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Light-emitting diode (LED) lamps are efficient elicitors of secondary metabolites. To investigate the influence of LED light on steviol glycosides (SGs) and phenolic compounds biosynthesis, stevia shoots were cultured under the following LED lights: white–WL, blue–B, red–R, 70% red and 30% blue–RB, 50% UV, 35% red and 15% blue–RBUV, 50% green, 35% red and 15% blue–RBG, 50% yellow, 35% red and 15% blue–RBY, 50% far-red, 35% red and 15% blue–RBFR and white fluorescent light (WFl, control). RBG light stimulated shoots' biomass production. RBFR had a beneficial impact on stevioside biosynthesis (1.62 mg/g dry weight, DW), while RBUV favoured the production of rebaudioside A (3.15 mg/g DW). Neochlorogenic, chlorogenic, caffeic, 4-feruloylquinic, isochlorogenic A, rosmarinic acids and the flavonoid quercitrin were identified in the obtained material. A stimulatory effect of RBFR and RBUV on the biosynthesis of phenolic compounds was noted. LED light also influenced stomata appearance, stomata density, photosynthetic pigments, soluble sugar content and antioxidant enzyme activities in stevia shoots. This is the first report to provide evidence of the stimulating effect of LED light on biomass yield, SGs production and phenolic compounds in stevia shoot cultures. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana (Bertoni), commonly known as stevia, is a sought-after natural sweetener, but its conventional propagation methods are slow and inefficient. This study aims to enhance the in vitro culture for stevia by investigating the impact of different Murashige and Skoog (MS) medium salt strengths and plant growth hormones on growth and rebaudioside A content. Apical bud-containing shoot segments were used as explants and cultured on various semi-solid and liquid MS media formulations, incorporating cytokinins (BAP and Kin), auxins (NAA and IAA), and different MS major salt concentrations (MS full, ½ MS, and ¼ MS). Assessments of shoot growth parameters, root formation, and HPLC analysis for rebaudioside A content were conducted. The optimal conditions for in vitro growth was found to be in the ¼ MS + Kin 3 mg/L + NAA 0.1 mg/L (semi-solid) medium, resulting in significantly improved shoot growth and enhanced 30.04% rebaudioside A content. Genetic fidelity of regenerated plants was confirmed using RAPD and ISSR markers. These findings offer valuable insights for optimizing in vitro propagation of stevia and potentially enhancing rebaudioside A content. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana (Bertoni) is an industrially important herb of immense importance due to its sweetening bioactive compounds, especially rebaudioside A and stevioside. The industrial demand for steviol glycosides is mainly fulfilled by conventional agricultural practices. This resulted in non-uniform steviol glycosides (SGs) accumulation in the produce of S. rebaudiana and it is primarily due to changes in varying climatic conditions, genotypes, and cultivars. Therefore, there is an utmost need for a robust protocol to provide quality planting material to produce metabolite-enriched S. rebaudiana. Ex-vitro acclimatization in fields/natural habitats is believed to be a vital part of determining the fate of regenerated plantlets through micropropagation. In the present investigation, elicitor-treated micropropagated plantlets were allowed to harden for one month; after which percentage survival, growth and leaf biomass yield, chlorophyll fluorescence, biochemical profiling of specialized metabolites, and transcriptional profiling of candidate genes were carried out. A high percentage (80–100%) of acclimatized plants was obtained after 30 days. The 1.5 mg L−1 polyethylene glycol (PEG) treated plants attained higher shoot length (14.10 cm) and maximum leaf biomass (537.60 mg FW and 111.03 mg DW). The Chl fluorescence parameters and pigment content indicated stable photosynthetic efficiency in all conditions. Maximum total SGs were accumulated in 1.5 mg L−1 PEG-treated plants. Expression profiling of SG biosynthetic pathway genes showed an apparent correlation with the metabolite content. The study unveils that 1.5 mg L−1 PEG-treated plants perform better in growth and yield attributes and metabolite accumulation parameters after 1 month of ex-vitro acclimatization. [ABSTRACT FROM AUTHOR]

Stevia plants are well-known for their ability to synthesize steviol glycosides (SGs), a natural sweetener blend. The principal SGs include stevioside (STV) and Rebaudioside-A (Reb-A), with the latter exhibiting superior sweetness and organoleptic properties. UDP glucosyltransferase-76G1 (UGT76G1) is responsible for converting STV to Reb-A, determining the intensity of sweetness. A better understanding of the structure/activity of SrUGT76G1 could provide insights into Reb-A production in stevia plants. To this end, a combination of enzymatic assays and sequencing analysis was performed using two stevia genotypes (Brazilian and Spanish) with contrasting Reb-A production capabilities (off/on). Relative expression of SrUGT76G1 gene showed remarkably higher expression (~ threefold) in Spanish samples compared to Brazilian ones. Foliar protein fractions (crude or partially purified extract) from Brazil plants were unable to convert STV into Reb-A under in vitro conditions, resulting in undetectable levels of Reb-A by HPLC. Molecular analyses revealed that the Brazilian SrUGT76G1 gene not only presents a premature stop codon, resulting in the absence of PSPG motif responsible for the binding of glycosyl groups, but also exhibits mutations affecting key amino acid residues in the acceptor-binding pocket. These alterations provide a plausible explanation for the Brazilian protein inability to catalyze the transformation of STV into Reb-A. [ABSTRACT FROM AUTHOR]

Simple Summary: Since foods rich in refined sugars promote obesity, the use of non-caloric sweeteners has gained popularity, and their consumption by pregnant women has increased. Stevia (a non-caloric sweetener) consumption was considered safe for humans by the European Food Safety Authority in a dose of up to 4 mg/kg body weight/day. However, the World Health Organization recommended in 2023 the restraint of these sweeteners at any life stage, highlighting the need for research on pregnant women and early stages of development. So, we aimed to study the effects of chronic consumption of the main sweetener compound of stevia (Rebaudioside A) during the reproductive stage. Female rats were treated with Rebaudioside A (4 mg steviol equivalents/kg body weight/day) in the drinking water from 4 weeks before mating until weaning. Food and water consumption, blood glucose and lipids, as well as heart structure, function and mitochondrial function, were assessed. Rebaudioside A decreased heart size, cardiomyocyte area and fibrosis without repercussions on cardiac or mitochondrial function. Both fasting blood glucose and cholesterol decreased. This work suggests that stevia consumption at this dose may be safe for females during the reproductive stage. However, more studies are mandatory to explore the effects of stevia consumption on offspring's health. The consumption of non-sugar sweeteners (NSS) has increased during pregnancy. The European Food Safety Agency suggested that steviol glycosides, such as Rebaudioside A (RebA), the major sweetener component of stevia, are safe for humans up to a dose of 4 mg/kg body weight/day. However, the World Health Organization recommended in 2023 the restraint of using NSS, including stevia, at any life stage, highlighting the need to study NSS safety in early periods of development. We aimed to study the mitochondrial and cardiometabolic effects of long-term RebA consumption during the reproductive stage of the life cycle. Female rats were exposed to RebA (4 mg steviol equivalents/kg body weight/day) in the drinking water from 4 weeks before mating until weaning. Morphometry, food and water consumption, glucose and lipid homeostasis, heart structure, function, and mitochondrial function were assessed. RebA showed an atrophic effect in the heart, decreasing cardiomyocyte cross-sectional area and myocardial fibrosis without repercussions on cardiac function. Mitochondrial and myofilamentary functions were not altered. Glucose tolerance and insulin sensitivity were not affected, but fasting glycemia and total plasma cholesterol decreased. This work suggests that this RebA dose is safe for female consumption during the reproductive stage, from a cardiometabolic perspective. However, studies on the effects of RebA exposure on the offspring are mandatory. [ABSTRACT FROM AUTHOR]

Rebaudioside A (Reb A) and neohesperidin dihydrochalcone (NHDC) are known as intense sweeteners. This study aimed to examine the anti-obesity effects of Reb A and NHDC. C57BL/6 J-ob/ob mice were supplemented with Reb A (50 mg/kg body weight [b.w.]), NHDC (100 mg/kg b.w.), or their combination (COMB) for 4 weeks. COMB-supplemented mice showed significant reduction in b.w. gain, food efficiency ratio, and fat mass. Additionally, mice in the COMB group showed suppressed levels of genes related to adipogenesis, lipogenesis, and lipolysis in the perirenal fat and the levels of hepatic triglyceride, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase. The lipogenesis and pro-inflammatory gene expressions were also downregulated in the liver, whereas β-oxidation related genes were upregulated in the COMB group. In addition, mice that received COMB showed distinct gut microbiota structure, enriched in Blautia and Parabacteroides, and depleted in Faecalibaculum and Mucispirillum, in relation to the control group. These results suggest that supplementation with Reb A and NHDC may be an effective treatment for obesity-related metabolic disorders. [ABSTRACT FROM AUTHOR]

