IngredientsGlucosylstevioside — also written as glucosyl-stevioside, glucosyl stevioside, or alpha-glucosyl stevioside — is the common commercial and scientific name for an enzymatically modified derivative of stevioside, the principal sweet glycoside of Stevia rebaudiana Bertoni. It is marketed under multiple designations, including glucosyl steviosides, enzymatically modified stevia glucosyl stevia, and enzymatically modified stevia (EMS). In Japanese pharmaceutical and food standards, enzymatically modified stevia is defined as the product obtained by addition of glucose to stevia extracts using α-glucosyltransferase, with α-glucosylstevioside as the principal component.
The broader category to which it belongs is often referred to in the regulatory literature as glucosylated steviol glycosides. These consist of a mixture of glucosylated steviol glycosides, containing 1–20 additional glucose units bound to the parent steviol glycosides. In the European Union the category was formally designated food additive E 960d. In March 2023, both Regulation (EC) No 1333/2008 and Commission Regulation (EU) No 231/2012 were amended by introducing the entry "glucosylated steviol glycosides" (E 960d), based on the evaluation completed by the EFSA Panel.
The precursor molecule, stevioside, originates from Stevia rebaudiana Bertoni, a small perennial shrub native to the highlands of Paraguay and southern Brazil. The extract of the Stevia rebaudiana plant contains a mixture of different sweet diterpene glycosides, which share a single base — steviol — and differ by the presence of carbohydrate residues at positions C-13 and C-19. These glycosides accumulate in stevia leaves and compose approximately 10%–20% of the total dry weight. At present, 43 different naturally occurring steviol glycosides from S. rebaudiana Bertoni have been identified. Stevioside itself is the most abundant of these. Stevioside is the most prevalent steviol glycoside in Stevia rebaudiana plant leaves.
Stevioside is 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 at C-13 with a β-sophorosyl disaccharide, chemically formulated as 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester. Chemically, stevioside features the aglycone steviol — a kaurene-type diterpene — glycosylated with three glucose units at the C-13 position and one glucose unit at the C-19 carboxyl group, giving the molecular formula C₃₈H₆₀O₁₈ and a molar mass of 804.87 g/mol.
Glucosylstevioside is formed when one or more additional glucose residues are enzymatically attached to this stevioside backbone. The optimized enzymatic α-glucosylation of stevioside using mutant glucansucrase Gtf180-ΔN-Q1140E and sucrose as donor substrate results in stevioside being mainly glucosylated at the steviol C-19 glucosyl moiety, as elucidated by NMR spectroscopy, mass spectrometry, and methylation analysis. Depending on the enzyme and reaction conditions used, the added glucose may be linked via α-(1→4), α-(1→6), or other linkages. The modification described in GRAS Notice GRN 000662, for example, involves treatment of steviol glycosides with a glucotransferase that results in additional glucose moieties conjugated to the parent steviol glycoside structures via α-(1–4) linkages, producing a final mixture of steviol glycosides containing up to twenty additional glucose moieties.
Glucosylstevioside is not a single compound but a mixture. Glucosylated steviol glycoside preparations have been described as a mixture containing 1–20 additional glucose units bound to the parent steviol glycoside via α-(1–4) linkages. EFSA noted that the mixtures on which data have been provided contain, on average, only three or four additional glucose units. Glucosylated steviol glycosides consist of not less than 95% (on dry, dextrin-free basis) of total steviol glycosides, comprised of glucosylated and parent steviol glycosides.
Commercially, glucosylstevioside is produced as a dried powder for use in food and beverage manufacturing. Glucosylated steviol glycosides are produced via enzymatic bioconversion using cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19), derived from a non-genetically modified strain of Anoxybacillus caldiproteolyticus, that catalyzes the transfer of glucose from starch to steviol glycoside mixtures isolated from the dried leaves of Stevia rebaudiana. A key chemical consequence of the additional glucosyl units is a dramatic increase in aqueous solubility. After 15 days of storage at 25 °C, 98% of glucosyl stevioside in an aqueous solution was present in a soluble form, compared to only 11% for stevioside or rebaudioside A.
The traditional history of glucosylstevioside as a distinct compound does not exist; it is an engineered derivative created through modern food biotechnology. However, its precursor — the leaf of Stevia rebaudiana with its native stevioside content — has a documented history of use spanning centuries. The Guaraní Indians had known for centuries about the unique advantages of kaa he-he (a native term which translates as "sweet herb") — long before European contact. These people knew the leaves of the wild stevia shrub (a perennial indigenous to the Amambay Mountain region) to have a sweetening power unlike anything else; they commonly used the leaves to enhance the taste of bitter mate (a tea-like beverage) and medicinal potions, or simply chewed them for their sweet taste.
