Identification of a 301 bp promoter core region of the SrUGT91D2 gene from Stevia rebaudiana that contributes to hormone and abiotic stress inducibility.

BACKGROUND: The UDP-glucuronosyltransferase 91D2 (SrUGT91D2) gene is a crucial element in the biosynthetic pathway of steviol glycosides (SGs) and is responsible for creating 1,2-ß-D glucosidic bonds at the C19 and C13 positions. This process plays a vital role in the synthesis of rebaudioside M (RM) and rebaudioside D (RD). The promoter, which regulates gene expression, requires functional analysis to understand gene expression regulation. However, investigations into the function of the promoter of SrUGT91D2 (pSrUGT91D2) have not been reported. RESULTS: The pSrUGT91D2 was isolated from six S. rebaudiana lines, and subsequent multiple sequence comparisons revealed the presence of a 26 bp inDel fragment (pSrUGT91D2-B1188 type) in lines GP, GX, 110, 1114, and B1188 but not in the pSrUGT91D2 of line 023 (pSrUGT91D2-023 type). Bioinformatics analysis revealed a prevalence of significant cis-regulatory elements (CREs) within the promoter sequences, including those responsive to abscisic acid, light, anaerobic conditions, auxin, drought, low temperature, and MeJA. To verify the activity of pSrUGT91D2, the full-length promoter and a series of 5' deletion fragments (P1-P7) and a 3' deletion fragment (P8) from various lines were fused with the reporter ß-glucuronidase (GUS) gene to construct the plant expression vector, pCAMBIA1300-pro∷GUS. The transcriptional activity of these genes was examined in tobacco leaves through transient transformation. GUS tissue staining analysis and enzyme activity assays demonstrated that both the full-length promoter and truncated pSrUGT91D2 were capable of initiating GUS expression in tobacco leaves. Interestingly, P8-pSrUGT91D2-B1188 (containing the inDel segment, 301 bp) exhibited enhanced activity in driving GUS gene expression. Transient expression studies of P8-pSrUGT91D2-B1188 and P8-pSrUGT91D2-023 in response to exogenous hormones (abscisic acid and indole-3-acetic acid) and light indicated the necessity of the inDel region for P8 to exhibit transcriptional activity, as it displayed strong responsiveness to abscisic acid (ABA), indole-3-acetic acid (IAA), and light induction. CONCLUSIONS: These findings contribute to a deeper understanding of the regulatory mechanism of the upstream region of the SrUGT91D2 gene and provide a theoretical basis for future studies on the interaction between CREs of pSrUGT91D2 and related transcription factors.

Nanocurcumin-Based Sugar-Free Formulation: Development and Impact on Diabetes and Oxidative Stress Reduction.

The objective of this study is the development of innovative nanocurcumin-based formulations designed for the treatment and prevention of oxidative stress and diabetes. Nanocurcumin was obtained through a micronization process and subsequently encapsulated within biopolymers derived from corn starch and fenugreek mucilage, achieving encapsulation rates of 75% and 85%, respectively. Subsequently, the encapsulated nanocurcumin was utilized in the formulation of sugar-free syrups based on Stevia rebaudiana Bertoni. The stability of the resulting formulations was assessed by monitoring particle size distribution and zeta potential over a 25-day period. Dynamic light scattering (DLS) revealed a particle size of 119.9 nm for the fenugreek mucilage-based syrup (CURF) and 117 nm for the corn starch-based syrup (CURA), with polydispersity indices PDIs of 0.509 and 0.495, respectively. The dissolution rates of the encapsulated nanocurcumin were significantly enhanced, showing a 67% improvement in CURA and a 70% enhancement in CURF compared with crude curcumin (12.82%). Both formulations demonstrated excellent antioxidant activity, as evidenced by polyphenol quantification using the 2.2-diphenyl 1-pycrilhydrazyl (DPPH) assay. In the evaluation of antidiabetic activity conducted on Wistar rats, a substantial reduction in fasting blood sugar levels from 392 to 187 mg/mL was observed. The antioxidant properties of CURF in reducing oxidative stress were clearly demonstrated by a macroscopic observation of the rats' livers, including their color and appearance.