Ethephon, as an ethylene-releasing compound, is frequently used for the regulation of plant growth and metabolism. The present study aimed to investigate the impact of ethephon application at various concentrations (0, 0.37, 0.75, 1.5, and 3 mM) and stages [(treatment on seed, and foliar application on seedling (6-leaf stage) and full-grown plant)] on growth and metabolites biosynthesis of stevia (Stevia rebaudiana L.). It was observed that ethephon had no significant effects on seed germination, while seed treated by 0.37 mM ethephon produced stevia plants with significant leaf biomass. The foliar application of ethephon on full-grown plants caused a significant loss in leaf production compared to non-ethephon-treated plants (control). The most significant increase (39% compared to control) in leaves dry weight was recorded at 0.75 mM ethephon applied on the seedling stage. A significant increase in steviol glycosides (SVglys) content was observed when 0.37 and 0.75 mM ethephon were applied to stevia seeds and seedlings, respectively. This result was equally certain regarding Rebaudioside (Reb) A and Stevioside (Stev). The SVglys production in stevia was significantly increased (50%, compared to control) when ethephon was applied to seeds and seedlings at 0.75 mM concentration. However, ethephon foliar application on full-grown plants sharply decreased the SVglys biosynthesis. The ethephon concentrations of 0.37 and 0.75 considerably increased the sucrose, glucose, and fructose content of the leaves. Based on the results presented, it can be concluded that stevia response to exogenous ethephon depends on ethephon concentration and time of application. In this respect, the application of 0.75 mM ethephon on seeds and seedlings could be an appropriate strategy for biosynthesis and accumulation of metabolites in stevia. [ABSTRACT FROM AUTHOR]

Low-calorie orange marmalade was produced using sugar and reb A (0.08%) or reb A-sugar free recipes with pectin (1%) and agar agar (AA) (1%) stabilizers. The physical, chemical, and sensory properties of the marmalades were evaluated. AA stabilizers increased the viscosity of the marmalades compared to pectin, but they did not affect samples with reb A. Low-calorie samples had higher L* and b* values and lower a* values than sugar-containing samples. Total sugar content in reb A samples was 8.30 to 9.28 g/100 g, and it was lower in agar samples produced in parallel with pectin. The hydroxymethyl furfural (HMF) value was lower in sugar-free recipes (3.03 to 3.62) than in sugar-containing samples. Pectin-reb A samples had favorable taste, while AA-reb A samples favored consistency among reb A-containing samples.

Low-calorie orange marmalade was produced using sugar and reb A (0.08%) or reb A-sugar free recipes with pectin (1%) and agar agar (AA) (1%) stabilizers. The physical, chemical, and sensory properties of the marmalades were evaluated. AA stabilizers increased the viscosity of the marmalades compared to pectin, but they did not affect samples with reb A. Low-calorie samples had higher L* and b* values and lower a* values than sugar-containing samples. Total sugar content in reb A samples was 8.30 to 9.28 g/100 g, and it was lower in agar samples produced in parallel with pectin. The hydroxymethyl furfural (HMF) value was lower in sugarfree recipes (3.03 to 3.62) than in sugar-containing samples. Pectin-reb A samples had favorable taste, while AA-reb A samples favored consistency among reb A-containing samples. [ABSTRACT FROM AUTHOR]

The present study is aimed to study the effect of SiO2 nanoparticles on in vitro morphogenesis and secondary metabolite biosynthesis potential of Stevia rebaudiana in solid and liquid culture media. Significant variations were recorded (in all the parameters) between plants regenerated on solid and liquid media, while no uniform pattern was obtained in the variations among different treatments. Morphological properties including shoot number (1.4 × to 1.8 ×), shoot length (1.7 × to 2 ×), node count (1.5 × to 1.8 ×), leaf area (2.6 × to 2.75 ×) and fresh weight (10.6 × to 14 ×) were higher in liquid cultures than solid cultures, irrespective of the SiO2 NP treatment. However, some concentrations of SiO2 NPs had positive impact on shoot morphology in both types of cultures increasing the parameters up to 2.4 folds in solid and 1.3 folds in liquid cultures. In case of biochemical parameters, inverse pattern was recorded such that chlorophyll and carotenoid contents were respectively 6 × to 10.6 × and 10 × to 12.5 × higher in solid cultures than in liquid cultures. Antioxidant activities also exhibited similar pattern which indicated higher stress in shoots regenerated on solid medium than on liquid medium. Further, content of reb-A was significantly higher in plants grown on solid medium than in liquid medium. In fact, the content increased up to 1.7 folds in the presence of SiO2 NPs in solid medium, wherein the content decreased in presence of SiO2 NPs in liquid medium. These findings provide first report which indicates that liquid cultures have growth promoting effect while in solid cultures the plants are comparatively under more stress even without any external stimuli and thus produce higher reb-A content. Moreover, SiO2 NPs are likely to have different uptake and action mechanism in solid and liquid medium which needs further investigation to gain a deeper insight into the mechanisms responsible for these effects. Key Message: Liquid medium promotes morphogenic response in Stevia, but solid cultures may provide a more conducive environment for inducing secondary metabolite biosynthesis in presence of SiO2 NPs, possibly due to comparatively slower & reduced nutrient diffusion into the plant cells. [ABSTRACT FROM AUTHOR]

Abstract Stevia rebaudiana Bertoni is a valuable medicinal plant and an essential source of natural sweetener, steviol glycosides (SGs), with rebaudioside A (RA) being one of the main components of SGs. bHLH transcription factors play a crucial role in plant development and secondary metabolism. In this study, 159 SrbHLH genes were identified from the S. rebaudiana genome, and each gene was named based on its chromosome location. The SrbHLH proteins were then clustered into 18 subfamilies through phylogenetic analysis. The analysis of conserved motifs and gene structure further supported the classification of the SrbHLH family. Chromosomal location and gene duplication events of SrbHLH genes were also studied. Moreover, based on the RNA-Seq data of different tissues of S. rebaudiana, 28 SrbHLHs were co-expressed with structural genes involved in RA biosynthesis. The expression pattern of candidate SrbHLH genes were confirmed by qPCR. Finally, dual luciferase reporter assays (DLAs) and subcellular localization analysis verified SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 are critical regulators of RA biosynthesis. This study provides new insights into the function of SrbHLHs in regulating SGs biosynthesis and lays the foundation for future applications of SrbHLH genes in molecular breeding of S. rebaudiana.

