Rebaudioside A

Stevia (Stevia rebaudiana Bertoni )

Stevia contains natural compounds, especially stevioside and rebaudioside A, that are estimated to be 150–400 times sweeter than saccharose. Used for centuries in parts of South America, stevia has been discovered in recent years by much of the calorie-conscious modern world. It is now widely and legally consumed by millions of people, from South Korea, Israel, and the People's Republic of China, but no country has done more to demonstrate stevia's dietary and economic potential than Japan, where the herb and its extract have been used since 1970s.

The Japanese, having subjected stevia extract to extensive safety testing and found it without health risk, now incorporate it in numerous food products, including soft drinks.

3.10.4.1.3(ii) ent-Kaurane

Rebaudioside A (4) has a branched trisaccharide unit at C-13 and is sweeter and more pleasant tasting than stevioside (5), with a C-13 sophorosyl disaccharide moiety. Removal of the C-19 sugar unit of rebaudioside A, so as to produce rebaudioside B (19), results in a less potently sweet-tasting compound. Rebaudioside C (20), having a terminal glucose unit at C-13 replaced by rhamnose, is not only less sweet than rebaudioside A (4), but is somewhat bitter. Sauvioside A (27) is unusual among the ent-kaurane sweet glycosides in that it contains no C-16, C-17-exomethylene group. Sauvioside B (28), which differs from rubusoside (24) only in the presence of a C-9 hydroxy group, has only half of the resultant sweetness potency (Table 1).^19,109

There is now a very large technical and patent literature on S. rebaudiana and its sweet steviol glycoside constituents. This information refers principally to methods for the purification of these substances, procedures for taste improvement, and biological test results.

Stevioside

Stevia species are members of the family of Asteraceae (qv). Stevia rebaudiana contains steviol glycosides, such as stevioside and rebaudioside A, used as artificial sweeteners and 100–300 times sweeter than sucrose [14,15].

Steviol glycosides have been reported to have mutagenic effects in some bacteria in vitro, but the results have been variable and are thought not to be relevant to daily use of stevioside as a sweetener [16].

In a multicenter, double-blind, randomized, placebo-controlled study, 106 Chinese subjects with hypertension aged 28–75 years were given stevioside 250 mg or placebo tds for 1 year [17]. After 3 months, the mean systolic and diastolic blood pressures in those who took stevioside fell significantly: systolic from 166 to 153 mmHg and diastolic from 105 to 90 mmHg, and the effect persisted for the whole year. There were no significant changes in lipids or glucose and no adverse reactions. In a similar 2-year study in 174 subjects by the same investigators, mean systolic pressure fell from 150 to 140 mmHg and diastolic pressure from 95 to 89 and there was a reduced incidence of left ventricular hypertrophy [18].

However, in a Brazilian randomized, placebo-controlled study in patients with previously untreated mild essential hypertension crude stevioside 3.75 mg/kg/day for 7 weeks, 7.5 mg/kg/day for 11 weeks, and 15 mg/kg/day for 6 weeks had no significant effect on blood pressure compared with placebo; there were no major adverse reactions during the trial [19]. In a randomized, double-blind, placebo-controlled, study in subjects with type 1 diabetes, subjects with type 2 diabetes, and subjects without diabetes and with normal/low normal blood pressures who were randomly allocated to stevioside 250 mg tds or placebo and were followed for 3 months, systolic and diastolic pressures, glucose, and glycated hemoglobin (HbA_1c) were not altered; there were no adverse reactions [20]. In a randomized, double-blind, placebo-controlled study of the stevia glycoside rebaudioside A 1000 mg/day for 4 weeks in 100 individuals with normal and low-normal blood pressures aged 18–73 there was no change in blood pressure or heart rate [21].

Abstract

The leaves of Stevia rebaudiana, a natural sweetener, contain more than 30 different steviol glycosides, of which stevioside (STV) and rebaudioside A are found in major proportion. The main chemistry structures of glycosides found in stevia are diterpenes with strong antioxidant and antiinflammatory properties. Most liver diseases involve nitrogen and oxygen free radicals that attack macromolecules such as lipids, proteins, and nucleic acids for the production and perpetuation of hepatic damage. Moreover, inflammation is a characteristic of acute hepatic damage and when it persists over time, chronic liver damage is established and fibrosis ensues with the consequent production of cirrhosis. Therefore, the well-known antioxidant and antiinflammatory properties of stevia make this medicinal herbal, mainly used to treat diabetes, an excellent therapeutic strategy to fight liver disease; in fact, some evidence demonstrates its actual beneficial effects on liver damage. However, this area remains almost unexplored and deserves further basic and clinical investigation.