The widespread native use of stevia was chronicled by the Spanish in historical documents preserved in the Paraguayan National Archives in Asunción. Historians noted that indigenous peoples had been sweetening herbal teas with stevia leaves "since ancient times." By the 1800s, daily stevia consumption had become well entrenched throughout the region — not just in Paraguay, but also in neighboring Brazil and Argentina. Called "Ka'a He'e" ("Sweet Herb") in Guaraní, the indigenous language of Paraguay and parts of Brazil, the stevia leaf was an important ingredient in the indigenous diet.
The indigenous peoples used stevia medicinally as well as for sweetening. They used liquid stevia for physical weakness, blood pressure, stomach and intestinal complaints as well as skin and fungal problems. In Latin America today, especially in Argentina, Brazil, Peru and Paraguay, medicines with stevia are offered for asthma, diabetes, and flu.
In 1884, the Swiss naturalist Moisés Giacomo Santiago Bertoni settled on the banks of the Paraná River in Paraguay, where he discovered the honey leaf, also known as Caa-hee. In 1905 the stevia plant was renamed Stevia rebaudiana Bertoni in honor of the discoverers Moisés Giacomo Santiago Bertoni and the chemist Ovidio Rebaudi. Rebaudi was the first to isolate and name the sweet ingredients from the leaves of the stevia plant. The description of steviosides in their present form goes back to the French chemists M. Bridel and R. Laveille in 1931.
Since Japan produced the first commercial stevia sweetener in 1971, the Japanese have been using stevia in a variety of food products from soft drinks to pickles, and stevioside has been approved and widely used in Japan for over 30 years. It was within this Japanese food-technology tradition that enzymatic modification of stevioside to produce glucosylstevioside was first developed, as a means of improving the taste profile and physical properties of the raw glycoside. In Asia, stevioside gained prominence in the 1970s, especially in Japan, where regulatory restrictions on artificial sweeteners prompted the development of natural alternatives. The Japanese pharmaceutical definition of enzymatically modified stevia (see Section 1.1 above) reflects this regulatory and commercial heritage.
All steviol glycosides — including glucosylstevioside — share the same aglycone, steviol. Steviol is substituted at C-19 and C-13 with single β-D-glucopyranose units or relatively small oligosaccharides composed of D-glucopyranose units, whether or not containing additional single D-fructofuranose, L-rhamnopyranose, D-quinovopyranose, or D-xylopyranose units. It has been established that the sweetness and taste quality of stevioside can be substantially improved via glycosylation: for instance, rebaudioside A (addition of one glucosyl group on the C-13 of stevioside), rebaudioside D (addition of one glucosyl group on the C-19 of rebaudioside A), and rebaudioside M each taste better than stevioside.
The relationship between glucosyl substitution and sensory quality has been confirmed across multiple studies. The addition of glucosyl groups generates stronger sweetness and less bitterness when the substituent number on C-13 is closer to that on C-19, and these findings convey insights into how to modify steviol glycosides to enhance their quality as sweeteners.
The dominant industrial method for producing glucosylstevioside uses cyclomaltodextrin glucanotransferase (CGTase; EC 2.4.1.19). 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. The purified steviol glycosides extract undergoes enzymatic treatment to achieve glucosylation via α-(1→4) linkages of the steviol glycosides.
Alternative enzyme systems have been explored. Enzymatic glucosylation is a strategy to reduce stevioside bitterness; the optimized efficient α-glucosylation of stevioside using the mutant glucansucrase Gtf180-ΔN-Q1140E employs sucrose as donor substrate. Dextransucrase from Leuconostoc citreum KM20 (LcDexT) has also been applied: glucosyl stevioside was synthesized via transglucosylation by LcDexT, forming α-D-glucosyl stevioside, with a production yield of 94% reached after 5 days at 30 °C, and glucosyl stevioside induced a 2-fold improved quality of taste and sweetness compared to stevioside. Enzymatic transglycosylation of stevioside using α-amylase from Aspergillus oryzae or Bacillus amyloliquefaciens, in the presence of soluble starch, has been shown to decrease the bitter aftertaste and improve sweetness potency.