Introduction Sesame paste is a product that is obtained by mixing Ardeh obtained from peeling of Sesame seeds with sugar or other sweeteners and flavorants such as cardamom and vanilla. Each 100 grams of this nutritious product contains 558 kcal and providing 17% of the daily calcium need, 100% for iron and 26% for protein. Sesame cream /Ardeh cream provides 478 kcal per 100 grams. Ardeh cream is also a good alternative to chocolates with artificial colors, so it is a very good choice in cases of hypoglycemia, such as before the exam session of students or during heavy excersize such as mountaineering. Sugar substitutes are compounds that, like sugars, are used to sweeten foods, except that they are not digested without the need for metabolized insulin, or are digested sparingly and therefore have little caloric value. The diet products are usually based on reducing calorie intake, which today, the growing interest of people in eating healthy diet has led to an increase in their desire to consume low-calorie products. Patients with diseases caused by sugar consumption such as diabetes, hypertension and hyperlipidemia, osteoporosis, obesity, etc. tend to use artificial sweeteners such as saccharin, aspartame, cyclamate and acesulfame. Materials and Methods Hydrogenated oil, Skim milk powder, Whey powder, Soy lecithin, Potassium dichromate, Ethanol, Ardeh , Erythritol, Isomalt Powder, Ribodioside a, Sugar Stable micro histometer systems, Ballmer, Memmert model digital scale, Autoclave, Oven, N Hexane, Desiccator, Soxhlet device, 500 cc balloon, Peptone water, Dichlorine-Rose Bengal Chloramphenicol Agar (DG18), coax reagent, EC broth culture medium, lauryl sulfate culture medium, pH meter with measuring accuracy of 0.1%, 0.1 N sulfuric acid solution, Potassium sulfate, dioxide Selenium, boric acid solution, crystallized copper sulfate, aluminum plate were used in this study . First, to prepare a control sample, raw materials such as flour, oil and sugar were added to the mixer and mixed together, then the rest of the ingredients were added and mixed inside the machine to make the cream uniform and homogeneous. Then it was transferred to the filter and packed in the desired containers. Sugar substitutes were used for the rest of the samples. Results and Discussion The prevalence of obesity and diabetes has increased dramatically in recent years, forcing consumers to look for low-calorie sweeteners. Replacing low-calorie sweeteners with sucrose results in low-calorie, and healthy foods. The aim of this study was to present a new formulation using isomalt, ribadioside a, erythritol as sucrose substituents and to investigate its effect on chemical, physical and sensory properties of substituted samples compared to the control sample. The use of sucrose substituents was effective in improving some physicochemical properties of Ardeh so that the moisture content of the substituted samples increased. The results of histological examination also showed that the replaced samples had softer tissue than the control sample, which was due to the weakening of tissue stability by sucrose substitutes. The results of sensory properties based on aroma, taste, odor and texture also showed that sample 2 with the formula 25% sucrose, 6.25% isomalt, 0.003 ribodiozide obtained the highest score in terms of overall acceptance. The reason for the decrease in the general acceptance of the samples replaced with complete elimination of sucrose is the lack of regulation of the desired sweetness by these alternatives. The results of microbial analysis of samples also showed that sugar substitutes have no effect on the microbial properties of Ardeh. According to the obtained results, sample 2 with 25% sucrose, 6.25% isomalt, 0.003% ribodioside a is the best combination and a healthy and low calorie snack.

Stevia rebaudiana Bertoni is a valuable medicinal plant and an essential source of natural sweetener, steviol glycosides (SGs), with rebaudioside A (RA) being one of the main components of SGs. bHLH transcription factors play a crucial role in plant development and secondary metabolism. In this study, 159 SrbHLH genes were identified from the S. rebaudiana genome, and each gene was named based on its chromosome location. The SrbHLH proteins were then clustered into 18 subfamilies through phylogenetic analysis. The analysis of conserved motifs and gene structure further supported the classification of the SrbHLH family. Chromosomal location and gene duplication events of SrbHLH genes were also studied. Moreover, based on the RNA-Seq data of different tissues of S. rebaudiana, 28 SrbHLHs were co-expressed with structural genes involved in RA biosynthesis. The expression pattern of candidate SrbHLH genes were confirmed by qPCR. Finally, dual luciferase reporter assays (DLAs) and subcellular localization analysis verified SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 are critical regulators of RA biosynthesis. This study provides new insights into the function of SrbHLHs in regulating SGs biosynthesis and lays the foundation for future applications of SrbHLH genes in molecular breeding of S. rebaudiana. [ABSTRACT FROM AUTHOR]

Purposes: Rebaudioside A (RA) is a natural sweetener whose key manufacturing step is crystallization. However, a rapid detection method suitable for this process is currently absent. With miniaturization becoming a trend in industrial applications, near-infrared (NIR) spectroscopy has become a powerful tool for understanding the manufacturing process of natural products. The potential of miniature NIR for quantification of RA content and visualization of the crystallization process was investigated in this study. Methods: The RA crystallization process was simulated in our laboratory. During the crystallization, the NIR spectra were collected using two micro-sensors (1350 ~ 1650 nm and 1550 ~ 1950 nm). To improve the performance of the model, convolution neural network (CNN) with sensor fusion was proposed. Results: The CNN model based on raw spectral fusion showed the best predictive capacity with RMSEP and RPD of 2.895 mg/mL and 3.919, respectively. Conclusions: Overall, the research showed that the CNN model with data fusion was promising for quantifying and visualizing RA crystallization based on NIR. This study promoted the popularization of portable spectrometers by providing a theoretical basis for instrument customization. [ABSTRACT FROM AUTHOR]

Steviol glycosides are non-caloric sweeteners found in high concentrations in the leaves of the Paraguayan perennial herb S. rebaudiana rebaudiana. In the present study, an attempt has been made to analyze promoter regions of SG-related biosynthesis genes of S. rebaudiana rebaudiana and Helianthus annuus. The close relationship between H. annuus, S. rebaudiana, and the SGs biosynthesis genes in the H. annuus genome provides a remarkable opportunity for SGs production in the H. annuus leaves. Several cis-acting regulatory elements associated with plant development, plant hormonal regulation, and stress response were identified and found in varying frequencies within the two kbp of 5′ regulatory regions, including A-box, MYB, MSB, ABRE, LTR, ERE, STRE, ABA, ARE, DRE, MBSI, TC-RICH REPEATS, WUN-MOTIF, MYC, MYB. The number of cis-elements in both plants is equal. However, S. rebaudiana's UGT genes have significantly more elements than H. annuus. These results suggest that the lack of H. annuus SGs is related to the plant's cis-elements of UGT genes. This finding indicates the potential cis-acting regulatory factors involved in the expression and regulation of the SG gene in H. annuus and S. rebaudiana during cellular development or environmental stress situations. Further, tandem repeats and CPG islands were detected in the promoter regions. RT-PCR analysis of seven SG biosynthesis key genes, including CDPS, KS, KO, KAH, and three glucosyltransferases, namely UGT85C2, UGT74G1, and UGT76G1, revealed that only KS and KO genes are expressed in H. annuus leaves. Futuristically, this data will be a helpful resource for upgrading H. annuus leaves to S. rebaudiana sweeteners producers and understanding the regulation of SG-related genes. [ABSTRACT FROM AUTHOR]

In this study, the interaction between nanoparticles (0, 50, 100, and 150 mg L−1) and light intensity (100, 200, and 400 μmol·m−2·s−1) was evaluated for effectiveness in improving stevia shoot induction by measuring morphological traits, nutrient absorption, total carbohydrates, steviol glycosides (SVglys), and DNA damage in two DNA sequence regions (promoter and sequence of the UGT76G1 gene). MWCNTs at a concentration of 50 mg L−1 in interaction with the light intensity of 200 μmol·m−2·s−1 improved the morphological traits and absorption of nutrients such as nitrogen (N), phosphorous (P), potassium (K), calcium (Ca), iron (Fe), and Manganese (Mn), compared to other treatments. Also, under this interaction, the accumulation of total carbohydrates and SVglys was elevated. Moreover, DNA damage in both regions of the DNA sequence under light intensity at low concentrations of MWCNTs (0 and 50 mg L−1) did not show a significant change but increased with increasing MWCNT concentration at high light intensities (200 and 400 μmol·m−2·s−1). These results demonstrate that the advantages and phytotoxicity of MWCNTs in the in vitro culture of stevia are dose-dependent and are affected by light intensity. Based on this, the interaction of 50 mg L−1 of MWCNTs with the light intensity of 200 μmol·m−2·s−1 is recommended to improve stevia micropropagation and subsequent growth and metabolism. [ABSTRACT FROM AUTHOR]

• Water deficit stress reduced the growth parameters of stevia. • Humic acid can improve plant biomass of stevia under water deficit stress. • Water deficit stress enhanced the contents of stevioside and rebaudioside A. • The influence of humic acid had not significant on the phytochemical traits. In sustainable agriculture, the foliar application of humic acid can be an effective solution in reducing the adverse effects of water deficit conditions in crops without damaging the environment. In order to investigate the effect of humic acid on the growth parameters and secondary metabolites of stevia (Stevia rebaudiana B.) under different soil moisture conditions, a study was done at the experimental filed of Western Tehran, Iran during the growing season 2018–2019. The experiment was performed as a factorial in a randomized complete block design. The experimental treatments included the foliar spraying of humic acid at three levels (0, 150 and 300 g/mL) and irrigation frequencies (3, 5, and 7 days based on irrigation at 80–85% of field capacity). The results indicated that with increasing interval irrigation, the plant height and stem fresh weight as well as the leaves dry and fresh weight decreased significantly: whereas, the use of humic acid (especially at the level of 300 g/mL) caused a significant improvement in these indices under reduced irrigation conditions. The highest amounts of the leaf area index (LAI) and carotenoid content were related to the application of 300 g/mL humic acid. The chlorophyll a and b contents and the concentrations of two main active substances such as stevioside and rebaudioside A contents in the leaves enhanced significantly under increasing interval irrigation. The highest amounts of the stevioside and rebaudioside A contents (41.5 and 11.6 mg/100 g dry matter, respectively) were obtained in the interval irrigation of 7 days and without humic acid. According to results of the interaction effects of the application of humic acid and irrigation found, the application of humic acid can improve plant biomass which can reduce the harmful impacts of deficit irrigation on the plant growth parameters of stevia:however, reduced irrigation without the foliar application of humic acid could increase the quality yield of stevia such as stevioside and rebaudioside A. [ABSTRACT FROM AUTHOR]