Ameliorative effect of Stevia rebaudiana Bertoni on sperm parameters, in vitro fertilization, and early embryo development in a streptozotocin-induced mouse model of diabetes.

Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.

Cold Plasma-Induced Changes in Stevia rebaudiana Morphometric and Biochemical Parameter Correlations.

Stevia rebaudiana Bertoni is an economically important source of natural low-calorie sweeteners, steviol glycosides (SGs), with stevioside (Stev) and rebaudioside A (RebA) being the most abundant. Pre-sowing seed treatment with cold plasma (CP) was shown to stimulate SGs biosynthesis/accumulation up to several fold. This study aimed to evaluate the possibility to predict CP-induced biochemical changes in plants from morphometric parameters. Principle component analysis (PCA) was applied to two different sets of data: morphometric parameters versus SGs concentrations and ratio, and morphometric parameters versus other secondary metabolites (total phenolic content (TPC), total flavonoid content (TFC)) and antioxidant activity (AA). Seeds were treated for 2, 5 and 7 min with CP (CP2, CP5 and CP7 groups) before sowing. CP treatment stimulated SGs production. CP5 induced the highest increase of RebA, Stev and RebA+Stev concentrations (2.5-, 1.6-, and 1.8-fold, respectively). CP did not affect TPC, TFC or AA and had a duration-dependent tendency to decrease leaf dry mass and plant height. The correlation analysis of individual plant traits revealed that at least one morphometric parameter negatively correlates with Stev orRebA+Stev concentration after CP treatment.

A New Substrate and Nitrogen Source for Traditional Kombucha Beverage: Stevia rebaudiana Leaves.

Recently, the use of different herbal products as carbon sources instead of black and green tea in the preparation of traditional kombucha has been investigated. In this study, functional kombucha was prepared by adding Stevia rebaudiana Bertoni leaves, which have special organoleptic properties, to kombucha medium, and some properties of the beverage were analyzed. Tea blends were determined as 100% green tea (control = C), 75% green tea (GT) + 25% Stevia (ST), 50% GT + 50% ST, and 100% ST. On the 15th day of fermentation, gluconic acid (43.12 ± 0.01 g/L) was detected as dominant organic acid in GT75 + ST25 samples compared to group C (p < 0.05). According to physicochemical parameters that determine the drinkability properties of prepared teas, the best results were in GT25 + ST75 compared to group C (p < 0.05). It proved that the highest activity was in GT25 + ST75 on the 10th day in the groups that applied different antioxidant tests (DPPH, MCA, and CUPRAC). The antimicrobial activities of kombucha at 25, 50, 75, and 100% concentrations of GT and ST reached the highest levels in the GT25 + ST75 group in samples after 10 days of fermentation for all selected microorganisms. The results prove that GT25 + ST75 kombucha is a functional product with high drinkability on the 10th day of fermentation and also more beneficial for health due to the phenolic compounds from both green tea and Stevia. Stevia rebaudiana leaves can be suggested that be used as a new substrate and nitrogen source for kombucha production.

Lelliottia steviae sp. nov. isolated from Stevia rebaudiana Bertoni.