7.7 β-Glycosyltransferase Glycosylation Systems Using UDP-Sugars

Only a few studies concerning the modification of steviol glycosides, using β-glycosyltransferases in combination with nucleotide sugars as donor substrates, have been reported.

Bovine colostrum β-1,4-galactosyltransferase has been tested as a biocatalyst for the β-galactosylation of stevioside (5) and steviolbioside (4) using UDP-Gal as galactose donor.²⁰⁷ In both cases, the terminal Glc residue of the C-13-β-sophorosyl disaccharide could be elongated with a Gal(β1-4) residue in high yields of 81% (106) and 95% (107) (Table 8), respectively. The synthesized galactosylated products showed improved sweetness and decreased aftertaste.

Recently, to improve the organoleptic properties of steviol glycoside products, an efficient (78%) enzymatic conversion of stevioside (5) into rebaudioside A (11) was reported, using recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana, together with the sucrose synthase AtSUS1 from Arabidopsis thaliana.²⁰⁸ The latter enzyme was used to catalyze the generation of the glucose donor, UDP-glucose, from sucrose and UDP. In a similar way, stevioside was converted to rebaudioside A using recombinant Saccharomyces cerevisiae, in which UGT76G1 was overexpressed as the whole-cell biocatalyst in a glucose-containing medium. The addition of citrate regulated the yeast metabolism toward enhanced synthesis of UDP-glucose.²⁰⁹

4.16.2.1.2 Stevioside

Several ent-kaurenoid diterpene glycosides with steviol as a common aglycon have been isolated from Stevia rebaudiana, which is native to subtropical and tropical South America and Central America.^23–27 Among the glycosides, stevioside is the most abundant followed by rebaudioside A. Stevioside is 140 times sweeter than sucrose, while rebaudioside is 240 times sweeter. Rebaudioside A has a better quality of sweetness. In Japan, stevia sweeteners have been produced commercially and are widely used in food products such as soy sauce, pickles, and boiled fish paste. Steviol glycosides are stable enough to remain sweet in processed foods.

4.18.3.4.2 Stevia Glycosides

Stevioside can be extracted using hot water leaching of the stevia leaf, using a Soxhlet apparatus.¹¹⁷ Stevioside and rebaudioside A were extracted using SC-CO₂ with or without the addition of ethanol–water as co-solvent.¹¹⁷ Stevioside extraction was significantly influenced by temperature and co-solvent addition, but not by pressure. The highest extraction yield was obtained at the highest temperature investigated (80 °C), and at ethanol co-solvent level of 20%. Rebaudioside A extraction was only affected significantly by ethanol co-solvent addition, with levels above 10%, resulting in the highest solubility.¹¹⁷ The highest yields, 41.10 mg g⁻¹ stevioside and 18.80 mg g⁻¹ rebaudioside A, were obtained at 25 MPa, 80 °C using 20% co-solvent. For traditional extraction methods, when water was used, the extract contained 41.96 mg g⁻¹ stevioside and 22.53 mg g⁻¹ rebaudioside A, whereas when ethanol was used the values were 33.76 mg g⁻¹ and 14.84 mg g⁻¹, respectively. Therefore, for stevia glycoside extraction, SC-CO₂ modified with ethanol is certainly viable in terms of the yields obtained.

History

The term steviol glycosides refers to mixtures of compounds extracted from the leaves of the plant Stevia rebaudiana. Several of these glycosides have the property of intense sweetness. Two of the major steviol glycosides contributing to the sweet taste of stevia extracts, namely, stevioside and rebaudioside A are reported to be 100–400 times as sweet as sucrose.

Purified stevia extracts have a long history of use as sweeteners in some countries (e.g., Japan), whereas regulatory approval of steviol glycosides in Europe, the USA, Australia, and New Zealand has been relatively recent. Steviol glycosides were approved as food additives in the USA, Australia, and New Zealand in 2008 and in Europe in 2011. Steviol glycosides are permitted in a large variety of foods and beverages over a wide range of maximum permitted levels. For example, in Australia and New Zealand, maximum permitted levels range from 50 (in fruit and vegetable juices) to 1100 mg kg⁻¹ (in sugar confectionary).

