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%.     

Effect of stevioside on PAH transport by isolated perfused rabbit renal proximal tubule

Stevioside, a non-caloric sweetening agent, is used as a sugar substitute. An influence of stevioside on renal function has been suggested, but little is known about its effect on tubular function. Therefore, the present study was designed to explore the direct effect of stevioside on transepithelial transport of p-aminohippurate (PAH) in isolated S2 segments of rabbit proximal renal tubules using in vitro microperfusion. Addition of stevioside at a concentration of 0.45 mM to either the tubular lumen, bathing medium, or both at the same time had no effect on transepithelial transport of PAH. Similarly, a concentration of 0.70 mM (maximum solubility in the buffer) when present in the lumen, had no effect on PAH transport. However, this concentration in the bathing medium inhibited PAH transport significantly by about 25-35%. The inhibitory effect of stevioside was gradually abolished after it was removed from the bath. Addition of 0.70 mM stevioside to both lumen and bathing medium at the same time produced no added inhibitory effect. Stevioside at this concentration has no effect on Na+/K+-ATPase activity as well as cell ATP content. These findings suggest that stevioside, at a pharmacological concentration of 0.70 mM, inhibits transepithelial transport of PAH by interfering with the basolateral entry step, the rate-limiting step for transepithelial transport. The lack of effect of stevioside on transepithelial transport of PAH on the luminal side and its reversible inhibitory effect on the basolateral side indicate that stevioside does not permanently change PAH transport and should not harm renal tubular function at normal human intake levels.     

Biological effects of stevioside on the survival of Escherichia colistrains and plasmid DNA

Stevioside is widely used daily in many countries as a non-caloric sugar substitute. Its sweetening power is higher than that of sucrose by approximately 250–300 times, being extensively employed as a household sweetener, or added to beverages and food products. The purpose of this study was to ascertain stevioside genotoxic and cytotoxic potentiality in different biological systems, as its use continues to increase. Agarose gel electrophoresis and bacterial transformation were employed to observe the occurrence of DNA lesions. In addition to these assays, Escherichia coli strains were incubated with stevioside so that their survival fractions could be obtained. Results show absence of genotoxic activity through electrophoresis and bacterial transformation assays and drop of survival fraction of E. coli strains deficient in rec A and nth genes, suggesting that stevioside (i) is cytotoxic; (ii) could need metabolization to present deleterious effects on cells; (iii) is capable of generating lesions in DNA and pathways as base excision repair, recombination and SOS system would be important to recover these lesions.     

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.     

Metabolism of stevioside by healthy subjects

Stevioside (250-mg capsules) was given thrice daily for 3 days to 10 healthy subjects. Blood samples were collected and blood pressure measured after nocturnal fasting, before and at different time points during the third day of the administration of stevioside. No significant differences were found between the control and the stevioside condition for blood pressure and blood biochemical parameters. The 24-hr urinary volume and urinary excretion of electrolytes were not significantly different. Likewise, no significant difference was found for mean blood glucose and insulin between control and stevioside conditions. Thus, oral stevioside is not directly effective as a hypotensive or hypoglycemic agent in healthy subjects at the dose administered in this study. Stevioside, free steviol, and steviol metabolites were analyzed in blood, feces, and urine after 3 days of stevioside administration. No uptake was found of stevioside by the gastrointestinal tract or the amounts taken up were very low and below the detection limit of the UV detector. Stomach juice did not degrade stevioside. All the stevioside reaching the colon was degraded by micro-organisms into steviol, the only metabolite found in feces. In blood plasma, no stevioside, no free steviol or other free steviol metabolites were found. However, steviol glucuronide (SV glu) was found in maximum concentrations of 33 micro g/ml (21.3 micro g steviol equivalents/ml). In urine, no stevioside or free steviol were present, but SV glu was found in amounts of up to 318 mg/24-hr urine (205 mg steviol equivalents/24 hrs). No other steviol derivatives were detected. In feces, besides free steviol, no other steviol metabolites or conjugates were detected. Steviol was excreted as SV glu in urine.     

Downstream processing of stevioside and its potential applications

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana Bertoni, which is commercially produced by conventional (chemical/physical) processes. This article gives an overview of the stevioside structure, various analysis technique, new technologies required and the advances achieved in recent years. An enzymatic process is established, by which the maximum efficacy and benefit of the process can be achieved. The efficiency of the enzymatic process is quite comparable to that of other physical and chemical methods. Finally, we believe that in the future, the enzyme-based extraction will ensure more cost-effective availability of stevioside, thus assisting in the development of more food-based applications.     