A Gram-negative, aerobic, chemoheterotrophic, rod-shaped, and motile bacterium, designated as LST-1T, was isolated from wild Stevia rebaudiana Bertoni and subjected to a polyphasic taxonomic analysis. The LST-1 strain grew optimally at 37 °C and pH 6.0-7.0 in the presence of 0.5% (w/v) NaCl. Phylogenetic analysis based on the 16S rDNA sequence indicated that LST-1 is closely related to Lelliottia jeotgali PFL01T (99.85%), Lelliottia nimipressuralis LMG10245T (98.82%), and Lelliottia amnigena LMG2784T (98.54%). Multi-locus sequence typing of concatenated partial atpD, infB, gyrB, and rpoB genes was performed to improve the resolution, and clear distinctions between the closest related type strains were observed. The results of average nucleotide identify analyses and DNA-DNA hybridization with four species (16S rDNA similarity > 98.65%) were less than 90 and 40%, respectively, verifying the distinct characteristics from other species of Lelliottia. The cellular fatty acid profile of the strain consisted of C16:0, Summed Feature3, and Summed Feature8 (possibly 16:1 w6c/16:1 w7c and 18:1 w6c) as major components. The major polar lipids included phosphatidylethanolamine, phosphatidylglycerol, an aminophospholipid, three non-characteristic phospholipids, and a non-characteristic lipid. The genome of LST-1T was 4,611,055 bp in size, with a G + C content of 55.02%. The unique combination of several phenotypic, chemotaxonomic, and genomic characteristics proved that strain LST-1T belongs to a novel species, for which the name Lelliottia steviae sp. nov. is proposed. The type strain is LST-1T (= CGMCC 1.19175T = JCM 34938T).Repositories: The genbank accession numbers for the 16S rRNA gene and genome sequences of strain LST-1T are MZ497264 and CP063663, respectively.

The Potential of Cold Plasma and Electromagnetic Field as Stimulators of Natural Sweeteners Biosynthesis in Stevia rebaudiana Bertoni.

Stevioside (Stev) and rebaudioside A (RebA) are the most abundant steviol glycosides (SGs) responsible for the sweetness of Stevia rabaudiana Bertoni. As compared to Stev, RebA has a higher sweetening potency, better taste and therefore is the most preferred component of the stevia leaf extracts. The aim of this study was to determine the effect of pre-sowing seed treatment with abiotic stressors cold plasma (CP) and electromagnetic field (EMF) on the amount and ratio of RebA and Stev in the leaves of stevia. Additionally, the effect on total phenolic content, flavonoid content and antioxidant activity was investigated. Seeds were treated 5 and 7 min with cold plasma (CP5 and CP7 groups) and 10 min with electromagnetic field (EMF10 group) six days before sowing. The germination tests in vitro demonstrated that all treatments slightly increased germination rate and percentage. HPLC analysis revealed that CP and EMF had strong stimulating effect on SGs accumulation. All treatments increased RebA concentration approximately 1.6-fold; however, the ratio of RebA/Stev decreased from 8.5 in the control to 1.9, 2.5 and 1.1 in CP5, CP7 and EMF10 groups respectively, since the concentration of Stev increased more than RebA, 7.1, 4.6 and 11.0-fold, respectively, compared to control. However, treatments had opposite effect on total phenolic content, flavonoid content, and antioxidant activity. We have demonstrated for the first time that short time pre-sowing treatment of stevia seeds with CP and EMF can be a powerful tool for the enhancement of biosynthesis of RebA and Stev, however it can have negative impact on the content of other secondary metabolites.

UHPLC coupled with charged aerosol detector for rapid separation of steviol glycosides in commercial sweeteners and extract of Stevia rebaudiana.

Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The Luna Omega Polar column featuring polar modification of the C18 stationary phase was employed with mobile phases composed of water and acetonitrile for the excellent separation of polar steviol glycosides. The flow rate of the mobile phase 0.35 mL/min, column temperature 50 °C and injection volume 2 µL were used. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6-105.0% and 93.8-111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0-12.3% higher than declared. The content was about 12.4% less than declared in one sample. But the difference from the labeled content corresponded to trueness and precision of the developed method together with variability of sweeteners production. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.

