Purple Phototrophic Bacterium Enhances Stevioside Yield by Stevia rebaudiana Bertoni via Foliar Spray and Rhizosphere Irrigation

This study was conducted to compare the effects of foliar spray and rhizosphere irrigation with purple phototrophic bacteria (PPB) on growth and stevioside (ST) yield of Stevia. rebaudiana. The S. rebaudiana plants were treated by foliar spray, rhizosphere irrigation, and spray plus irrigation with PPB for 10 days, respectively. All treatments enhanced growth of S. rebaudiana, and the foliar method was more efficient than irrigation. Spraying combined with irrigation increased the ST yield plant ^-1 by 69.2% as compared to the control. The soil dehydrogenase activity, S. rebaudiana shoot biomass, chlorophyll content in new leaves, and soluble sugar in old leaves were affected significantly by S+I treatment, too. The PPB probably works in the rhizosphere by activating the metabolic activity of soil bacteria, and on leaves by excreting phytohormones or enhancing the activity of phyllosphere microorganisms.     

Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni

To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-O-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction I, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-O-methylsteviol, but not to iso-steviol and 13-O-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-O-glucopyranoside. Enzyme fraction II, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP-glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13, 19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-O-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-O-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A.     

Efficient micropropagation and chlorocholine chloride induced stevioside production of Stevia rebaudiana Bertoni

A promising method of micropropagation of Stevia rebaudiana Bertoni has been developed with an aim to increase the biomass, survivability of the plantlets and stevioside production, using chlorocholine chloride (CCC). Microshoots transferred to the MS medium containing different combinations CCC and IBA were found to be most effective in terms of growth pattern, hardening ability of the plantlets and stevioside content, compared to MS medium containing either IBA or CCC. Among other combinations tested, MS medium supplemented with 3 mg/l CCC and 3 mg/l IBA was found most effective in inducing significant changes like reduced shoot length, increased number of roots, higher leaf size, increased biomass and chlorophyll retaining capacity, higher survival percentage and most importantly the elevated stevioside content. Collectively, the major observations of this research indicate that application of CCC in micropropagation of S. rebaudiana Bertoni is a promising approach and has commercial prospects.     

Enrichment of the rebaudioside A concentration in Stevia rebaudiana extract with cyclodextrin glycosyltransferase from Bacillus licheniformis DSM 13

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.     

Stevioside mediated chemosensitization studies and cytotoxicity assay on breast cancer cell lines MDA-MB-231 and SKBR3

Cancer is one of the most impacting life-threatening disease for the human populace. Hence, over the years we have seen a consistent interest to study and investigate new treatments to cure and prevent this disease. Medicinal plants have played a progressive part in treatment since many years. In this research study, we have explored the cytotoxicity effect of purified bioactive compound isolated from Stevia rebaudiana leaves and the key mechanism responsible for apoptosis in human breast cancer cells. The anticancer properties of Stevia rebaudiana leaves has been suggested in earlier literature. Hence, the aim of this study was to investigate the cytotoxicity of purified stevioside in human breast cancer cell lines MDA-MB-231 and SKBR3. Results showed that purified stevioside inhibited the growth of cancerous cell lines. The IC50 obtained after treatment with stevioside on cancer cells MDA-MB-231 and SKBR3 are 55 µM and 66 µM respectively. This shows purified stevioside is capable of inducing apoptosis indicating its promising anticancer activity. However, so far chemosensitization effects of stevioside on breast cancer have not been fully explained by other studies. Hence, additionally, this study also evaluates the chemosensitization potential of stevioside in combination with 5-FU. This research study shows the importance of Stevia rebaudiana as a good source of bioactive compounds with high anti-cancer property.     

Efficient enzymatic production of rebaudioside A from stevioside

Stevioside and rebaudioside A are the chief diterpene glycosides present in the leaves of Stevia rebaudiana. Rebaudioside A imparts a desirable sweet taste, while stevioside produces a residual bitter aftertaste. Enzymatic synthesis of rebaudioside A from stevioside can increase the ratio of rebaudioside A to stevioside in steviol glycoside products, providing a conceivable strategy to improve the organoleptic properties of steviol glycoside products. Here, we demonstrate the efficient conversion of stevioside to rebaudioside A by coupling the activities of recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana and sucrose synthase AtSUS1 from Arabidopsis thaliana. The conversion occurred via regeneration of UDP-glucose by AtSUS1. UDP was applicable as the initial material instead of UDP-glucose for UDP-glucose recycling. The amount of UDP could be greatly reduced in the reaction mixture. Rebaudioside A yield in 30?h with 2.4?mM stevioside, 7.2?mM sucrose, and 0.006?mM UDP was 78%.     