Background: Stevia rebaudiana is a medicinal herb that accumulates non-caloric sweeteners called steviol glycosides (SGs) which are approximately 300 times sweeter than sucrose. This study used alginate (ALG) as an elicitor to increase steviol glycosides accumulation and elucidate gene transcription in the steviol glycosides biosynthesis pathway. Methods and Results: To minimize the grassy taste associated with stevia sweeteners, plantlets were grown in complete darkness. ALG was applied to stevia plants grown in suspension culture with a Murashige and Skoog (MS) medium to determine its effect on SGs' content and the transcription profile of SG-related genes using the HPLC and RT-qPCR methods, respectively. Treatment with alginate did not significantly affect plantlet growth parameters such as shoot number, dry and fresh weight. Rebaudioside A (Reb A) content increased approximately sixfold in the presence of 1g L-1 alginate and KS, KAH, and UGT74G1 genes showed significant up-regulation. When the concentration was increased to 2g L-1, the transcription of KO and UGT76G1, responsible for the conversion of stevioside to Reb A, was increased about twofold. Conclusions: The current study proposes that adding alginate to the MS suspension medium can increase Reb A levels by altering the SG biosynthesize pathway's transcription profile. The present experiment provides new insights into the biochemical and transcriptional response mechanisms of suspension-cultured stevia plants to alginate. [ABSTRACT FROM AUTHOR]

In this study, a new strain of Pantoea vagans, SRS89, was isolated from surface-sterilized stevia seeds. The isolate was evaluated using morphological, molecular, and biochemical methods. The bacterium was 1.5 μm long, yellowish in color, and classified as Gram-negative. Whole genome sequencing of our strain revealed the presence of a 4,610,019 bp chromosome, and genome annotation resulted in the detection of 4283 genes encoding 4204 putative coding sequences. Phylogenic analysis classified the genome of our strain close to the MP7 and LMG 24199 strains of P. vagans. Functional analysis showed that the highest number of genes within the analyzed bacterium genome were involved in transcription, amino acid transport and metabolism, and carbohydrate transport and metabolism. We also identified genes for enzymes involved in the biosynthesis of carotenoids and terpenoids. Furthermore, we showed the presence of growth regulators, with the highest amount noted for gibberellic acid A3, indole-3-acetic acid, and benzoic acid. However, the most promising property of this strain is its ability to synthesize rebaudioside A; the estimated amount quantified using reversed-phase (RP)-HPLC was 4.39 mg/g of the dry weight of the bacteria culture. The isolated endophytic bacterium may be an interesting new approach to the production of this valuable metabolite. [ABSTRACT FROM AUTHOR]

Rebaudioside A is used in the food and beverage industries owing to its sweetness, even in extremely small amounts, as well as its natural origin. Liquid chromatography is commonly used to quantify rebaudioside A; however, it suffers from complicated sample pretreatment and time consumption. External-calibration quantitative nuclear magnetic resonance (qNMR) analysis of probiotic-fermented milk has thus far not been reported. Moreover, it involves more factors contributing to errors than internal standard approaches used in the official methods, such as those recommended by the International Organization for Standardization. In this study, we developed a method for quantifying rebaudioside A in probiotic-fermented milk using internal-calibration qNMR. This method entails a simple pretreatment step with dilution and solid-phase extraction, enabling rebaudioside A determination in five types of beverages including probiotic-fermented milk. The results revealed a recovery rate and relative standard deviation of 85.4–104 % and 1.57–4.09 %, respectively, satisfying the international guidelines. The working range of qNMR was 0.80–26.7 mg/100 g of product, and the analysis time was 10–20 min. Therefore, the proposed internal-calibration qNMR method facilitates easy, rapid, and accurate quantification of rebaudioside A in probiotic-fermented milk and is applicable for analyzing various other beverages and foods. [Display omitted] • The steviol glycoside rebaudioside A is generally recognized as a safe sweetener. • Rebaudioside A within probiotic-fermented milk was determined using qNMR. • The recovery rate and RSD of the qNMR method satisfied the international guidelines. • Pretreatment was simple, and a rapid, highly accurate analysis was possible. • This method can be applied in the quality control of various beverages and foods. [ABSTRACT FROM AUTHOR]

Steviol glycosides produced by Stevia rebaudiana Bertoni are regarded as the most promising substitute for sucrose with a wide range of potential applications and high economic value due to their high sweetness, low caloric energy, non-participation in human metabolism and health care functions. Three key genes from Stevia leaves, SrUGT85C2, SrUGT91D2m and SrUGT76G1 in the biosynthetic pathway of steviol glycosides, were cloned, and overexpression vector of plant genes were constructed. In individual or in combined way, these genes were transferred into the S. rebaudiana, and the transgenic plants were obtained. Compared with the control of wild-type plant, the content of steviolmonoside increased in the transgenic plant transferred SrUGT85C2 alone, despite little changes in the total content of steviol glycosides, rebaudioside A, and the content ratio of rebaudioside A to the total steviol glycosides. In the transgenic plant transferred SrUGT91D2m alone, the content of steviolmonoside decreased, while the content of steviolbioside increased significantly. In the other hand, in the transgenic plant transferred SrUGT76G1 alone, the total amount of steviol glycosides significantly increased, especially the content of rebaudioside A reached to 10%, which was nearly two times than that in the control, whereas the content of stevioside reduced by half. The transgenic plant transferred the three genes in combination was similar to the transgenic plant transferred SrUGT76G1 alone, and the total content of steviol glycosides, rebaudioside A, and the content ratio of rebaudioside A to the total steviol glycosides increased significantly. These results provides the theoretical basis and technical methods for regulating the expressions of key genes in the biosynthesis of steviol glycosides and cultivating new high-quality S. rebaudiana strains with high content of rebaudioside A through molecular biology techniques. [ABSTRACT FROM AUTHOR]

In this study, it is aimed to produce low sugar orange marmalade containing Reb A and to determine some quality characteristics. For this purpose, recipes with different amounts of Reb A, sugar and pectin were used to produce marmalades. Water-soluble dry matter (WSDM), color and sensory analyzes of the produced marmalades were made. In the determination of optimum pectin ratio, panelists liked marmalades with 1.00% pectin the most, among 0.50, 0.75 and 1.00% pectin ratios. They also liked the 6. recipe (400 g mixture with 0.48 g Reb A and 1.00% pectin) of marmalades produced with only Reb A. They stated that the 11. recipe (400 g mixture with 58.4 g sugar, 0.30 g Reb A and 1.00% pectin) was the closest recipe to the standard marmalade among all recipes. As the amount of sugar decreased in the recipe, the content of WSDM also decreased. L* and b* values decreased while browning index (BI) increased by an increase in the sugar content of marmalades. The highest color change (E) was determined in recipes 1 and 2 (400 g mixture with 97.6 g sugar containing 1.00 and 0.75% pectin, respectively). In sensory analysis, the sample produced with only Reb A was more vivid, yellow and close to fruit color. It also had a higher orange scent; however, its consistency and gummy structure decreased with a decrease in the amount of sugar. As the amount of Reb A increased, chemical odor, bitter taste and sweetness increased. As a result, the panelists stated that they could purchase marmalades produced with Reb A when they were on a diet, but they liked the recipes produced with Reb A-sugar mixture more than others. [ABSTRACT FROM AUTHOR]