Descriptores sensoriales de preparaciones culinarias en base a Stevia Rebaudiana (Bertoni) de producción agroecológica mediante un grupo focal / Sensory descriptors of culinary preparations based on stevia rebaudiana (bertoni) of agroecological production through a focus groups

Fundamentos: La Stevia Rebaudiana (Bertoni) es un cultivo ancestral, cuyas hojas contienen glucósidos esteviol diterpenos que le confieren poder edulcorante, siendo factible en base aellas el desarrollo de preparaciones culinarias. Métodos: Se llevó a cabo un grupo focal con integrantes dela comunidad de la Universidad Nacional de Lanús (n = 8), con la finalidad de definir descriptores sensoriales del edulcorante y de las preparaciones con Stevia desarrolladas en etapas previasde la investigación. Estas preparaciones fueron degustadas durante el transcurso del estudio, como parte de la obtenciónde las cualidades sensoriales. El grupo focal fue registrado en formato auditivo y escrito y categorizado con posterioridad.Resultados: En base a los resultados emergentes del grupo focal se pudieron caracterizar las percepciones, impresiones y sensaciones tanto a la Stevia como a las preparaciones culinarias desarrolladas. Conclusiones: Los descriptores sensoriales obtenidos podrán ser utilizados para el fomento del consumo de Stevia y para el análisis sensorial cuantitativo de los productos. (AU)

Background: Stevia Rebaudiana (Bertoni) is an ancestral crop, whose leaves contain steviol diterpene glycosides, which give it sweetening power, being feasible based on them thedevelopment of culinary preparations.Methods: A focus group was carried out with members of the community of the National University of Lanús (n = 8), inorder to define sensory descriptors of the sweetener and preparations with Stevia developed in previous stages of research. They were tasted during the course of the study, as part ofobtaining sensory qualities. The focus group was recorded in auditory and written format and subsequently categorized. Results: Based on the emerging results focus group, it waspossible to characterize both Stevia and the culinary preparations developed from the point of view of perceptions, impressions and sensations. Conclusions: The sensory descriptors obtained can be used to promote the consumption of Stevia and for the quantitative sensory analysis of the products. (AU)

An insight on the future therapeutic application potential of Stevia rebaudiana Bertoni for atherosclerosis and cardiovascular diseases.

Stevia rebaudiana Bertoni is a native plant to Paraguay. The extracts have been used as a famous sweetening agent, and the bioactive components derived from stevia possess a broad spectrum of therapeutical potential for various illnesses. Among its medicinal benefits are anti-hypertensive, anti-tumorigenic, anti-diabetic, and anti-hyperlipidemia. Statins (3-hydro-3-methylglutaryl-coenzyme A reductase inhibitor) are a class of drugs used to treat atherosclerosis. Statins are explicitly targeting the HMG-CoA reductase, an enzyme in the rate-limiting step of cholesterol biosynthesis. Despite being widely used in regulating plasma cholesterol levels, the adverse effects of the drug are a significant concern among clinicians and patients. Hence, steviol glycosides derived from stevia have been proposed as an alternative in replacing statins. Diterpene glycosides from stevia, such as stevioside and rebaudioside A have been evaluated for their efficacy in alleviating cholesterol levels. These glycosides are a potential candidate in treating and preventing atherosclerosis provoked by circulating lipid retention in the sub-endothelial lining of the artery. The present review is an effort to integrate the pathogenesis of atherosclerosis, involvement of lipid droplets biogenesis and its associated proteins in atherogenesis, current approaches to treat atherosclerosis, and pharmacological potential of stevia in treating the disease.

Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana.

BACKGROUND: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. RESULTS: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results. CONCLUSION: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

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.

Supplementation of diets with tannins from Chestnut wood or an extract from Stevia rebaudiana Bertoni and effects on in vitro rumen fermentation, protozoa count and methane production.

The aim of the trial was to evaluate the effect of dietary additions of Stevia rebaudiana Bertoni extract (SB) and Chestnut wood tannin (CWT) on the in vitro rumen fermentability, protozoal population and methane yield. Both plant products were tested at 3 different levels of inclusion (0.75, 1.50 and 3.00% of incubated dry matter, DM) in a total mixed ration (TMR) for ruminants by using rumen batch culture systems and a rumen inoculum collected from sheep. Total volatile fatty acid concentration, their proportions and gas production were not modified by the plant extracts inclusion, except a significant linear increment of gas production at 24 hr for SB (p = .049). Ammonia concentration decreased (p < .05) of about 17% when 1.50 or 3.00% of CWT were included into TMR. Rumen protozoa population was depressed by the SB inclusion (p = .002) with a maximum reduction of 40% at the highest SB dosage, whereas CWT negatively affected total protozoa counts (-19%) only at the dose of 3.00%. In vitro DM and NDF degradability were not affected by the supplementation of SB and CWT, as well as the methane yield. Thus, the addition of SB and CWT decreases the in vitro protozoa population of the rumen with different intensity and without effects on fermentation parameters, apart from a reduction of nitrogen degradability caused by CWT. Despite the effect on protozoa, no decreasing effect on methane production was detected.

