds play an essential role in human health. Their usage provides a positive effect on preventing diseases related to sugar consumption, including diabetes mellitus, cancer, and lipid metabolism disorders. The traditional extraction of SGs is performed by means of solvent extraction, which limits their application since the removal of residual solvents is a challenging task requiring further downstream purification steps. In addition, the presence of residual solvents negatively affects the quality of such compounds. Today, food technicians are looking for innovative and improved techniques for the extraction, recovery and purification of SGs. Membrane-based technologies, including microfiltration, ultrafiltration, and nanofiltration, have long been proven to be a valid alternative for efficient extraction and purification of several high added-value molecules from natural sources. Such processes and their possible coupling in integrated membrane systems have been successfully involved in recovery protocols of several compounds, such as metabolites, polyphenols, anthocyanins, natural pigments, proteins, from different sources (e.g., agro-food wastes, plant extracts, fruits, fermentation broths, among others). Herein, we aim to review the current progresses and developments about the extraction of SGs with membrane operations. Our attention has been paid to the latest insights in the field. Furthermore, key process parameters influencing the extraction and purification of SGs are also discussed in detail.

Biotechnological interventions of in vitro propagation and production of valuable secondary metabolites in Stevia rebaudiana.

Plant cell and tissue culture makes provision of a sustainable and nature-friendly strategy for the production of secondary metabolites, and modern progress in gene editing and genome engineering provides novel possibilities to improve both the qualitative and quantitative aspects of such phytochemicals. The ever-expanding quest for plant-based medicine to treat diabetes facilitates large-scale cultivation of Stevia rebaudiana to enhance the yield of its much-coveted low-calorie sweetener glycosides. The potential to process stevia as a "natural" product should enhance the acceptance of steviosides as a natural calorie-free sweetener especially suitable for use in diabetic and weight control drinks and foods. Besides sweetener agents, S. rebaudiana is a potent source of many antioxidant compounds and is used to cure immunodeficiencies, neurologic disorders, inflammation, diabetes mellitus, Parkinson's disease, and Alzheimer's disease. This comprehensive review presents the research outcomes of the many biotechnological interventions implicated to upscale the yield of steviol glycosides and its derivatives in in vitro cell, callus, tissue, and organ cultures with notes on the use of bioreactor and genetic engineering in relation to the production of these valuable compounds in S. rebaudiana. KEY POINTS: • Critical and updated assessment on sustainable production of steviol glycosides from Stevia rebaudiana. • In vitro propagation of S. rebaudiana and elicitation of steviol glycosides production. • Genetic fidelity and diversity assessment of S. rebaudiana using molecular markers.

Metabolic engineering for the synthesis of steviol glycosides: current status and future prospects.

With the pursuit of natural non-calorie sweeteners, steviol glycosides (SGs) have become one of the most popular natural sweeteners in the market. The SGs in Stevia are a mixture of SGs synthesized from steviol (a terpenoid). SGs are diterpenoids. Different SGs depend on the number and position of sugar groups on the core steviol backbone. This diversity comes from the processing of glycoside steviol by various glycosyltransferases. Due to the differences in glycosylation, each SG has unique sensory properties. At present, it is more complicated to extract high-quality SGs from plants, so the excavation of the metabolic pathways of engineered microorganisms to synthesize SGs has been extensively studied. Specifically, the expression of different glycosyltransferases in microbes is key to the synthesis of various SGs by engineered microorganisms. To trigger more researches on the functional characterization of the enzymes encoded by these genes, this review describes the latest research progresses of the related enzymes involved in SG biosynthesis and metabolic engineering.Key points• Outlines the research progress of key enzymes in the biosynthetic pathway of SGs• Factors affecting the catalytic capacity of stevia glucosyltransferase• Provide guidance for the efficient synthesis of SGs in microbial cell factories.

Salt Stress Mitigation via the Foliar Application of Chitosan-Functionalized Selenium and Anatase Titanium Dioxide Nanoparticles in Stevia (Stevia rebaudiana Bertoni).

