IngredientsSteviol glycosides are the chemical compounds responsible for the sweet taste of the leaves of the South American plant Stevia rebaudiana (Asteraceae). Steviol glycosides accumulate in Stevia leaves where they may comprise from 10 to 20% of the leaf dry weight. At present, 43 different naturally occurring steviol glycosides from S. rebaudiana Bertoni have been identified.
Glycosylsteviosides — also rendered as glycosyl steviosides, glucosylated steviol glycosides, or alpha-glucosyl steviosides — are a semi-synthetic, enzymatically modified subclass of steviol glycosides. αG Sweet, a well-known commercial preparation, is described as an enzymatically glycosylated stevioside (also known as enzyme-modified steviol glycosides) developed using the extract of Stevia rebaudiana (Bertoni), an herbal shrub of the Asteraceae family. They are not isolated directly from plant leaves but are produced post-extraction through enzymatic transglycosylation of naturally extracted steviosides.
The structure of stevioside was established to be a tetracyclic diterpene (ent-kaurene) glycoside built up from steviol, ent-13-hydroxykaur-16-en-19-oic acid, as the aglycone, esterified at the C-19 carboxylic acid function with a β-D-glucopyranosyl unit and substituted at the tertiary hydroxyl function at C-13 with a β-sophorosyl disaccharide. These steviol glycosides, which are ent-kaurene-type diterpenes, are connected to various sugars such as glucose, rhamnose, xylose, fructose, and deoxy glucose at C-13 and C-19 positions via 1,2-; 1,3-; 1,4- or 1,6- α or β-glycosidic linkages.
In glycosylsteviosides, additional glucose units (typically one to three) are attached to the pre-existing glucose residues of stevioside or rebaudioside A through α-glucosidic bonds — a modification absent in the native leaf compounds. Structural analysis showed that 1 to 3 glucosyl groups were respectively linked with α-1,4 glycosidic bonds at the C-13 site of stevioside; when 1 or 2 glucosyl groups were linked at the C-13 site, the taste quality was improved compared with stevioside; and when three glucosyl groups were linked at the C-13 site, the sweetness was reduced.
The ingredient is known under multiple overlapping terms in the scientific and regulatory literature: glycosyl steviosides, glucosyl steviosides, alpha-glucosyl steviosides, glucosylated steviol glycosides (GSGs), and enzyme-modified stevia. The EFSA Panel on Food Additive and Flavourings (FAF) has assessed the safety of glucosylated steviol glycosides proposed for use as a new food additive in different food categories. In Japan, the commercial product class is commonly designated by trade names such as αG Sweet.
Stevia extracts generally contain a high percentage of the glycoside diterpenes stevioside (CAS no. 57817-89-7) and rebaudioside A (CAS no. 58543-16-1), the principal sweetening compounds, and smaller amounts of other steviol glycosides. Glycosylsteviosides are produced by adding glucose units to these principal compounds. Steviol glycosides from Stevia rebaudiana have been reported to be between 30 and 320 times sweeter than sucrose.
Steviol glycosides are typically isolated further by different methods, e.g., selective precipitation, ultrafiltration, or column chromatography, using adsorption or ion-exchange resins. The final product is commonly spray-dried. After enzymatic transglycosylation, the glucosylated product is similarly purified and spray-dried for use as a food-grade ingredient. One industrial process discloses simultaneous transglycosylation of the steviol glycosides mixture by acting with a CGTase produced by Bacillus stearothermophilus and converting the remaining maltodextrins to fructose-terminated oligosaccharides; the glucosylated mixture was purified on macroporous resin and deionized to high purity.
The commercial glycosylated stevioside αG Sweet is reported to be nearly 250 times as sweet as sucrose. It is available as a dry powder suitable for incorporation into beverages, dairy products, confectionery, and other food systems.
For hundreds of years, people in Brazil and Paraguay have used the leaves of stevia to sweeten teas and foods and have used it as herbal medicine for diabetes, obesity, hypertension, and other conditions. The plant was cultivated and used by the Guaranà people of Paraguay, who called it ka'a he'ê ("sweet herb"). Stevia has been traditionally employed as a sweetener in South America; currently, this use is spread all over the world.
Chemical characterization studies of the major sweet components in the leaves have been carried out since 1908. A first preliminary structure of a steviol glycoside, called stevioside, appeared in 1955, followed by several additional structural studies and its organic synthesis starting from steviol in 1980.