Effect of Growth Regulators on Stevia rebaudiana Bertoni Callus Genesis and Influence of Auxin and Proline to Steviol Glycosides, Phenols, Flavonoids Accumulation, and Antioxidant Activity In Vitro.

Stevia is a plant containing many active compounds, but usually propagated by stem cuttings because of low seed-yield-germination ability. The aim of this study was to investigate the impact of plant-growth regulators on stevia callus induction and growth from somatic tissue, as well as to determine the effect α-naphthalene acetic acid (NAA) and proline (PRO) on the amount of stevioside, rebaudioside A, phenols, flavonoids, and antioxidant activity. Stem and leaf segments were inoculated on a Murashige and Skoog (MS) medium supplemented with different concentrations of NAA and 6-benzylaminopurine (BAP) for callus genesis. The amount of steviol glycosides (SGs) was evaluated using high-performance liquid chromatography (HPLC), and the amounts of total phenols, flavonoids, and antioxidant activity by spectrophotometric methods. The highest callus-induction frequency and callus-mass increase were obtained from the leaf explants in MS medium supplemented with 2.0 µM NAA. The highest amount of SGs, phenols, and flavonoids, and stronger antioxidant activity were determined in the cellular compounds of callus from leaf explant. PRO reduced the amount of SGs and flavonoids. The significantly highest amount of total phenolic compounds was obtained in the callus from leaf explants in the medium supplemented with 2.0 µM NAA and 2.0 µM PRO.

Stevia rebaudiana extract attenuate metabolic disorders in diabetic rats via modulation of glucose transport and antioxidant signaling pathways and aquaporin-2 expression in two extrahepatic tissues.

Today, plant-based therapies have been attracted attention to overcome diabetes complications. This study was an attempt to evaluate whether antidiabetic and nephroprotective effects of Stevia Rebaudiana Bertoni (SRB) can be exerted via upregulation of GLUT-4, SNAP23, and Stx4 in skeletal muscles or modulation of AQP2 mRNA expression and antioxidant signaling pathway activity (Nrf2/Keap1) in kidneys. To achieve this aim, diabetes was induced via STZ-nicotinamide (STZ-NA). Diabetes increased the level of Blood Urea Nitrogen (BUN), serum creatinine, Fasting Blood Sugar (FBS), and Keap1 mRNA expression, which was coincide with reduction in mRNA levels of Nrf2, GLUT4, SNAP23, and Stx4. SRB and metformin compensate mentioned variables. However, SRB extract was more effective than metformin to increase the levels of GLUT4 and Nrf2 mRNA. It seems that SRB might attenuate the diabetic complications via manipulating the glucose uptake components in peripheral tissues and might exert the nephroprotective effects by modulation of AQP2, and Nrf2/Keap1 mRNA expression. PRACTICAL APPLICATIONS: Synthetic antidiabetic drugs have been only partially successful in controlling the diabetic complications. Moreover, use of these drugs is associated with a number of adverse effects. Over the past few years, a renewed attention has been paid to the prevention and treatment of diabetes using medicinal plants and functional foods. SRB that have been known as natural sweetener for centuries, is a such natural agent that has high source of various phytochemicals with antidiabetic, renal protective, antitumor, and antioxidant properties. In the current study, possible molecular mechanisms of insulin-mimetic and nephroprotective effects of SRB extract was evaluated in diabetic rats. Due to powerful antihyperglycemic and nephroprotective effects of SRB extract that were showed in this study and previous studies, hence the fact that SRB is to be highlighted for future research as a new therapeutic agent for diabetes.