A 2023 study identified a novel high-efficiency CGTase from Alkalihalobacillus oshimensis. CGTase-13 demonstrated optimal transglycosylation activity with 10 g/L steviol glycoside and 50 g/L soluble starch as substrates at below 40 °C, and the conversion rate of stevioside and rebaudioside A reached 86.1% and 90.8%, respectively. Compared with the commercially used Toruzyme® 3.0 L products, the glucosylated steviol glycoside product produced by CGTase-13 exhibited weaker astringency and unpleasant taste, faster sweetness onset, stronger sweetness intensity, and higher overall preference.
Fungal-derived beta-glucans have also been investigated as glucosyl donors. The commercial β-glucanase Finizym 250L® was employed for the transglycosylation of stevioside; after optimization of reaction parameters, the maximal reaction yield obtained was 19%, with barley β-glucan as the glycosyl donor. Alternative glucosyl donors from fungal sources reached up to 15.5% conversion yield from Pleurotus-extracted β-glucan.
A high-efficiency CGTase from Paenibacillus sp. CGMCC 5316 has been shown to produce a single specific product. With starch as glycosyl donor, this CGTase can transform stevioside into a single specific product that is an isomer of rebaudioside A and identified as mono-glycosylated stevioside; the taste of stevioside is improved noticeably by this transformation, which possesses a sucrose-like taste and has sweetness increased significantly by 35.4%.
Enzymatic transglycosylation of stevioside is a solution to improve edulcorant quality, but highly derivatized stevioside coming with high conversion is undesired. Using a commercial CGTase and cornstarch hydrolyzate with controlled parameters, the product can be mainly composed of mono- and di-glucosylated stevioside, while the highest stevioside conversion reached 77.11%.
The number and position of additional glucose units critically determines the physicochemical and sensory profile. A promising procedure is to subject steviol glycosides to enzymatic glycosylation, thereby introducing additional monosaccharide residues into the molecules; depending on the number and positions of the monosaccharide units, the taste quality and sweetness potency of the compounds will vary. One of the main obstacles for the successful commercialization of stevia sweeteners, especially in food, is their slight bitter aftertaste and astringency. These undesirable properties may be reduced or eliminated by modifying the carbohydrate moieties of the steviol glycosides. A promising procedure is to subject steviol glycosides to enzymatic glycosylation, thereby introducing additional monosaccharide residues into the molecules.
The primary scientifically documented and commercially recognized application of glucosylstevioside is as a high-intensity sweetener with an improved sensory profile compared to native stevioside or even rebaudioside A. This is the area with the most robust published evidence.
Bitterness reduction: Sensory analysis of α-glucosylated stevioside products by a trained panel revealed a significant reduction in bitterness compared to stevioside, resulting in significant improvement of edulcorant/organoleptic properties.
Taste quality improvement: Glucosyl stevioside induced a 2-fold improved quality of taste and sweetness compared to stevioside.
Aqueous solubility: After 15 days of storage at 25 °C, 98% of glucosyl stevioside in an aqueous solution was present in a soluble form, compared to only 11% for stevioside or rebaudioside A.
Stability in acidic beverages: A study investigating stability under conditions simulating beverage storage showed that under mild conditions (at a pH range of 2–6.5 over 72 hours at 50°C) stevioside and mono- and di-glucosyl-stevioside showed good stability; degradation of up to 55% was observed at pH 3 and 80°C after 72 hours, and stevioside was less stable than mono-glucosyl-stevioside. Furthermore, glucosyl stevioside exhibited a similar or improved stability in commercially available soft drinks compared to stevioside and rebaudioside A, suggesting that glucosyl stevioside could serve as a highly pure and stable sweetener in soft drinks.
Evidence strength: The evidence for taste improvement, bitterness reduction, and enhanced solubility is strong and consistent across multiple independent studies using diverse enzymatic approaches, trained sensory panels, and physicochemical analyses. This is the best-evidenced area of research for glucosylstevioside.
Research into glycemic and antidiabetic effects has focused primarily on the parent class of steviol glycosides (particularly stevioside and rebaudioside A) rather than specifically on glucosylstevioside. The evidence for glucosylstevioside per se in this area is indirect.
The natural sweetener from Stevia rebaudiana Bertoni, steviol glycoside (SG), has been proposed to exhibit a range of antidiabetic properties; the objective of one 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 of outcomes including BMI, blood pressure, fasting blood glucose, lipids, and glycated hemoglobin (HbA1c) revealed an overall significant reduction in systolic blood pressure in favor of SGs compared to placebo, with a mean difference of −6.32 mm Hg.