CuO nanoparticles significantly influence in vitro culture,steviol glycosides,and antioxidant activities of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni

Copper oxide (CuO) nanoparticles (NPs) synthesized through co-precipitation method were employed in MS media during in vitro culture of Stevia rebaudiana. Physiological characteristics, production of steviol glycosides, and antioxidative parameters were investigated in regenerated plants. CuO NPs had crystalline monoclinic cubic cuprous oxides with average size 47 nm. The NPs were applied at 0, 0.1, 1.0, 10, 100 and 1000 mg/L in MS media for direct organogenesis of S. rebaudiana from nodal segments. Shoot organogenesis was found highest (88.5%) at 10 mg/L CuO and average shoot length, mean number of shoot per explant, and fresh weight were also found significantly higher at the same concentration. High performance liquid chromatography (HPLC) illustrated significant rise of bioactive major steviol glycosides (rebaudioside A and stevioside) at 10 mg/L CuO NPs in MS media. The oxidative stress produced by CuO nanoparticles on S. rebaudiana was affirmed by antioxidant activities i.e. total antioxidant activity (TAC), total reducing power (TRP) and 2,2-diphenyl-1-picryl hydrazyl (DPPH)-free radical scavenging activity. The oxidative stress generated by NPs involved production of antioxidative molecules total phenolic content (TPC), total flavonoid content (TFC) depending on NPs concentration. The study concludes that copper oxide nanoparticles functions as a stimulator of bioactive components productions, and can be employed in in vitro batch cultures.     

Production of rebaudioside D from stevioside using a UGTSL2 Asn358Phe mutant in a multi-enzyme system

Rebaudioside D is a sweetener from Stevia rebaudiana with superior sweetness and organoleptic properties, but its production is limited by its minute abundance in S. rebaudiana leaves. In this study, we established a multi-enzyme reaction system with S. rebaudiana UDP-glycosyltransferases UGT76G1, Solanum lycopersicum UGTSL2 and Solanum tuberosum sucrose synthase StSUS1, achieving a two-step glycosylation of stevioside to produce rebaudioside D. However, an increase in the accumulation of rebaudioside D required the optimization of UGTSL2 catalytic activity towards glucosylation of rebaudioside A and reducing the formation of the side-product rebaudioside M2. On the basis of homology modelling and structural analysis, Asn358 in UGTSL2 was subjected to saturating mutagenesis, and the Asn358Phe mutant was used instead of wild-type UGTSL2 for bioconversion. The established multi-enzyme reaction system employing the Asn358Phe mutant produced 14.4 g l⁻¹ (1.6 times of wild-type UGTSL2) rebaudioside D from 20 g l⁻¹ stevioside after reaction for 24 h. This system is useful for large-scale rebaudioside D production and expands our understanding of the pathways involved in its synthesis.     

Contributions to the biotechnological production of sweeteners from stevia rebaudiana bertoni. I. A method for the serial analysis of diterpene glycosides by HPLC

An existing HPLC-method for the analysis of stevioside from Stevia rebaudiana plants was adapted to analyse plant cell culture material. A new extraction method with methanol was developed. Exhaustive extraction of many samples (10 with our apparatus) can be done simultaneously. The quality of extraction is better than soxhlet extraction because the cold methanol was used. Samples with low stevioside content can be prepared with minimal loss. Substances which interfere with stevioside in HPLC analysis using UV detection are common in plant cell culture samples and hence need a special technique of elimination. This was accomplished by TLC-purification of the samples.     