Production of low-calorie apricot nectar sweetened with stevia: Impact on qualitative, sensory, and nutritional profiles.

This study aimed to develop a low-calorie apricot nectar by replacing sucrose with different amount of Stevia rebaudiana bertoni (Rebaudioside A, 98%). Stevia has become very popular as sweetener for the production of low-calorie products but its addition could be a challenge for industry, since it could modify sensory features of the product and consumers' acceptance. To this end, apricot nectars without sugar, with sucrose 10%, and with different amounts of stevia were produced and evaluated for microbiological quality using the pour-plate technique, and physicochemical (pH, TTA, and a w) and nutritional (moisture, fat, protein, carbohydrates, and ash) characteristics. Furthermore, a sensory analysis of the samples was performed by a panel of trained judges using quantitative descriptive analysis. The effect of stevia addiction on the consumers' acceptance was investigated by 102 consumers of fruit juices that evaluated the overall acceptability of the samples using a structured 9-point hedonic scale. Levels of microbial groups in nectars were under the detection limit confirming a good hygienic practice within the production. Nectars produced with stevia resulted in significant reduction in caloric value from 86 kcal (nectar with 10% sucrose) to 49 kcal (nectars with stevia), without altering its typicality. Different sensory profiles among samples were pointed out; all the products are liked, but with a different level of pleasantness. The study highlighted that the apricot nectars with 0.07% stevia are characterized for sweet and liquorice aroma notes and received the same level of consumer acceptability of nectars produced with 10% sucrose.

A beta-glucosidase gene from Stevia rebaudiana may be involved in the steviol glycosides catabolic pathway.

We herein report the preparation of a full-length raucaffricine-O-beta-D-glucosidase gene of stevia rebaudiana Bertoni (named SrRG1, GenBank accession number MK920450). Sequence analysis indicated SrRG1 consists of a 1650 bp open reading frame encoding a protein of 549 amino acids. Its deduced amino acid sequence showed a high identity of 82% with a raucaffricine-O-beta-D-glucosidase from H. annuus of glycoside hydrolase family 1. The expression pattern analyzed by real-time quantitative PCR showed no significant difference among different tissues, developmental stages, and cultivars under normal growth conditions. Furthermore, the gene function of SrRG1 was preliminarily studied by agrobacterium-mediated transformation on instantaneous expression. In the test of agrobacterium-mediated transformation on instantaneous expression, it was observed that overexpression of SrRG1 increased the accumulation of steviol content and decreased the major components and total SGs contents. Such results demonstrated that SrRG1 may participate in the steviol glycosides catabolic pathway. However, the effect of silencing construct infiltration on steviol and SGs content was not significant and its expression pattern was constitutive, which most probably, attributed the hydrolysis of SGs to the secondary activity of SrRG1. This study firstly identified the bate-glucosidase in stevia and advances our understanding of steviol glycosides hydrolyzation.

In vitro rumen fermentation of feed substrates added with chestnut tannins or an extract from Stevia rebaudiana Bertoni.