Not until the 1970s were the extracts — the steviol glycosides — first commercialized as a sweetener in Japan. Japan was the earliest adopter of stevia-based sweeteners on a commercial scale. Because the use of artificial sweeteners such as dulcin, sodium cyclamate, and saccharin had been banned or restricted in view of food sanitation concerns, harmless natural sweeteners were receiving ever-increasing demands. In response to these demands, a drive for production increase of stevioside rapidly mounted among agriculturally-concerned parties and sweetener manufacturers.
The enzymatic conversion of stevioside to alpha-glycosyl stevioside was first patented in Japan in the early 1980s as a solution to the organoleptic limitations of native stevioside. U.S. Patent 4,219,571 relates to a process for producing a sweetener, characterized in allowing alpha-glucosyltransferase to act on an aqueous solution containing stevioside and alpha-glucosyl sugar compound to form alpha-glycosyl stevioside. This biotechnological modification, developed primarily in Japan, created what is now broadly called glycosylsteviosides or glucosylated steviol glycosides. The rationale was to overcome known deficiencies of native stevioside: besides being sweet, stevioside imparts bitterness and astringency; the sweetness of stevioside effects slower in the mouth than that of sucrose and gives a lingering, unpleasant aftertaste; and stevioside is difficultly soluble in water, with a solubility of only 0.12% at 20°C.
Today, stevia is cultivated in many countries and China is the largest grower of stevia as well as exporter of steviol glycosides.
Transglycosylation is the in-vivo or in-vitro process of transferring glycosyl groups from a donor to an acceptor, which is usually performed by enzymatic reactions because of their simplicity, low steric hindrance, high region-specificity, low production cost, and mild processing conditions.
The enzymatic transglycosylation of steviol glycosides can improve the edulcorant quality of steviol glycosides. Cyclodextrin glucanotransferase (CGTase) is one of the most popular glucanotransferases applied in this reaction. Cyclomaltodextrin-glucanotransferase (CGTase) efficiently catalyzes intermolecular glycosylation to transfer α-glucosyl units from starch to 4-OH of a glucosyl moiety (trans-α-1,4-glucosylation). CGTase (EC 2.4.1.19) produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli have been used for transglycosylating stevioside and rebaudiosides A with the use of starch or cyclodextrin as donor.
Cyclodextrins and starches provided the best transglucosylation yield, while mono- and disaccharides were not effective glycosyl donors to stevioside with the CGTase. The enzyme used for grafting steviol glycosides is mainly glycosyltransferase, such as cyclodextrin glucosyltransferase, and there are also reports about glucosidase, fructofuranosidase, and galactosidase.
CGTases produced by Bacillus stearothermophilus B-5076 and B. macerans BIO-4m were among the most effective biocatalysts. The CGTase-producing strain Alkalihalobacillus oshimensis CGMCC 23164, isolated from Stevia-planting soil, was found to produce a high-efficiency CGTase that converted steviol glycosides to glucosylated steviol glycosides. This CGTase demonstrated optimal transglycosylation activity with 10 g/L steviol glycoside and 50 g/L soluble starch as substrates at <40 °C, with conversion rates of stevioside and rebaudioside A reaching 86.1% and 90.8%, respectively.
The transglycosylated products catalyzed by CGTase are widely used in food additives, supplements, and personal care and cosmetic products. This is due to improvements in the solubility, stability, bioactivity, and length of the synthesized products. The glycosylated steviol glycosides can improve the solubility and bioavailability of water-insoluble drugs. Glucosyl stevioside (Stevia-G) is a safe food additive made from natural stevioside that has been enzymatically modified to disguise the bitter taste of the aglycone's core structure.
To achieve better sweetness with less astringency, suppression of glycosylation at the 19-O-glucosyl moiety is necessary. Industrial process refinements therefore often include a β-amylase treatment step to trim overly long oligoglucosyl chains generated at the C-19 position.
The shared pharmacological scaffold of all steviol glycosides — including glycosylsteviosides — is the diterpene aglycone steviol. Stevioside dose-dependently decreased protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and PEPCK mRNA after 15 days of treatment. The steviol aglycone is the final common metabolite released after gut microbial deglycosylation and is thought to mediate most systemic bioactivities.
Commercial glycosylstevioside preparations typically contain mixtures of mono-, di-, and tri-glucosylated derivatives of stevioside and rebaudioside A. Rebaudioside C can be glucosylated by the action of Bacillus stearothermophilus CGTase using tapioca starch as a source of glucose unit; the reaction mixture treated with activated carbon, deionized, and spray-dried, may contain at least 20% glucosylated Rebaudioside C. Similar preparations have been described for rebaudiosides B and M.