For health and safety concerns, traditional high‐calorie sweeteners and artificial sweeteners are gradually replaced in food industries by natural and low‐calorie sweeteners. As a natural and high‐quality sugar substitute, steviol glycosides (SvGls) are continually scrutinized regarding their safety and application. Recently, the cultivation of organic stevia has been increasing in many parts of Europe and Asia, and it is obvious that there is a vast market for sugar substitutes in the future. Rebaudioside A, the main component of SvGls, is gradually accepted by consumers due to its safe, zero calories, clear, and sweet taste with no significant undesirable characteristics. Hence, it can be used in various foods or dietary supplements as a sweetener. In addition, rebaudioside A has been demonstrated to have many physiological functions, such as antihypertension, anti‐diabetes, and anticaries. But so far, there are few comprehensive reviews of rebaudioside A. In this review article, we discuss the physicochemical properties, metabolic process, safety, regulatory, health benefits, and biosynthetic pathway of rebaudioside A and summarize the modification methods and state‐of‐the‐art production and purification techniques of rebaudioside A. Furthermore, the current problems hindering the future production and application of rebaudioside A are analyzed, and suggestions are provided. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana Bertoni (Asteraceae) is a South American perennial herb native to the Paraguay region. It is high-value commercial crop in the food industry because of an array of bioactive diterpenoid glycosides, the most important of which are the steviol glycosides, stevioside (Stev) and rebaudioside A (Reb A). Standardized extract from Stevia leaves, which is high in steviol glycosides, can be used as a high-potency sweetener. The current study proposes a high-performance thin layer chromatography (HPTLC) approach for estimating stevioside and rebaudioside that is rapid, verified, and repeatable. A collection of 32 chemotypes of Stevia rebaudiana were collected from 32 districts across nine Indian states. Stevioside and rebaudioside A content ranged between 1.43 - 5.88 % and 0.77 - 1.95 % respectively across 32 chemotypes of S. rebaudiana. Among all the studied samples, SrP1 chemotype was found to possess the highest quantity of stevioside (5.88 %) and rebaudioside A (1.95 %), followed by SrH11 (5.28 % Stev; 1.89 % Reb A) chemotypes obtained from Jalandhar, Punjab and Palampur, Himachal Pradesh respectively. Chemotype SrWb32 (1.43 % Stev; 0.77 % Reb A) collected from Bardhaman, West Bengal exhibited least amount of stevioside and rebaudioside A. The current fast, reproducible, and validated HPTLC method presents an efficacious toolkit to explore the quantitative variation in the stevioside and rebaudioside A contents in natural S. rebaudiana chemotypes. This methodology may be employed to screen high stevioside and rebaudioside A yielding elite genotypes for future propagation as well as commercialization of these highly potent sweeteners to deal with the ever-expanding demand for alternative yet natural sweeteners in food industries. [Display omitted] • Thirty two populations or chemotypes of Stevia rebaudiana were analysed to detect steviol glycosides. • Chemotypes of the plant revealed altitudinal variation in steviosides and rebaudisides A content. • Elite chemotypes can further be used for propagation and commercial utilization. [ABSTRACT FROM AUTHOR]

Abstract Stevia rebaudiana is a sweet herbaceous perennial plant, which is frequently used in the preparation of plant‐based sweeteners. The demand for such sweeteners continues to increase due to purposeful nutrition and modern‐day metabolic syndromes. More than 20 types of steviol glycosides provide a sweet taste, which are more than 300 times sweeter than sucrose. They are formed of two main components, namely stevioside and rebaudioside A. Only a handful of studies have dealt with Stevia rebaudiana leaf extracts, the conversion of pure stevioside into the preferred rebaudioside A is more common. The aim of this study was to enrich the rebaudioside A content of Stevia rebaudiana leaf extract using enzymatic bioconversion by applying fermented cyclodextrin glycosyltransferase from Bacillus licheniformis DSM13. Two differently processed plant materials, namely dried and lyophilized Stevia rebaudiana plants, were extracted and compared. Following the bioconversion, the rebaudioside A content was on average doubled. The maximum increase was fivefold with a 70–80% conversion of the stevioside.

The separation and removal of stevioside from natural product steviol glycosides to obtain high‐purity rebaudioside A is of great significance for the application of steviol glycosides in food, medicine, and other fields. Here, in order to explore the adsorbent pore structure suitable for the separation of stevioside and rebaudioside A, a hierarchically porous amino‐functionalized metal–organic framework (HP‐NH2‐MIL‐53) with an appropriate and narrow pore size distribution was prepared using a modulator‐induced defect‐formation strategy. The results showed that the hierarchically porous structure with micropores and mesopores increased the specific surface area and exposed amino groups compared with original metal organic framework (NH2‐MIL‐53), and the maximum adsorption capacity of HP‐NH2‐MIL‐53 for stevioside and rebaudioside A was 233.89 mg/g. The narrow pore size distribution close to 3.80 nm promoted the screening effect, resulting in a maximum adsorption selectivity of 4.13. This work proves that when the pore size of the adsorbent is between 1.41 and 3.80 nm, it has a certain pore size screening effect on stevioside and rebaudioside A, and the hierarchically porous metal‐organic frameworks provide a pre‐design idea of adsorbent structure for the separation of natural products with molecular weight of 800–1000 Da. [ABSTRACT FROM AUTHOR]

A plant tissue culture protocol from stevia was optimized for the production of planting materials and the natural sweetener, rebaudioside A. The highest survivability (88.90% ± 5.55) of explants was achieved at 15 and 30 days after culture initiation (DACI) on Murashige and Skoog (MS) media by sterilization with 30% Clorox (5 min) and 10% Clorox (10 min), respectively. Supplementation of MS with 0.50 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.10 mg/L zeatin produced 50% callus at 15 DACI while 1.50 mg/L 2,4-D and 0.10 mg/L zeatin at 30 DACI increased callus production to 76.67%. The highest shoot proliferation per callus was achieved with 10.00 mg/L 6-benzyl amino purine (BAP) in MS at 15 DACI (5.80) and 30 DACI (12.33). The longest shoots of 4.31 cm and 6.04 cm at 15 and 30 DACI, respectively, were produced using BAP (10.00 mg/L) and 1.00 mg/L naphthalene acetic acid (NAA). MS media (0.50 strength) induced 2.86 and 6.20 roots per shoot and produced 3.25 cm and 7.82 cm long roots at 15 and 30 DACI, respectively. Stevia grown on 0.25 MS accumulated the highest concentration of rebaudioside A (6.53%), which correlated with the expression level of its biosynthetic gene uridine-diphosphate-dependent (UDP)-glycosyltransferase (UGT76G1). [ABSTRACT FROM AUTHOR]

Two field experiments were conducted in 2019 and 2020 seasons at Desert Research Center, Mataria, Cairo, Egypt, and its experimental farm at Ras Sudr Experimental Station in South Sinai, in cooperation with the Department of Horticulture, Faculty of Agriculture Benha University, to study the effect of biogas slurry fertilizer and natural extracts on biomass, chemical constituents, and quality of Stevia rebaudiana L. plant. Results showed that biogas fertilizer along with the different natural extracts exerted a significant influence on yield, chemical constituents, stevioside and rebaudioside A., of stevia. The highest values in most of the parameters i.e. shoot fresh and dry weights biomass, leaves fresh and dry weights biomass, N, P, K and pigments in the first cut by recommended dose of chemical fertilizer (T1) combining with garlic as drench addition, Yucca extract as a foliar spray or Azolla extract as drench addition (M4, M5 or M2,respectively). On the other hand, the second cut took the same line as the first cut but with 50% NPK + 5000 kg h-1 (T3) as either garlic extract or Azolla extract as drench addition (T3×M4 or T3×M2) as registered the highest values of parameters mention before in most cases. As for the highest values of stevioside and total stevioside content were recorded by 50% NPK + 3000 kg h-1 combining with Azolla aqueous extract as drench addition (T4×M2). Rebaudioside A., registered by combination of the recommended dose of biogas fertilizer and yucca aqueous extract as a foliar spray (T2×M5). Conclusively, the use of half (50%) of the recommended dose of chemical fertilizer with its equivalent of the recommended nitrogen dose and replaced with biogas fertilizer, in addition to the use of both extracts of Azolla and garlic as a drench addition, led to reducing nitrate accumulation as a problem of the use of chemical fertilizers on the human health and economic damages.