In animal research, lipid profile, lipid peroxidation biomarkers, and levels of enzymatic and non-enzymatic antioxidants were comparable to those of healthy rats in the treated group; the experiment confirmed beneficial effects of treatment with rebaudioside A on glucose management and lipid metabolism in diabetes. These experiments used stevioside and rebaudioside A (not glucosylstevioside specifically), so extrapolation to glucosylstevioside requires caution.
Evidence strength: Moderate-quality clinical evidence exists for the parent steviol glycosides in blood glucose and blood pressure outcomes, but there are no clinical trials specifically evaluating glucosylstevioside as a pharmacologically active compound. Animal data are preclinical and cannot be directly extrapolated to human dietary use.
Research into antioxidant properties of steviol glycosides yields mixed conclusions that depend heavily on the purity and composition of the preparation tested. Purified steviol glycoside compounds (95–98% purity) did not exhibit antioxidant activity — they were neither able to decrease basal reactive oxygen species levels at the highest concentration used nor to counteract intracellular ROS raise due to exogenous oxidative stress. This lack of antioxidant activity may be because compounds used in such studies are commercial sweeteners containing 95–98% steviol glycosides with no appreciable amounts of polyphenols, which are naturally present in stevia leaves and likely responsible for the antioxidant activity attributed to crude stevia preparations.
Evidence strength: Antioxidant activity should not be attributed to pure glucosylstevioside or purified steviol glycoside preparations based on available in vitro evidence. Any antioxidant effects observed with whole stevia leaf preparations are attributed to polyphenolic co-constituents rather than to the glycosides themselves.
Multiple pharmacological activities have been reported for the broader steviol glycoside class. Scientific reports indicate that stevia preparations or isolated compounds can exert several health-promoting effects, including antimicrobial/antifungal, antioxidant, antihypertensive, anti-inflammatory, anti-caries, and antidiabetic activity. However, these findings come from studies of the broader glycoside class — primarily stevioside and rebaudioside A — using in vitro or animal models. No studies specifically testing glucosylstevioside in these contexts were identified in the peer-reviewed literature reviewed here, and claims about glucosylstevioside sharing these properties would require independent verification.
Evidence strength: These pharmacological properties are reported for steviol glycosides generally and are not established for glucosylstevioside specifically. The evidence base for these activities is predominantly preclinical.
Glucosylstevioside is formulated almost exclusively for use as a food-grade additive, flavoring modifier, or sweetener ingredient. It is commercially produced as a dried, water-soluble powder. Stevia-based sweeteners are heat-stable, pH-stable, and not fermentable.
No specific clinical dose–response trials for glucosylstevioside as a therapeutic agent or dietary supplement have been identified in the peer-reviewed literature reviewed here. Exposure in food applications is governed by regulatory maximum permitted levels expressed as steviol equivalents.
For the broader class of steviol glycosides, including glucosylated forms, the regulatory acceptable daily intake (ADI) was established as follows. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established an ADI for steviol glycosides of 0–4 mg/kg body weight per day, expressed as steviol. The existing acceptable daily intake (ADI) for steviol glycosides (E 960) of 4 mg/kg body weight per day expressed as steviol can also be applied to glucosylated steviol glycosides.
In animal pharmacology studies of the parent glycosides, doses reported included pure stevioside or rebaudioside A administered at two levels (500 or 2500 mg/kg body weight) to Wistar rats for 5 weeks. These are preclinical research doses, not human dietary recommendations.
In the enzymatic production research context, CGTase-13 demonstrated optimal transglycosylation activity with 10 g/L steviol glycoside and 50 g/L soluble starch as substrates at below 40 °C. These are manufacturing process parameters, not human intake doses.
The EFSA Panel on Food Additives and Flavourings (FAF) assessed the safety of glucosylated steviol glycosides proposed for use as a new food additive in different food categories. The path to regulatory approval involved an initial rejection. The EFSA Panel on Food Additives and Nutrient Sources added to Food (EFSA ANS Panel) had previously evaluated the safety of glucosylated steviol glycosides to be used as a food additive and concluded that the submitted data were insufficient to assess its safety (EFSA ANS Panel, 2018). After a new application with additional metabolic and toxicological data, the Panel concluded that there is no safety concern for the use of glucosylated steviol glycosides as a new food additive at the proposed use and use levels.
In the United States, methods for the enzymatic modification of steviol glycosides are described in GRAS notices, such as GRN 000662, and are referred to as enzyme-modified steviol glycosides. The modification described involves treatment of steviol glycosides with a glucotransferase that results in additional glucose moieties conjugated to the parent steviol glycoside structures via α-(1–4) linkages, yielding a mixture containing up to twenty additional glucose moieties. Enzyme-modified steviol glycosides for which the FDA does not have questions following review of a GRAS notification are among the principal steviol glycosides considered permissible for food use.