From the common terpene pathway, kaurenoic acid 13-hydroxylase (KAH) produces steviol by hydroxylation of (−)-kaurenoic acid in the endoplasmic reticulum. Subsequent transfer of glucose units is performed by plant UDP-glucosyltransferases (UGT) from UDP-glucose to steviolmonoside. Three types of UGTs are involved in the pathway: UGT85C2, UGT74G1, and UGT76G1, located in the cytosol.
In studies of two rat models of diabetes, stevioside (0.5 mg/kg) lowered blood glucose levels in STZ-induced diabetic rats, peaking at 90 min; stevioside administered twice daily also demonstrated dose-dependent effects in lowering glucose levels in both diabetic rat models. Stevioside was reported to regulate blood glucose levels in diabetic rat models by increasing insulin secretion through downregulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression. The PEPCK protein is an enzyme that activates the metabolic pathway of gluconeogenesis and converts oxaloacetate into phosphoenol pyruvate and carbon dioxide. Inhibition of this enzyme or reduction in its gene expression can decrease glucose production from non-sugar sources.
Compared to control, stevioside reduced the incremental area under the glucose response curve by 18% (P = 0.013). The insulinogenic index was increased by approximately 40% by stevioside compared to control (P < 0.001).
The hypotensive effect of intravenously administered stevioside on both systolic and diastolic blood pressure was dose-dependent for doses of 50, 100, and 200 mg/kg in conscious spontaneously hypertensive rats (SHR). In animal pharmacology, the mechanism appears to involve vasodilation without significant alteration of plasma catecholamines.
The anti-inflammatory properties of stevioside were confirmed in vitro by decreasing TNF-α, IL-1β, and IL-6 synthesis and inhibiting of NF-κB transcription factor, and in vivo by inhibiting NF-κB and MAPK in laboratory animals. Stevioside also prevented in vitro upregulation of genes involved in liver inflammation. In silico assays demonstrated its antagonistic action in two pro-inflammatory receptors: tumor necrosis factor receptor (TNFR)-1 and Toll-like receptor (TLR)-4-MD2.
Stevia rebaudiana Bertoni and its glycosides are known not only as natural, noncaloric sweeteners but also for their antihypertensive, anti-inflammatory, immune-modulatory, and antihyperglycaemic effects. In in vitro studies on cardiac fibroblasts, steviol glycoside mixtures were found to modulate levels of catalase (CAT) and superoxide dismutase (SOD1) mRNA, suggesting a role in cellular antioxidant defense.
The metabolism of steviol glycosides is dependent upon gut microbiota, which breaks down glycosides into steviol that can be absorbed by the host. Several in vitro studies mimicking the anaerobic conditions of the colon have confirmed the ability of the gut microbiota from mice, rats, hamsters, and humans to hydrolyze steviol glycosides completely to steviol.
For the glucosylated derivatives specifically, about 74–78% deglycosylation of the parent glucosylated steviol glycoside (GSG) occurred within 4 h in both adult male and female fecal homogenates. Within 12 h of incubation, almost complete deglycosylation of GSG to steviol was achieved. However, the total molar equivalent of steviol formed was only observed at a mean of 41% in males and 39% in females at the 4 h time point. This relatively low formation of total molar equivalent of steviol at 4 h suggested the stepwise occurrence of deglycosylation involving partially deglycosylated intermediates such as stevioside and rebaudioside A.
Results clearly demonstrate steviol glycosides produced by extraction from stevia leaf, or enzymatic conversion of stevia leaf extract, share the same metabolic fate in the human gut microbiota from adults and children. Considering a common metabolite structure and a shared metabolic fate in all ages, safety data for individual steviol glycosides can be used to support safety of all steviol glycosides produced by extraction and enzymatic conversion of stevia leaf extract.
The natural sweetener from Stevia rebaudiana Bertoni, steviol glycoside (SG), has been proposed to exhibit a range of antidiabetic properties. The objective of a 2019 systematic review was to critically evaluate evidence for the effectiveness of SGs on human health, particularly type 2 diabetic (T2D) biomarkers, collecting data from randomized controlled trials (RCTs). Seven studies, nine RCTs, including a total of 462 participants were included. A meta-analysis was performed to assess the effect of SGs on BMI, blood pressure (BP), fasting blood glucose (FBG), lipids, and glycated hemoglobin (HbA1c). The meta-analysis revealed an overall significant reduction in systolic BP in favour of SGs between SG and placebo, mean difference (MD): −6.32 mm Hg. The overall effect of BMI, diastolic BP, FBG, total cholesterol, and high-density lipoprotein cholesterol (HDL-C) was a non-significant reduction in favour of SGs, and a non-significant increase in low-density lipoprotein cholesterol and triglyceride, while no significant effect of HbA1c was found.