In this study, we hypothesize that flavor compounds, volatile as well as non-volatile, can be transferred from the oral cavity to the nasal cavity in the form of aerosol particles. To test the potential of aerosol as the carrier of flavor compounds during a drinking process, the aerosol particles of sweetener solutions (sucrose or rebaudioside A) have been quantified at both the in situ and simulated conditions, with or without saliva mixing. As observed by an optical particle counter, shaking or splashing of test samples can generate huge numbers (1000–10000) of aerosol particles with most of them smaller than 1 μm. It was further observed that addition of sucrose, rebaudioside A, saliva, thickener (konjac glucomannan) and defoamer (simethicone emulsion) can significantly alter the generated aerosol particles of water both in situ and in vitro. As estimated by the intensity of fluorescence, the amount of transferred mass within the in vitro prepared aerosol can be 4.9 μg/min. The shear viscosities, surface tension, extensional break time and relaxation time were found to correlate with the aerosol generation of test sample. These results suggest that aerosol particles have the potential in carrying substantial amount of flavor compounds during oral processing, and their generation can be sensitively affected by the presence of food components and saliva. [Display omitted] • Aerosol particles can be generated during oral processing. • Aerosol particles function as an independent mechanism of oral-nasal mass transfer. • Taste compounds can be transferred from the oral cavity to nasal cavity during oral processing. • Generation and particle size distribution of aerosol particles during oral processing influenced by a number of factors. [ABSTRACT FROM AUTHOR]

Owing to zero-calorie, high-intensity sweetness and good taste profile, the plant-derived sweetener rebaudioside D (Reb D) has attracted great interest to replace sugars. However, low content of Reb D in stevia rebaudiana Bertoni as well as low soluble expression and enzymatic activity of plant-derived glycosyltransferase in Reb D preparation restrict its commercial usage. To address these problems, a novel glycosyltransferase YojK from Bacillus subtilis 168 with the ability to glycosylate Reb A to produce Reb D was identified. Then, structure-guided engineering was performed after solving its crystal structure. A variant YojK-I241T/G327N with 7.35-fold increase of the catalytic activity was obtained, which allowed to produce Reb D on a scale preparation with a great yield of 91.29%. Moreover, based on the results from molecular docking and molecular dynamics simulations, the improvement of enzymatic activity of YojK-I241T/G327N was ascribed to the formation of new hydrogen bonds between the enzyme and substrate or uridine diphosphate glucose. Therefore, this study provides an engineered bacterial glycosyltransferase YojK-I241T/G327N with high solubility and catalytic efficiency for potential industrial scale-production of Reb D.

BACKGROUND Rebaudioside A (RA) and its monoglucosyl derivative, as like rebaudioside D (RD) are the most popular stevia glycosides but possess poor solubility in water, which limited their application as edible surfactants, the applications as in micellar solubilization and drug delivery. Meanwhile, effect of the monoglucosyl attached to RA moiety remains unclear. RESULTS: Monoglucosyl rebaudioside A (RAG1) was synthesized via hydrolyzing the transglycosylation product of RA with 95% of RA converted. RAG1 content in raw reaction mixture was as high as 69.5% of total glycosides, and harvested with a content of 88.2% by simple filtration. The RAG1 exhibited an aqueous solubility of 87 folds of RA or 391 folds of RD at 25 °C. The surface activity of RAG1 solution was higher than RA and invincible to RD. The RAG1 micelles promoted aqueous solubility of idebenone (IDE) up to 500 folds higher at 25 °C. The cumulative release rate of IDE encapsulated in RAG1 micelles was 777.5% or 456.7% higher of that of free IDE in simulated gastric/intestinal fluids in 14 h, respectively. The RAG1‐IDE remained the same in 98 days at 25 °C. CONCLUSION: The α‐linked glucosyl to RA induced higher hydrophilicity and surface activity than that resulted by β‐linked glucosyl, making RAG1 not only dramatically raise the aqueous solubility of RA, but also endow IDE folds higher in bioaccessibility, yet making the capsule stable at storage. The results would provide a new edible delivery nanocarrier for encapsulation of hydrophobic bioactive components. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana Bertoni is famous for sweetness due to the content of steviol glycosides (SG). This study aimed to determine the effect of SG (stevioside, rebaudioside A) and steviol on adipogenesis and lipogenesis in the 3T3-L1 model. Test compounds were also analysed for their ability to affect glucose uptake in hypertrophied insulin-resistant 3T3-L1 adipocytes. The most active compound in modulating adipogenesis, lipogenesis and insulin resistance was steviol, SG metabolism product. Steviol (10 μM, 100 μM) significantly down-regulated the expression of adipogenic transcription factors (PPARγ, C/EBPα, SREBP1) and lipogenic genes (FAS, aP2, LPL), which caused decreased lipid accumulation and triglyceride content in adipocytes. Treatment of insulin-resistant adipocytes with steviol (1 μM) and stevioside (1 μM, 10 μM) increased GLUT-4 transcript level and improved glucose uptake. Steviol also lowered resistin gene expression, which may mitigate insulin resistance in hypertrophied adipocytes. This study promotes SG as potential health-promoting ingredients in low-calorie functional foods.

Uridine diphosphate glycosyltransferases (UGTs) as fine catalysts of glycosylation are increasingly used in the synthesis of natural products. Sucrose synthase (SuSy) is recognized as a powerful tool for in situ regenerating sugar donors for the UGT-catalyzed reaction. It is crucial to select the appropriate SuSy for cooperation with UGT in a suitable way. In the present study, eukaryotic SuSy from Arabidopsisthaliana (AtSUS1) helped stevia glycosyltransferase UGT76G1 achieve the complete conversion of stevioside (30 g/L) into rebaudioside A (RebA). Position of the individual transcription units containing the genes encoding AtSUS1 and UGT76G1 in the expression plasmid has an effect, but less than that of the fusion order of these genes on RebA yield. Fusion of the C-terminal of AtSUS1 and the N-terminal of UGT76G1 with rigid linkers are conducive to maintaining enzyme activities. When the same fusion strategy was applied to a L637M-T640V double mutant of prokaryotic SuSy from Acidithiobacillus caldus (AcSuSym), 18.8 ± 0.6 g/L RebA (a yield of 78.2%) was accumulated in the reaction mixture catalyzed by the fusion protein Acm-R3-76G1 (the C-terminal of AcSuSym and the N-terminal of UGT76G1 were linked with (EAAAK)3). This work would hopefully reveal the potential of UGT-SuSy fusion in improving the cascade enzymatic glycosylation. [ABSTRACT FROM AUTHOR]

In order to expand the types of sweeteners used for cigarette tipping paper to include natural flavors, the application technology of a natural sweetener rebaudioside A on cigarette tipping paper was evaluated and the formulation of water-based ink and the fully coating technique of tipping paper were studied in order to keep the current style of tipping paper with sweet taste unchanged. The physical properties and hygienic indexes of finished cigarette tipping paper were tested, and the sensory evaluation of finished cigarette tipping paper and finished cigarettes were conducted. The results showed that: 1) Rebaudioside A had the advantages of high sweetness and good taste quality and was suitable for cigarette tipping paper. 2) Adopting fully coating technique effectively avoided the color difference issue and ensured the machine adaptability of the tipping paper. 3) The water-based ink formula containing rebaudioside A with the mass ratio of citric acid:rebaudioside A: water-based ink of 1:20:1 000 effectively improved the taste and met the production needs. 4) The finished tipping paper met the technical requirements in terms of physical indexes, hygiene indexes and the requirements of machine adaptability and consumer experience. The overall sensory quality of the finished cigarette samples featured a purer sweet taste, deep aftertaste, and prominent sweet scent. [ABSTRACT FROM AUTHOR]

BACKGROUND: Stevia rebaudiana is a high value crop due to the strong commercial demand for its metabolites (steviol glycosides) but has limited geographical cultivation range. In non‐native environments with different daylength and light quality, Stevia has low germination rates and early flowering resulting in lower biomass and poor yield of the desired metabolites. In this study, artificial lighting with light‐emitting diodes (LEDs) was used to determine if different light quality within and outside of the photosynthetically active radiation (PAR) range can be used to improve germination rates and yields for production of steviol glycosides for the herbal supplement and food industry. RESULTS: Plants treated with red and blue light at an intensity of 130 μmol m−2 s−1 supplemented with 5% of UV‐A light under a 16‐h photoperiod produced the most desirable overall results with a high rate of germination, low percentage of early flowering, and high yields of dry leaf, stevioside and rebaudioside A, 175 days after planting. CONCLUSION: While red and blue light combinations are effective for plant growth, the use of supplemental non‐PAR irradiation of UV‐A wavelength significantly and desirably delayed flowering, enhanced germination, biomass, rebaudioside A and stevioside yields, while supplemental green light improved yield of biomass and rebaudioside A, but not stevioside. Overall, the combination of red, blue and UV‐A light resulted in the best overall productivity for Stevia rebaudiana. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana is a sweet herbaceous perennial plant, which is frequently used in the preparation of plant‐based sweeteners. The demand for such sweeteners continues to increase due to purposeful nutrition and modern‐day metabolic syndromes. More than 20 types of steviol glycosides provide a sweet taste, which are more than 300 times sweeter than sucrose. They are formed of two main components, namely stevioside and rebaudioside A. Only a handful of studies have dealt with Stevia rebaudiana leaf extracts, the conversion of pure stevioside into the preferred rebaudioside A is more common. The aim of this study was to enrich the rebaudioside A content of Stevia rebaudiana leaf extract using enzymatic bioconversion by applying fermented cyclodextrin glycosyltransferase from Bacillus licheniformis DSM13. Two differently processed plant materials, namely dried and lyophilized Stevia rebaudiana plants, were extracted and compared. Following the bioconversion, the rebaudioside A content was on average doubled. The maximum increase was fivefold with a 70–80% conversion of the stevioside. [ABSTRACT FROM AUTHOR]