Antibacterial activity of stevioside towards food‐borne pathogenic bacteria

This study determines the inhibitory effect of Stevia rebaudiana leaf extracts and its purified bioactive compound ‘stevioside’ against food‐related pathogens. The S. rebaudiana solvent extracts (1000 μg/mL) displayed antibacterial activity to Serratia marcescens, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, B. subtilis, Alcaligenes denitrificans and Salmonella typhimurium. Of the six solvents, ethanol and acetone extracts displayed the highest zone of inhibition. The bioactive compound from S. rebaudiana was purified by solvent extraction, thin‐layer chromatography followed by structural characterization by spectroscopy evidence. Purified stevioside prevented the growth of tested bacterial species, i.e. B. subtilis, K. pneumoniae and S. typhimurium. Significant zone of inhibition (12 mm) was observed against B. cereus which proposes potential application of stevioside in foods to increase their shelf life.     

Contributions to the biotechnological production of sweeteners from stevia rebaudiana bertoni. II. Induction of stevioside accumulation in cell cultures by variation of the nutrient medium and the analysis of small amounts of stevioside

Cell cultures of Stevia rebaudiana in general do not contain stevioside when grown on their basal nutrient medium. No stable and prolonged stevioside accumulation was achieved by varying the medium components. However, in some cases a transient state was observed where more stevioside was synthesized than catabolized. The dynamic behaviour of product synthesis which led to accumulation of stevioside during only a short period was demonstrated. For accurate determination of very small amounts of stevioside, a combined TLC-HPLC method was used [1].     

The Effect of Long and Short Day Length upon the Growth of Whole Plants and the Level of Soluble Proteins, Sugars, and Stevioside in Leaves of Stevia rebaudiana Bert.

Long-day conditions increase internode length, leaf area, anddry weight and reduce the interval between the appearance ofsuccessive leaf pairs in S. rebaudiana as compared with shortdays. Total soluble leaf sugars, protein, and stevioside contentare also augmented in both absolute and relative terms and thebiosynthesis of steviol, the aglucone present in stevioside,is increased by 45%. The interaction between dry matter, steviosidecontent, and day length is discussed and a theory for the biologicalrole of stevioside as a possible defence mechanism against insectherbivory is proposed: as such this is the first known caseof a tetracyclic diterpene glycoside assuming this function.     

In Vitro Regeneration of Stevia rebaudiana (Bert) from the Nodal Explant

Procedure for micropropagation of Stevia rebaudiana Bertoni, containing stevioside, a natural noncaloric sweetner, has been developed using nodal segments as explant. Higher proliferation of shoots and multiplication was obtained on Murashige and Skoog basal medium (MS) supplemented with 1.0 mg l-1 indoleacetic acid (IAA) plus 10.0 mg l^-1 kinetin and 30.0 mg l-1 adenine sulphate. Sprouting of 90% of the axillary buds was observed within 4 weeks of inoculation, producing >10.0 shoots per explant within 12 weeks. Profuse roots were induced from 90% of the regenerated shoots within 4 weeks of inoculation on half strength MS solid medium supplemented with 1.0 mg l^-1 IAA. High survival rate, > 60%, was obtained when the plantlets were transferred to field conditions. The survival rate of taller plants was always higher. The in vitro regenerated plants were morphologically indistinguishable from the donor plants and leaves were of intense sweet taste upon chewing. The heterogenic nature of S. rebaudiana necessitates establishment of protocol for every genotype independently.     

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l^?1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH₄NO₃ (24.7 mM), 3.0× KNO₃ (56.4 mM), 3.0× MgSO₄ (4.5 mM) and 3.0× KH₂PO₄ (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day^?1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.     