Previous studies have confirmed the digestion of enzymatically modified stevia (EMS) to steviol glycosides, which are then degraded to steviol by gut microflora, metabolized to glucuronide in the liver, and finally excreted in the urine. In simulated gastric and intestinal fluid assays, there were no significant changes in glucosylated steviol glycosides (GSG) and steviol glycosides. The anaerobic metabolism study using human fecal homogenates showed complete decomposition of GSG into steviol, similar to common SG such as rebaudioside A, rebaudioside M, and stevia extract. The results of the in vitro digestion study show that ingested GSGs can be digested similarly to other SGs that have established safety for consumption.
The EFSA Panel considered that the metabolism of glucosylated steviol glycosides is sufficiently similar to that of the already authorized steviol glycosides, and thus, the toxicological data previously assessed for steviol glycosides (E 960) were considered to support their safety as a food additive. The critical conclusion is that the ultimate metabolite — the aglycone steviol — is the same regardless of whether glucosylated or non-glucosylated precursors are consumed.
The metabolic fate of steviol glycosides leads to the aglycone which is absorbed; given that all steviol glycosides follow the same metabolic pathways, the EFSA Panel considered that glucosylated steviol glycosides would fall within the same group of substances and the group approach to toxicological assessment would be applicable.
The 2018 EFSA opinion identified specific concerns regarding incomplete data. The limited evidence provided in the 2018 application dossier did not demonstrate the complete hydrolysis of the glucosylated steviol glycosides. No toxicological studies on glucosylated steviol glycoside preparations had been provided for assessment. The Panel concluded at that time that the submitted data were insufficient to assess the safety of the preparations. These gaps were subsequently addressed in the 2022 resubmission, leading to the positive EFSA opinion.
The EFSA Panel noted that the proposed glucosylated steviol glycoside preparations may contain up to 20 glucose moieties per molecule of steviol glycoside, and that the proposed permitted level for this new food additive is expressed on a steviol equivalent basis. The Panel therefore considered that this could lead to an additional exposure to glucose from a sweetener proposed to have a technological function of replacing sugars in food. This observation — that glucosylated preparations carry extra glucose that is released upon hydrolysis — was considered in the safety evaluation.
The stability profile is relevant to safety. The stability of stevioside and mono- and di-glucosyl-stevioside (produced via Leuconostoc citreum SK24.002 alternansucrase acceptor reaction), and the possible formation of steviol at elevated temperature and different pH levels, was assessed, covering a typical pH range that simulated both relevant and extreme beverage storage conditions. Complete degradation was observed at pH 2 and 80°C after 72 hours. The principal degradation concern at extreme conditions is the generation of the aglycone steviol, whose safety profile is well characterized in the existing regulatory dossiers for steviol glycosides.
No specific allergenicity signals for glucosylated steviol glycosides have been identified in the EFSA assessment reviewed here. The safety of steviol glycosides has been reviewed by several scientific bodies and regulatory agencies, including JECFA, the European Commission's Scientific Committee on Food, EFSA, Food Safety Australia/New Zealand, and Health Canada. None of these reviews have identified specific drug interaction profiles or allergenic potential for purified steviol glycoside preparations, including the glucosylated form, under normal dietary use conditions.
Individuals who are allergic to plants in the family Asteraceae (Compositae) — the botanical family to which Stevia rebaudiana belongs — may potentially show cross-sensitivity, but this pertains to the whole leaf and crude extracts and has not been specifically characterized for purified glucosylstevioside preparations in the sources reviewed.
Under EU food law, glucosylated steviol glycosides (E 960d) are listed alongside steviol glycosides from stevia (E 960a) and enzymatically produced steviol glycosides (E 960c) in Annex II to Regulation (EC) No 1333/2008. These food additives have combined maximum permitted levels for use in foods, expressed as steviol equivalents, and are listed in the functional group of sweeteners.
JECFA reviewed the safety of steviol glycosides at four separate meetings (51st, 63rd, 68th, and 69th) in 1998, 2004, 2007, and 2008, establishing specifications and an ADI for stevia extract as a high-potency sweetener. In 2011, the Codex Alimentarius Commission (CAC) adopted proposed draft maximum levels for steviol glycosides in foodstuffs; Codex standards are developed by committees from United Nations member states and these standards are often used by many countries to support their own approval process.
Health conditions that Glucosylstevioside may help support.
Body systems that Glucosylstevioside may help support.
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