Stevia Sweeteners

Stevia rebaudiana Bertoni (Compositae) is a herb native to Paraguay but cultivated in South-east Asia, Japan, Paraguay, Brazil, Israel, and USA. Stevia sweeteners are extracted from the dried leaves, clarified and crystallized. The sweet constituents of stevia include eight diterpene glycosides: stevioside, steviolbioside, rebaudiosides A, B, C, D, and E, and dulcoside A, which collectively are 100–300 times sweeter than sucrose. They are similar in structure in that a steviol aglycon is connected at C-4 and C-13 to mono-, di-, or trisaccharides consisting of glucose and/or rhamnose residues, as shown in Figure 5. Stevioside is the major constituent, whereas dulcoside A and rebaudiosides A and C are the other main components. Stevioside is 300 times sweeter than sucrose (Table 1). It shows a sweetness profile similar to that of sucrose, except that it has an unpleasant, persistent, menthol, and bitter aftertaste. However, the development of new cultivars, derivatization and incorporation of cyclodextrin, l-histidine, potassium phosphate, glucono-δ-lactone and maltose in formulations has eliminated undesirable aftertastes. Rebaudioside A is more stable, sweeter, and has a better taste profile than stevioside. The remaining diterpene glycosides are not as sweet as stevioside.

Stevioside is a white powder, highly soluble in water, ethanol, and methanol, and is nonfermentable. When heated at 100 °C for 1 h, solutions of stevioside at pH 3–9 show little loss in sweetness and no change at 22 °C for 5 months. However, considerable decomposition occurs at pH 10. Some degradation of stevioside and rebaudioside has been observed in carbonated beverages acidified with phosphoric and citric acids during storage at 37 °C. Heating at 60 °C for 6 days has resulted in 0–6% loss of sweetness. Exposure to 1 week of sunlight does not affect stevioside, but results in 20% loss of rebaudioside A. The high stability of stevioside makes it a suitable sweetener for cooked and baked foods and for beverages.

Stevioside suppresses the growth of oral microorganisms, and both stevioside and rebaudioside A provide very few calories. In Paraguay, S. rebaudiana is used for the treatment of diabetes because of its hypoglycemic activity. Studies have suggested that stevioside is not toxic, mutagenic, or teratogenic in a number of animal species. In addition, the product has been used for more than 10 years in South America and Japan, but there are contradictory reports on the in-vivo metabolism to steviol, which is mutagenic for Salmonella typhimurium TM677.

Stevioside is available in three purity ranges: crude extract, and 50% and 90% purity. Since 1970, stevia sweeteners have been used in a wide range of food and beverage applications in Japan, including soft drinks, candies, chocolate, chewing gum, icecream, yogurt, jam, pudding, and table-top sweeteners. It is commonly used in combination with sucrose and fructose and also with other sweeteners such as aspartame, cyclamate, and acesulfame-K, but not with saccharin. It is currently approved for use in Japan, Taiwan, and Mercosur. An acceptable daily intake of stevioside for humans of 7.94 mg per kilogram of body weight has been suggested.

Adjusting Recipes and Meals for People with Diabetes

Like cooking and baking for people with cardiovascular disease, adapting favorite recipes for diabetics requires scrutinizing ingredients and techniques and experimenting with different products and cooking methods. In some cases, taste and texture will hardly be affected. In other instances, changes to the original recipes may be significant. Sugar and fat substitutes do not taste the same or react the same way to the ingredients they mimic. Using real ingredients but less of them, just like heart-healthy cooking, is also a good strategy for diabetic cooking.

Portion size is critical because it reduces calories, and fewer calories may help blood sugar management. However, smaller portions may look insignificant on a plate. That is where the use of fresh and cooked vegetables with only 5 grams of carbohydrates per serving comes in. Free foods (such as salad greens) with virtually no calories come in handy.

Draw an imaginary line through the center of the lunch or dinner plate and then another line that dissects one-half of the plate into two sections. One of these sections (about one-fourth of the plate) should be filled with whole grains, such as cooked brown rice or quinoa, or starchy vegetables, such as corn, peas or root vegetables. The other section (also about one-fourth of the plate) should be filled with lean protein foods, such as lean meats, seafood, skinless poultry or tofu.