A comprehensive utilization of rebaudioside A (RA), stevioside (ST), and rebaudioside C (RC) from natural stevia resources was proposed. The influence of the solvent, solvent concentration, solid-liquid ratio, temperature, and time on the purity and recovery were investigated with response surface methodology. A 99% purity and 81% recovery of the RA were achieved by one crystallization of crude stevia under optimized conditions. Around a 95% purity and 80% recovery of the ST were easily achieved by the recrystallization of less value of mother liquor sugar (MLS) with a certain concentration of isopropyl alcohol-methanol aqueous solution. During the crystallization of the ST, the enriched RC in the liquid phase was more than three times higher than that in the crude stevia, which can provide cheaper RC raw material for the subsequent preparation of very expensive and high-purity RC. [ABSTRACT FROM AUTHOR]

Saccharin sodium and rebaudioside A are extensively used as non-nutritive sweeteners (NNSs) in daily life. NNSs elicit a multitude of endocrine influences on animals, differing across species and chemically distinct sweeteners, whose exposure induce activation of sweet taste receptors in oral and extra-oral tissues with consequences of metabolic changes. To evaluate the influence of NNSs on histologic change and expression of sweet taste receptors in testis and epididymis of young male guinea pigs, thirty 4-week-old male guinea pigs with body weight 245.73 ± 6.02 g were randomly divided into five groups (n = 6) and received normal water (control group) and equivalent sweetness low dose or high dose of sodium saccharin (L-SS, 1.5 mM or H-SS, 7.5 mM) or rebaudioside A (L-RA, 0.5 mM or H-RA, 2.5 mM) solution for 28 consecutive days. The results showed that the relative testis weight in male guinea pig with age of 56 days represented no significant difference among all groups; in spite of heavier body weight in L-SS and H-RA, NNS contributes no significant influence on serum testosterone and estradiol level. Low-dose 0.5 mM rebaudioside A enhanced testicular and epididymal functions by elevating the expressions of taste receptor 1 subunit 2 (T1R2) and gustducin α-subunit (GNAT3), and high-dose 7.5 mM sodium saccharin exerted adverse morphologic influences on testis and epididymis with no effect on the expression of T1R2, taste receptor 1 subunit 2 (T1R3), and GNAT3. In conclusion, these findings suggest that a high dose of sodium saccharin has potential adverse biologic effects on the testes and epididymis, while rebaudioside A is a potential steroidogenic sweetener for enhancing reproductive functions.

Herein, we report on the performance of graphene oxide (GOx) and zinc oxide nanoparticles (ZnONPs) on a platinum (Pt) electrode, immobilized with the human T1R2 sweet taste receptor subunit for the detection of rebaudioside A (Reb-A). The characterization studies performed in this work confirmed the thin-layered structure of GOx and the polydispersed nature of ZnONPs. The elucidation of the mass loss observed by TGA demonstrates the stability of GOx. The cyclic voltammetry results for Pt/GOx revealed good catalytic activity over Pt/ZnONPs for adsorption of the T1R2-Reb-A complex. In addition, a series of computational modelling studies were carried out to better understand the surface adsorption phenomena of GOx and ZnONPs to mimic the layer-by-layer electrode modification strategies independently. The strongest interaction energy observed (−573 kcal mol−1) for the direct interaction of ZnONPs onto the Pt electrode surface, demonstrates a stronger adsorption in contrast to the GOx modified Pt electrode (−23 kcal mol−1). However, the overall results for the layered-nanocomposite revealed that the GOx (−256 kcal mol−1) were more strongly adsorbed in contrast to ZnONPs (−231 kcal mol−1) for the detection of the T1R2-ReB-A complex, demonstrating the reliability of our GOx electrode functionalization strategy. The results of this study can potentially be used to improve the design of rapid Reb-A sensors for the food and beverage industry. [ABSTRACT FROM AUTHOR]

Background: Rebaudioside A is one of the major diterpene glycosides found in Stevia had been reported to possess anti-hyperlipidemic effects. In this study, we explore the potential cholesterol-regulating mechanisms of Rebaudioside A in the human hepatoma (HepG2) cell line in comparison with simvastatin. Methods: Cells were incubated with Rebaudioside A at several concentrations (0-10 μM) to determine the cytotoxicity by the MTT assay. Cells were treated with selected dosage (1 and 5 μM) in further experiments. Total cellular lipid was extracted by Bligh and Dyer method and subjected to quantitative colorimetric assay. To illustrate the effect of Rebaudioside A on cellular lipid droplets and low-density lipoprotein receptors, treated cells were subjected to immunofluorescence microscopy. Finally, we investigated the expression of experimental gene patterns of cells in response to treatment. Results: In this study, cytotoxicity of Rebaudioside A was determined at 27.72 μM. Treatment of cells with a higher concentration of Rebaudioside A promotes better hepatocellular cholesterol internalization and ameliorates cholesterol-regulating genes such as HMGCR, LDLR, and ACAT2. Conclusions: In conclusion, our data demonstrated that Rebaudioside A is capable to regulate cholesterol levels in HepG2 cells. Hence, we proposed that Rebaudioside A offers a potential alternative to statins for atherosclerosis therapy. [ABSTRACT FROM AUTHOR]

Diabetes mellitus, a metabolic disorder rapidly spreading worldwide affects multiple organs and body functions. Stevia rebaudiana (Bertoni), belonging to the Asteraceae family is a herb with medicinal and commercial importance to cure chronic diseases like diabetes mellitus. The present study was aimed to explore the antidiabetic effect of two reference standards like Steviol Glycosides System Suitability (SGSS, a mixture of nine steviol glycosides) and Rebaudioside A in alloxan-induced diabetic rats. In this regard, diabetes was induced in rats by administration of a single intraperitoneal (i.p.) injection of alloxan monohydrate (150 mg/kg body weight). After exposure to alloxan monohydrate, the overall intracellular antioxidant functions get disturbed which significantly increase the blood glucose levels, plasma malondialdehyde and sialic acid content. The oral administration of SGSS and Reb A at a dose of (20 and 30 mg/kg b.wt.) for a period of 21 days to diabetic rats significantly (P >0.05) reduced the blood glucose levels when compared with alloxan-induced diabetic control (DC) rats of group II and restore the antioxidant potential by decreasing the oxidative damage and also normalizes hyperlipidemic indices. The study demonstrated that bioactive components (steviol glycosides) of S. rebaudiana have potential therapeutic prospects to attenuate diabetes and its related complications. [ABSTRACT FROM AUTHOR]

The leaves of the stevia (Stevia rebaudiana Bertoni) plant, a perennial plant from the Asteraceae family, contain glycosides such as Stevioside, Rebaudioside A, B, C, D, E and Dulcoside A, which are sweeter than sucrose. However, among these glycosides, Stevioside (Stv) and Rebaudioside A (Reb A) give the plant the main sweetening properties. Especially, the higher the amount of Reb A in the leaves, the higher the sweetener quality of the products. This sweetener is known to be 200-300 times sweeter than sugar. Since the sweetener obtained from the stevia does not need insulin secretion during digestion, it can be used by diabetics. It is also preferred in diet products due to its zero-calorie feature. Adaptation studies carried out in Antalya conditions have shown that stevia can be cultivated perennially. It is important to know the change of Reb A amount and Reb A / Stv ratio, which are very important for the sweetener quality of the stevia leaves, according to the plant's development period (ontogenetic) and the position of the leaves in the plant. In order to determine the most suitable harvest time, trial samples were taken on 20 July, 5 August, 20 August, 5 September and 20 September 2020 and the amounts of Reb A and Stv were determined. Also, Reb A and Stv analyses were performed in the lower, middle and upper leaf samples on the same plant. It is found that the 4th harvest time (September 5th) is superior to other harvest times in terms of both high leaf yield and high SGs amount in Antalya conditions. At this harvest date, Reb A / Stv ratio, which is important for leaf quality, was found to be high as well. On the other hand, the change in Reb A / Stv ratios in lower, middle and upper leaves at different harvest times was found to be significant. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana Bertoni is a plant cultivated worldwide due to its use as a sweetener. The sweet taste of stevia is attributed to its numerous steviol glycosides, however, their use is still limited, due to their bitter aftertaste. The transglycosylation of steviol glycosides, aiming at the improvement of their taste, has been reported for many enzymes, however, glycosyl hydrolases are not extensively studied in this respect. In the present study, a β-glucosidase, MtBgl3a, and a β-galactosidase, TtbGal1, have been applied in the transglycosylation of two steviol glycosides, stevioside and rebaudioside A. The maximum conversion yields were 34.6 and 33.1% for stevioside, while 25.6 and 37.6% were obtained for rebaudioside A conversion by MtBgl3a and TtbGal1, respectively. Low-cost industrial byproducts were employed as sugar donors, such as cellulose hydrolyzate and acid whey for TtbGal1- and MtBgl3a- mediated bioconversion, respectively. LC-HRMS analysis identified the formation of mono- and di- glycosylated products from stevioside and rebaudioside A. Overall, the results of the present work indicate that both biocatalysts can be exploited for the design of a cost-effective process for the modification of steviol glycosides.