Formation of a transfer product from stevioside by the cultures of Actinomycete.

An Actinomycete, strain K-128, which was isolated from soil, formed a transfer product when the strain was cultured in a medium containing both stevioside and curdlan. The transfer product in culture broth was separated by DIAION HP-20 column and TOYOPEARL HW-40F column chromatographies. From structural studies, the transfer product was identified as C13-O-beta-6(2)-beta-glucosylsophorosyl-C19-O-beta-glucopyranosyl steviol. This product was the first to be obtained by a culture of an Actinomycete.     

Increase of insulin sensitivity by stevioside in fructose-rich chow-fed rats.

The intake of dietary fructose has undergone a marked increase around the world, especially the developed countries, in recent times. Stevioside, a glycoside contained in the leaves of Stevia rebaudiana Bertoni (Compositae), was used to screen the effect induced by a diet containing 60% fructose on insulin resistance in rats. Single oral administration of stevioside for 90 min decreased plasma glucose concentrations in a dose-dependent manner in rats receiving fructose-rich chow for four weeks. In addition, insulin action on glucose disposal rate was measured using the glucose-insulin index, the product of the areas under the curve of glucose, and insulin during the intraperitoneal glucose tolerance test. Oral administration of stevioside (5.0 mg/kg) in rats given four weeks of fructose-rich chow for 90 min reversed the value of glucose-insulin index, indicating that stevioside has the ability to improve insulin sensitivity in this insulin-resistant animal model. Time for the loss of plasma glucose lowering response to tolbutamide (10.0 mg/kg, i. p.) in fructose-rich chow fed rats was also markedly delayed by repeated stevioside treatment three times daily compared to the vehicle-treated group. The plasma glucose-lowering activity of tolbutamide was introduced to account for varying levels of endogenous insulin secretion, and is widely used as the indicator of insulin resistance development. Thus, it provided the supportive data that repeated oral administration of stevioside delayed the development of insulin resistance in rats on a high-fructose diet. Increased insulin sensitivity by stevioside administration was further identified using the plasma glucose-lowering action of exogenous insulin in streptozotocin-induced diabetic rats (STZ-diabetic rats). Oral administration of stevioside at 0.2 mg/kg three times daily into STZ-diabetic rats for ten days increased the response to exogenous insulin. Taken together, this demonstrated that oral administration of stevioside improves insulin sensitivity, and seems suitable as an adjuvant for diabetic patients and/or those that consume large amounts of fructose.     

Rapidin Vitro propagation and enhanced stevioside accumulation inStevia rebaudiana Bert

A very rapid and efficient regeneration method has been established using mature expiants ofStevia rebaudiana Bert. Adventitious shoots were induced from nodal expiants of field-grown plants on four basal media supplemented with various combinations of auxins and cytokinins. The best performance (23.4 ± 2.1 shoots per expiant) was obtained on a Murashige and Skoog (MS) medium containing 2 mg L^-1 IAA and 0.5 mg L^-1 kinetin. Roots were then produced when thesein vitro- regenerated shoots were transferred to an MS medium supplemented with 3% sucrose and 2 mg L^-1 IBA. When acclimatized to soil, the rooted plants had a 98.4% survival rate. Following transplantation in the field, stevioside contents were similar between the regenerated plants (10.68 mg g^-1 dry weight) and the mother plants (12.01 mg g^-1 dw).     

Enzymatic modification of stevioside by cell-free extract of Gibberella fujikuroi

Stevioside is a natural sweetener obtained from the leaves of Stevia rebaudiana. It is a glycoside of steviol and other glycosides of the same aglycone are also found in the plant. Stevioside is usually the major component in commercial products, but it is not the one with the best organoleptic properties. It has a bitter aftertaste and, for this reason, attempts have been made in order to modify its molecule. In this work, the commercial and purified stevioside were modified by hydrolytic enzymes from Gibberella fujikuroi. A screening was carried out on six strains of the fungus in order to select the most active. The production of the enzymes by the fungi was induced by its culture in a medium containing stevioside as the sole carbon source and the enzymatic extract was then used in the experiments. The products obtained were analyzed by HPLC-UV and HPLC-MS/MS. The results showed a significant increase in the concentration of rebaudioside A in the final product, which has better organoleptic properties than stevioside.     