Stevioside biosynthesis by callus,root, shoot and rooted-shoot cultures in vitro

Leaf explants of Stevia rebaudiana Bertoni (Compositae), an herb which produces the sweet ent-kaurene glycoside stevioside, were cultured in Murashige and Skoog medium with vitamins, sucrose (30 g l^–1), agar (0.9% w/v) and supplemented with naphthaleneacetic acid (NAA, 0.5 mg l^–1) and benzylaminopurine (BAP, 0.5 mg l^–1). These conditions yielded friable callus cultures. Differentiation of the callus tissue was then achieved by eliminating the agar and modulating the medium's hormone concentrations. Thus, medium containing increased auxin concentration (1.0 mg l^–1) and no cytokinin or increased cytokinin (1.0 mg l^–1) and no auxin yielded root or shoot cultures respectively. Supplementation of the shoot medium with NAA (1.0 mg ml^–1) induced shoot cultures to grow roots thereby differentiating into rooted-shoot cultures. Only the rooted-shoot cultures tasted sweet. Feedings of [2-¹⁴C]acetic acid to callus, shoot or rooted-shoot cultures demonstrated that only the rooted-shoot cultures are capable of de novo biosynthesis of the aglycone moiety of stevioside (steviol). In addition, [methyl-³H(N)steviol feedings to shoot or rooted-shoot cultures illustrated that both types of cultures are capable of the glycosylation reaction. The ability of these tissues to glycosylate steviol to stevioside was also demonstrated employing crude enzyme preparations derived from shoot or rooted-shoot cultures. These results suggest that stevioside biosynthesis is a function of tissue differentiation since both roots and leaves are required for cultured S. rebaudiana to biosynthesize stevioside from acetate, while the final biosynthetic steps can be performed at all levels of differentiation.     

New sweet diterpene glucosides from Stevia rebaudiana

From the leaves of Stevia rebaudiana, two new sweet glucosides, rebaudiosides A and B, were isolated besides the known glucosides, stevioside and steviolbioside. On the basis of IR, MS, ¹H and ¹³C NMR as well as chemical evidences, the structure of rebaudioside B was assigned as 13-O-[β-glucosyl(1-2)-β-glucosyl(1-3)]-β-glucosyl-steviol and rebaudioside A was formulated as its β-glucosyl ester.     

Effect of nitrogen and phosphate on in vitro growth and metabolite profiles of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni (Asteraceae)

The commercialization of Stevia rebaudiana Bertoni (Asteraceae) extracts as a natural sweetener is driving interest in the use of in vitro propagation systems as an alternative source of steviol glycosides. Out of this suite of chemicals, stevioside is the most abundant but rebaudioside A is the sweetest. We established an in vitro propagation method from germinated seedlings on a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium with aims to study the effects of nitrogen and phosphate on the growth and metabolite profiles of S. rebaudiana plants. Generally, NH₄NO₃ is supplied at a concentration of 20.61 mM in MS medium and together with 18.79 mM KNO₃, provide nitrogen to in vitro growing plants. In this study, we used a range of 0.3–72.1 mM NH₄NO₃ and 9.4–65.8 mM KNO₃ and generated six different media with altered nitrogen. Similarly, six different concentrations of KH₂PO₄, ranging from 0.6 to 4.4 mM were tested for the phosphate treatments and the control medium had 1.25 mM KH₂PO₄. By reducing the nitrogen and phosphate levels to half, respectively, this led to the tallest plants. Increasing concentrations of nitrogen in the medium significantly lowered the amount of rebaudioside A as plants on the control medium accumulated 270 mg g^?1 rebaudioside A compared to those that were on a medium with 3.5 times the nitrogen supply (30 mg g^?1 rebaudiose A). Steviol increased with increasing nitrogen available to the microplants. The highest levels of stevioside (740 mg g^?1) quantified was linked to microplants on a medium with half the phosphate concentration. To further assess changes to the metabolomic profiles of treated microplants, LC–MS/MS was used in combination with multivariate statistical analyses. Two distinct clusters were revealed after principal component analysis. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the separation of phosphate-treated plants from those with variable nitrogen concentrations. Chlorogenic acid and its derivatives were linked to changing phosphate concentrations. The clustering suggests different molecular mechanisms at play that are affected by nitrogen and phosphate supply which serve to alter secondary metabolic flux, resulting in different chemical profiles.     

Contributions to the biotechnolgical production of sweeteners from stevia rebaudiana bertoni. III. Accumulation of secondary metabolites by means of a precursor and by elicitation of cell cultures

Gibberellin A₃ fed to cell suspension cultures of Stevia rebaudiana showed a fast conversion to stevioside. The product was detected within one day after gibberellin addition and achieved its maximum concentration after one week. However, using special production media (without precursor or elicitor), stevioside was produced only two to seven weeks after inoculation [1]. Elicitation of suspension cultures was performed with Stevia specific and non-specific fungi and with yeast extract. Although production of some secondary metabolites was induced, stevioside was not synthesized.