Twelve type 2 diabetic patients were included in an acute, paired cross-over study. A standard test meal was supplemented with either 1 g of stevioside or 1 g of maize starch (control). Blood samples were drawn at 30 minutes before and for 240 minutes after ingestion of the test meal. Compared to control, stevioside reduced the incremental area under the glucose response curve by 18% (P = 0.013), and the insulinogenic index was increased by approximately 40% by stevioside compared to control (P < 0.001).
Long-term human trials have been conducted investigating the effect of 200–1500 mg/day orally administered stevioside and 500–1000 mg/day rebaudioside A in time periods ranging from 3 days to 2 years on glycated hemoglobin and other glycemic parameters.
The evidence for glycemic benefits is preliminary to moderate. The only robust meta-analysis of RCTs (9 trials, 462 participants) found significant systolic blood pressure reduction but no significant effect on HbA1c or fasting blood glucose across the aggregate. Substantial variations were observed in trial design, variation in daily doses of stevioside, duration of intervention, and differences in protocols regarding lifestyle, including continuation of antihypertensive and antidiabetic medications across included studies. Most trials used native stevioside rather than glycosylsteviosides specifically; extrapolation to the enzymatically modified form requires caution.
The landmark placebo-controlled study enrolled 106 randomized Chinese subjects (53 women, 53 men) with a mean age of 54 years. Mean systolic BP was 160 mmHg, and diastolic BP was 102 mmHg. This was a multicenter, randomized, double-blind, placebo-controlled trial in Chinese men and women aged between 20 and 75 years with mild essential hypertension (SBP 140–159 mmHg and DBP 90–99 mmHg). Patients took capsules containing 500 mg stevioside powder or placebo 3 times daily for 2 years. After 2 years, the stevioside group had significant decreases in mean SBP and DBP compared with baseline (SBP, from 150 [7.3] to 140 [6.8] mm Hg; DBP, from 95 [4.2] to 89 [3.2] mm Hg; P < 0.05) and compared with placebo (P < 0.05).
Results from long-term clinical trials (1–2 years) in China studying men and women with mild to moderate essential hypertension suggested antihypertensive effects of stevioside at intakes of 750 and 1500 mg/day.
There is moderate-quality RCT evidence that oral stevioside (750–1500 mg/day for 1–2 years) reduces systolic and diastolic blood pressure in patients with mild essential hypertension. However, these trials were conducted in Chinese populations with native stevioside and have not been replicated with glycosylsteviosides specifically. Heterogeneity was significant for several analyses in the systematic review, limiting the strength of pooled conclusions.
Evidence is largely preclinical. The active compounds isolated from Stevia rebaudiana possess interesting medicinal activities, including antidiabetic, antihypertensive, anti-inflammatory, antioxidant, anticancer, and antidiarrheal activity. The described bioactivities of steviol glycosides deserve special attention based on their dose dependence and specific pathological situations. Further clinical research is needed to understand underlying mechanisms of action, therapeutic indexes, and pharmacological applications. No clinical trials specifically with glycosylsteviosides for inflammatory endpoints have been identified in the published literature to date.
Evidence is in vitro and in vivo (animal) only. In vitro and in vivo studies showed that stevia has antiglycemic action and antioxidant effects in adipose tissue and the vascular wall, reduces blood pressure levels and hepatic steatosis, stabilizes the atherosclerotic plaque, and ameliorates liver and kidney damage. These findings have not been confirmed in adequately powered human RCTs specifically for glycosylsteviosides.
Due to the lack of randomized clinical trials in humans, available reviews included in vitro studies using certain microbial strains and in vivo animal studies. Results indicated that stevia consumption has a potential benefit on the microbiome's alpha diversity. Alterations in the colonic microenvironment may depend on the amount and frequency of stevia intake, as well as on the simultaneous consumption of other dietary components. Evidence specific to glycosylsteviosides' effects on human gut microbiota is at an early stage.
Steviol glycosides exhibit a superior sweetener proficiency to that of sucrose and are noncaloric, noncariogenic, and nonfermentative. Because glycosylsteviosides are not fermented by oral bacteria, they do not promote dental caries — a property shared by all steviol glycosides and supported by mechanistic plausibility and regulatory acceptance.