High salt levels are one of the significant and major limiting factors on crop yield and productivity. Out of the available attempts made against high salt levels, engineered nanoparticles (NPs) have been widely employed and considered as effective strategies in this regard. Of these NPs, titanium dioxide nanoparticles (TiO2 NPs) and selenium functionalized using chitosan nanoparticles (Cs-Se NPs) were applied for a quite number of plants, but their potential roles for alleviating the adverse effects of salinity on stevia remains unclear. Stevia (Stevia rebaudiana Bertoni) is one of the reputed medicinal plants due to their diterpenoid steviol glycosides (stevioside and rebaudioside A). For this reason, the current study was designed to investigate the potential of TiO2 NPs (0, 100 and 200 mg L-1) and Cs-Se NPs (0, 10 and 20 mg L-1) to alleviate salt stress (0, 50 and 100 mM NaCl) in stevia. The findings of the study revealed that salinity decreased the growth and photosynthetic traits but resulted in substantial cell damage through increasing H2O2 and MDA content, as well as electrolyte leakage (EL). However, the application of TiO2 NPs (100 mg L-1) and Cs-Se NPs (20 mg L-1) increased the growth, photosynthetic performance and activity of antioxidant enzymes, and decreased the contents of H2O2, MDA and EL under the saline conditions. In addition to the enhanced growth and physiological performance of the plant, the essential oil content was also increased with the treatments of TiO2 (100 mg L-1) and Cs-Se NPs (20 mg L-1). In addition, the tested NPs treatments increased the concentration of stevioside (in the non-saline condition and under salinity stress) and rebaudioside A (under the salinity conditions) in stevia plants. Overall, the current findings suggest that especially 100 mg L-1 TiO2 NPs and 20 mg L-1 Cs-Se could be considered as promising agents in combating high levels of salinity in the case of stevia.

A Novel Urinary Biomarker Approach Reveals Widespread Exposure to Multiple Low-Calorie Sweeteners in Adults.

BACKGROUND: Observational investigations into the health impacts of low-calorie sweeteners (LCSs) in humans fail to adequately identify or fully characterize LCS consumption. OBJECTIVES: We aimed to utilize a novel biomarker approach to investigate exposure to 5 LCSs and to test whether reported low-calorie sweetened beverage (LCSB) consumption effectively identifies exposure to LCSs in adults. METHODS: In this cross-sectional analysis, 2 population studies were conducted in adults. Urinary excretions of 5 LCSs, namely acesulfame-K, saccharin, cyclamate, sucralose, and steviol glycosides, were simultaneously determined using LC tandem-MS. In Study 1, previously collected 24-h urine samples (n = 357) were analyzed. In Study 2, previously collected 24-h urine samples (n = 79) were analyzed to compare urinary excretions of LCSs with self-reported LCSB consumption for identifying LCS exposure. Exposure to LCSs was characterized using descriptive statistics and chi-square tests were performed to assess associations between age-groups and LCS excretion, and to assess the proportion of individuals identified as LCS consumers using biomarker data or reported LCSB consumption. RESULTS: A total of 341 adults (45% men) and 79 adults (39% men) were included in the final analysis of Studies 1 and 2, respectively. In Study 1, >96% of samples contained ≥1 LCS and almost 60% contained ≥3 LCSs. A greater proportion of younger adults (<40 y old) excreted ≥3 LCSs than older adults (>40 y old) (P < 0.001). In Study 2, a much higher prevalence of LCS consumption was observed using biomarker data (92%) than reported LCSB consumption (6%) (P < 0.001). CONCLUSIONS: This work indicates widespread exposure to LCSs, suggesting that population-based research to date into LCS exposure and health may be flawed. Therefore, a urinary biomarker approach offers considerable potential for more robust investigations in this area.

Identification of GH1 gene family fgt members in Stevia rebaudiana and their expression when grown in darkness.

Stevia rebaudiana Bertoni is an important economic crop that is well known for its secondary metabolites, steviol glycosides (SGs), found in leaves. Because the enzymes of deglycosylation (glycoside hydrolases) play important roles in SGs biosynthetic processes, our study is focused on the functions of ß-glucosidases in SGs catabolism in stevia. We cloned and characterized 19 stevia GH1 genes based on transcriptomic sequences. The 19 genes were divided into five putative subfamilies in Arabidopsis. Conserved motifs in the SrGH1 proteins were analysed using the online motif-based sequence analysis tool, MEME. Most of the identified proteins contain the conserved 'TFNEP' motif (contains the catalytic acid/base) and 'ITENG' motif (contains the catalytic nucleophile). Furthermore, the steviol glycoside content and expression of these 19 genes were characterized under constant darkness. The dark treatment lowered the steviol glycoside content significantly, while SrBGLU16 responded to darkness and was markedly upregulated. This study is the first transcriptome-wide analysis of the GH1 family in Stevia rebaudiana. The sequences of 19 SrGH1 members and their expression when grown in darkness were characterized. Among the 19 genes, SrBGLU16 was markedly upregulated by darkness. Thus, we identified SrBGLU16 for further investigation as a possible steviol glycoside beta-glucosidase.