Rumen fermentation parameters and microbiota were evaluated in 3 in vitro rumen fermentation experiments after addition of chestnut tannins (CWT) or an extract from Stevia rebaudiana Bertoni (SB) to substrates. A control (CTR) substrate was fermented alone or added with 1.5% of CWT or SB extracts in a batch culture system (Exp. 1, fermentation in 500 mL for 24 h) and in a subsequent continuous culture system (Exp. 2, fermentation in 2 L bottles for 9 d). Experiment 3 used the fermentation system of Exp. 1 and tested 7 doses of each extract added to CTR (additions of 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2% and 1.4% for 48 h). The addition of CWT lowered (P < 0.01) the in vitro rumen ammonia concentration in all experiments and reduced the protozoa counts in Exp. 1 (P < 0.05). In contrast, the SB extract did not modify the ammonia concentrations, but significantly lowered the protozoa counts in all 3 experiments (reduction of 47% and 20% in Exp. 1 and 2, P < 0.05; and a quadratic reduction in Exp. 3, R 2 = 0.63, P < 0.01). Neither extract affected the fermentation in terms of gas production (Exp. 1 and 3) nor volatile fatty acids (VFA) yield (Exp. 1 and 2), if we exclude a reduction at the highest CWT concentration in Exp. 3. Changes in VFA profile were induced by CWT and were limited to reductions in the iso-valerate (P < 0.01, in Exp. 2) and iso-butyrate levels (P < 0.01, Exp. 2). The CWT increased the abundance of Prevotella ruminicola and Selenomonas ruminantium and decreased that of Ruminobacter amylophilus (P < 0.01, P < 0.05 and P < 0.05, respectively). The SB extract increased the relative abundance of Treponema saccarophylum (P < 0.05). Both of the studied substances had an impact on rumen metabolism, with SB reducing protozoa counts and CWT lowering the rumen ammonia concentration. The effects of both extracts on the rumen were appreciable at low dietary doses, and the negative impacts on fermentation were limited to the reduction in protein degradation with the addition of CWT.

Growth and viability of Streptococcus mutans in sucrose with different concentrations of Stevia rebaudiana Bertoni.

OBJECTIVE: To evaluate total absorbance, planktonic growth, biofilm formation, viability, metabolic activity, and pH of Streptococcus mutans UA159 cultures when different dilutions of Stevia rebaudiana Bertoni were applied and to determine the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) of Stevia on S. mutans. MATERIALS AND METHODS: The effects of different dilutions of Stevia (0-400 mg/ml) on S. mutans total growth, planktonic growth, biofilm formation, viability, metabolic activity, and pH during a 72-h growth period were evaluated in this in vitro study. A stock solution was prepared by mixing 10 ml of tryptic soy broth (TSB) supplemented with 1% sucrose (TSBS) and 4 g of Stevia. RESULTS: S. mutans total growth and biofilm formation decreased with reduced concentrations of Stevia. Furthermore, the MIC was 25 mg/ml and the MBIC was 6.25 mg/ml. Complete eradication of S. mutans was not observed with any of the Stevia concentrations. Planktonic growth of S. mutans was not repressed by high concentrations of Stevia and most of the Stevia concentrations generated an increased pH. CONCLUSION: Because Stevia reduces biofilm and acid production, Stevia can be considered a non-cariogenic sweetener. CLINICAL RELEVANCE: This study confirms the anticariogenic effect of Stevia, like it has been previously reported, but more studies on the most effective concentration are needed, and in the present study, the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) was determined in the presence of sucrose. Additionally, this is the first study to evaluate the effect of Stevia on S. mutans metabolic activity.

Stevia Rebaudiana Bertoni, a Source of High-Potency Natural Sweetener-Biochemical and Genetic Characterization.

Stevia rebaudiana is a natural sweetener herb that is increasingly used in herbal medicines in the food and cosmetics industries. Molecular methods can be combined with morphological techniques to identify stevia genotypes as a starting material to produce more reliable bioproducts. This study evaluated the level of the genetic and biochemical diversity in various stevia genotypes using HPLC (high performance liquid chromatography) analysis and random amplified polymorphic DNA (RAPD) markers. Stevia genotypes collected from different locations of the world showed clear variations at the biochemical and genetic level in Polish climate conditions. The influence of the genotypes on the content of steviol glycosides, antioxidants, phenols, flavonoids, and tannins was analyzed using phytochemical assays. Genotypes from Morocco, Poland, Egypt, and Nigeria can be defined as samples of higher quality compared to other genotypes analyzed in terms of the amount of steviol glycosides. Considering the rebaudioside A/stevioside ratio as a selection criterion, genotypes from Australia, China, India, and Pakistan should be considered to be valuable in terms of suitability for obtaining new varieties. The present results of RAPD marker analysis indicated differential banding pattern and considerable polymorphism among all ten stevia genotypes. Genotypes from Morocco, Egypt, Poland, Nigeria, China, and India, as genetically different, can be selected for further stevia breeding programs.