From clinical trials, it has been suggested that administration of stevia and steviol glycosides does not exhibit any body weight or BMI-lowering effect compared with placebo treatment. The use of glycosylsteviosides as a low-calorie sweetener may indirectly support caloric reduction, but direct evidence of weight loss effects from the ingredient itself is lacking.
The following dosages are reported in the cited literature and pertain to stevioside or steviol glycosides (not always specifically to glycosylsteviosides); they are provided here as the direct comparator data from clinical research:
For glycosylsteviosides specifically as a food ingredient, there is no separately established therapeutic dose in the published clinical literature. The ingredient is used primarily as a sweetener at levels determined by food technology requirements rather than by therapeutic trials.
The EFSA Panel set an Acceptable Daily Intake (ADI) of 4 mg per kg body weight per day for steviol glycosides, a level consistent with that already established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Toxicological testing showed that the substances are not genotoxic, nor carcinogenic, or linked to any adverse effects on the reproductive human system or for the developing child.
Considering the available toxicity data (in vitro and in vivo animal studies and some human tolerance studies), the Panel concluded that steviol glycosides, complying with JECFA specifications, are not carcinogenic, genotoxic, or associated with any reproductive/developmental toxicity. The ADI for steviol glycosides was established at 4 mg/kg bw/day expressed as steviol equivalents, based on application of a 100-fold uncertainty factor to the NOAEL in the 2-year carcinogenicity study in the rat of 2.5% stevioside in the diet. This is equal to 967 mg stevioside/kg bw/day.
Conservative estimates of steviol glycosides exposures both in adults and in children suggest that it is likely that the ADI would be exceeded at the maximum proposed use levels.
The EFSA Panel considered that the metabolism of glucosylated steviol glycosides is sufficiently similar to the already authorised steviol glycosides, and thus the toxicological data previously assessed for steviol glycosides (E 960a) were considered to support their safety as a food additive. The existing ADI for steviol glycosides (E 960a) of 4 mg/kg bw per day expressed as steviol can also be applied to glucosylated steviol glycosides.
No additional toxicological data were therefore required by EFSA when evaluating glucosylated steviol glycosides as a new food additive category.
It was concluded by JECFA that steviol glycosides did not demonstrate any toxic effect on genes nor cancer-causing potential. There is no safety concern upon normal consumption. These conclusions apply to steviol glycosides meeting JECFA and EFSA purity specifications.
In 2010, the EFSA ANS Panel adopted a scientific opinion on the safety of steviol glycosides (E 960) and established an ADI of 4 mg/kg body weight per day. Conservative estimates of exposure, in both adults and children, suggested that it is likely that the ADI would be exceeded at the maximum proposed use level. This remains a regulatory concern especially for children and heavy consumers of multiple steviol-glycoside-sweetened products.
Studies suggest that stevioside can reduce blood glucose levels in type II diabetics and can reduce blood pressure in mildly hypertensive patients. Because steviol glycosides and their glucosylated derivatives share these pharmacological actions with antidiabetic (e.g., insulin, metformin) and antihypertensive medications, additive pharmacodynamic effects are theoretically possible. Several included RCTs reported variations in protocols regarding the continuation of antihypertensive and antidiabetic medications across studies, indicating this has been recognized as a confounding variable. Formal interaction studies using glycosylsteviosides specifically have not been identified in the published literature.
Data from in vitro and animal studies suggest that steviol glycosides have minimal or no effect on gut microbiota. Results indicated that stevia consumption has a potential benefit on the microbiome's alpha diversity. Alterations in the colonic microenvironment may depend on the amount and frequency of stevia intake, as well as on the simultaneous consumption of other dietary components.
Stevioside apparently interferes with secretory transport systems at very high doses. Further studies of secretory-transport-system inhibition are needed in order to understand whether stevioside might delay drug clearance. This has not been demonstrated at ADI-level exposures.
To date, steviol glycosides are permitted in many countries including Mainland China, Japan, Korea, the USA, Australia, and New Zealand. On the basis of EFSA scientific opinion, the European Commission authorised steviol glycosides for their proposed use in food and drinks in the European Union. Currently, steviol glycosides (E 960a–d) are authorised in the EU in 32 different food categories.
Glucosylated steviol glycosides have received specific EFSA evaluation as a food additive distinct from the naturally extracted forms. The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of steviol glycoside preparations obtained by enzymatic bioconversion of highly purified stevioside and/or rebaudioside A stevia leaf extracts. JECFA similarly continues to review updated specifications. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established an ADI for steviol glycosides of 0–4 mg/kg bw per day, expressed as steviol.
Health conditions that Glycosylsteviosides may help support.
Body systems that Glycosylsteviosides may help support.
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