Steviol glycosides are well‐known food sweeteners; their consumption has steadily increased over time. A pretreatment method was developed and validated to better separate rebaudioside A and stevioside from various protein‐rich and fatty foods for quantification. This method was applied to soy sauce in liquid type and fish cake and coffee in solid type. Parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision were calculated. Calibration curves were linear in the working range of 5–100 mg/l, with coefficients of determination ≥0.99. The LOD and LOQ were in the ranges of 0.16–0.39 and 0.52–1.28 mg/kg, respectively. The percentage recoveries of the fortified samples were in the 88.01%–103.09% range, and the relative standard deviation was <10%. Method validation predicted a desirable accuracy, linearity, and precision. Therefore, the developed method can be practically applied for the quantitation of steviol glycosides in various foods, including soy sauce in liquid type and fish cake and coffee in solid type. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana (Bert.) produces steviol glycoside sweeteners that are sweeter than sucrose. Stevioside and rebaudioside A are important steviol glycosides used in food production. This study was conducted to investigate the effect of different concentrations of cadmium chloride (0, 20, 40, 60 and 80 mg/L) and silver nitrate (0, 15, 30, 45 and 60 mg/L) and different sampling times on stevioside and rebaudioside A production and Ent-kaurenoic acid 13-hydroxylase, uridine diphosphate-glycosyltransferase 74G1 and uridine diphosphate-glycosyltransferase 76G1 genes expression in in vitro conditions. The results showed that the application of cadmium nitrate and silver nitrate decreased stevioside and rebaudioside A production. Among various concentrations of cadmium chloride, the highest concentrations of stevioside and rebaudioside A were respectively 39.76 mg/g and 2.27 mg/L dry weight 96 and 72 h after elicitation with 20 mg/L cadmium chloride. Among different concentrations of silver nitrate, the highest amounts of stevioside and rebaudioside A were respectively 33.33 mg/g dry weight was 2.19 mg/L obtained at control condition after 96 h and elicitation with 60 mg/L for 24 h. In elicitation with cadmium chloride the expression all three genes were increased but silver nitrate elicitation increased uridine diphosphate-glycosyltransferase 76G1 gene expression and decreased the Ent-kaurenoic acid 13-hydroxylase and uridine diphosphateglycosyltransferase 74G1 genes expression. The correlation analysis showed that Ent-kaurenoic acid 13-hydroxylase gene expression was not correlated with uridine diphosphate-glycosyltransferase 74G1 and uridine diphosphate-glycosyltransferase 76G1 genes expression, while uridine diphosphateglycosyltransferase 74G1 was inversely correlated with uridine diphosphate-glycosyltransferase 76G1. Also, positive correlations were observed between uridine diphosphate-glycosyltransferase 74G1 gene expression with stevioside synthesis and uridine diphosphate-glycosyltransferase 76G1 gene expression with rebaudioside A. [ABSTRACT FROM AUTHOR]

Nimodipine (NMD), a calcium channel blocker, has demonstrated benefits in treating glaucoma. However, its ocular therapeutic application remains limited due to its poor aqueous solubility, which restrains the development of an ophthalmic formulation. Thus, the present study aimed to formulate an NMD micelle ophthalmic solution to enhance the potential of NMD in an ocular topical formulation to treat glaucoma. The NMD micelle ophthalmic solution was formulated with nanocarriers composed of rebaudioside A and d-α-tocopheryl polyethylene glycol 1000 succinate. Spherical mixed micelles were optimized and obtained at a small micelle size 13.429 ± 0.181 nm with a narrow size distribution (polydispersity index 0.166 ± 0.023) and high encapsulation efficiency rate (99.59 ± 0.09%). Compared with free NMD, NMD in micelles had much greater in vitro membrane permeability and antioxidant activity. The NMD micelle ophthalmic solution was well tolerated in rabbit eyes. It profoundly improved the in vivo intraocular permeation of NMD, and in vivo intraocular pressure reduction and improved miosis were also observed. Accordingly, this NMD micelle ophthalmic solution might be a promising ocular formulation to treat glaucoma. [ABSTRACT FROM AUTHOR]

Plants under different environmental regimes exhibit phenotypic plasticity, sometimes producing more secondary metabolites when microenvironmental conditions are manipulated but these responses may be species, cultivar and/or genotype dependent. To test the hypothesis of whether in vitro plants of S. rebaudiana Bertoni would accumulate higher amounts of steviol glycosides when plants were growing under salt and drought stress, cultivar ST2100 plants were used. We thus applied 25 to 100 mM NaCl and polyethylene glycol 6000 (PEG) at 2.5% to 10.0% (w/v) to generate different Murashige and Skoog (Physiol Plant 15:473–497, 1962) media. Microplant cultures were also profiled for stevioside, rebaudioside A and steviol via LC–MS. Essential oil chemicals and fatty acids were assessed using GC–MS. Finally, a chemometric analysis of ethanolic extracts produced from treated and control plants is presented from MSE fragmentation data and various phenolic acids were tentatively identified using ion fragmentation patterns. Increasing amounts of both NaCl and PEG led to poor growth and development in cultures of S. rebaudiana. For example, the 25 and 50 mM NaCl-treated plants had fewer roots in comparison to controls and at even higher concentrations (75 and 100 mM NaCl), plants did not to root. Poor in vitro organogenesis was more pronounced with PEG. For instance, when plants were placed on a 10% PEG-medium, the ability for shoot regeneration was lost and callus became more apparent. Increasing levels of NaCl and PEG were also correlated to lowered levels of rebaudioside A and stevioside. In relation to the control plants that had 0.054 mg g−1 FW of steviol, the 25 mM NaCl treatment group had highest levels of this compound, recorded at 0.156 mg g−1 FW. All other salt treatments led to trace amounts of this chemical (0.005–0.009 mg g−1 FW) and it was not detected in any of the PEG-treated plants, except for the controls. The PCA loadings plots exposed stevioside, rebaudioside E and a steviol glycoside derivative as the MS signals that contributed to discriminant clusters segregating controls from the NaCl-treated groups. For PEG, segregation in the PCA is mostly influenced by dicaffeoylquinic acid as a marker ion, separating the controls from the treatment groups. PEG-treatments caused more prominent changes to the essential oil chemistry of Stevia plants. This was evident when 7.5 or 10% PEG was applied as sabinene, α-terpinolene, n-amyl isovalerate, 7-octen-4-ol, α-bergamotene, junipene, (+)-calarene, α-cadinol, β-pinene, α-bergamotene, (+)-calarene and junipene became undetectable. Changes of this nature may be undesirable when aromatic oils of S. rebaudiana are targetted for commercial markets as our data suggest adjustment to stresses may negatively impact volatile compounds leading to a loss of bioactive aromatic compounds. This study reports, for the first time, the effects of salinity and drought conditions in vitro on changed essential oil profiles of S. rebaudiana, providing new insights into the effects of stress on the essential oil chemistry of S. rebaudiana. Key message: Stevia responses to salt and drought stresses in vitro lead to lowered measured steviol glycosides (i.e stevioside, rebaudioside A and steviol) and significantly changes the essential oil terpenoid profiles [ABSTRACT FROM AUTHOR]