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.     

Enzymatic determination of stevioside in Stevia rebaudiana

A simple enzymatic method is described for the determination of stevioside in Stevia rebaudiana. The method is based on the hydrolysis of stevioside with crude hesperidinase. The reaction is followed by monitoring the production of glucose with a glucose oxidase-peroxidase-2, 2′-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) system. The results for the stevioside content in S. rebaudiana leaves correlate with those obtained by other methods. The stevioside content in S. rebaudiana plants showed large variation.     

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.     

Effects of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant growth,stevioside, NPK,and chlorophyll content of Stevia rebaudiana

Stevia rebaudiana Bertoni is a unique medicinal plant which is mostly utilized as a sugar substitute for diabetic patients. In this research, regenerated plantlets of stevia in tissue culture is transferred to pots in greenhouse and inoculated with plant-growth-promoting rhizobacteria (PGPRs) (Bacillus polymixa, Pseudomonas putida, and Azotobacter chroococcum) and arbuscular mycorrhizal fungus (AMF) (Glomus intraradices). The results showed that in comparison to control, inoculation with a single microorganism, significantly increased root and shoot biomass as well as stevioside, chlorophyll, and NPK content in plants. However, such increased effects have been found to be further enhanced significantly due to dual compatible mixtures of inoculants resulting from their strong synergistic relationships among themselves. All growth parameters recorded the highest in 60-days-old plants in the treatment of Glomus+Azotobacter and followed with Glomus+Bacillus and Azotobacter+Pseudomonas treatments, respectively. Furthermore, high-performance liquid chromatography (HPLC) chromatograms revealed that the highest stevioside content have been produced in same treatments. Triple treatments had less positive effects compared to dual inoculations. Probably competence between microorganisms in triple inoculations has reduced their efficiency. Thus, suitable combination of mycorrhizal fungi and PGPR as biotic elicitors can enhance growth and stevioside content in tissue culture-regenerated plantlets of stevia.     

Molecular docking analysis of stevioside with Akt and PPARγ

Stevioside is a diterpenoid glycoside consisting of an aglycone (steviol) and three glucose molecules. It is commonly used as an anti-hyperglycemic food because of its non-caloric property. Therefore, it is of interest to document the interactions of stevioside with AKT & PPAR-γ proteins using Autodock Vina PyRx docking techniques. Results of the docking studies indicate that stevioside had more than two hydrogen bond interactions with the AKT and PPAR γ protein for further consideration.     

Immune up regulatory response of a non-caloric natural sweetener, stevioside

Immunomodulation is a process, which alters the immune system of an organism by interfering with its functions. This interference results in either immunostimulation or immunosuppression. An immunomodulator is any substance that helps to regulate the immune system. This “regulation” is a normalization process, so that an immunomodulator helps to optimise immune response. Immunomodulators are becoming very popular in the worldwide natural health industry as these do not tend to boost immunity, but to normalize it. Keeping this in view, major efforts have to be directed to modulate the immune responses, to permit effective treatment of various ailments associated with immune system and thus the development of a safe and effective immunomodulator for clinical us.

Leaves of Stevia rebaudiana are a source of several sweet glycosides of steviol. The major glycoside, stevioside, diterpenoid glycoside—is used in oriental countries as a food sweetener. Its medical use is also reported as a heart tonic. Besides, it is used against obesity, hypertension, and stomach burn and to lower uric acid levels. Here in this study, stevioside was tested for its immunomodulatory activity on different parameters of the immune system at three different doses (6.25, 12.5 and 25 mg/kg p.o.) on normal as well as cyclophosphamide treated mice. Stevioside was found effective in increasing phagocytic activity, haemagglutination antibody titre and delayed type hypersensitivity. In parallel, stevioside substantially increase proliferation in the LPS and Con A stimulated B and T cells, respectively. Present study, therefore, reveals that the drug holds promise as immunomodulating agent, which acts by stimulating both humoral as well as cellular immunity and phagocytic function.     

Combined enzymatic modification of stevioside and rebaudioside A

Cyclodextrin glucanotransferases (CGTases, EC 2.4.1.19) produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli were used for transglycosylating stevioside and rebaudiosides A with the use of starch as a donor. CGTases produced by Bacillus stearothermophilus B-5076 B. Macerans BIO-4m were the most effective biocatalysts. This method can be used successfully for direct transglycosylation of stevia extract without purification of its individual components.     

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.     

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.