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Standard and Specification
> Natural Additives
> Steviol glycoside
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| Definition |
Stevioside is obtained from leaves of Stevia rebaudiana Bertoni. Its major component is stevioside. It contains sweetening component of trees which have isosteviol as its framework.
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| [Compositional Specifications of Steviol glycoside] |
| Content |
When Stevioside is dried for 2 hours at 100°, it should contain not less than 98.0% of stevioside (C38H60O18). |
| Description |
Stevioside is white or grayish white powder or crystalline powder with sweet taste. |
| Identification |
(1) 1 g of Stevioside is dissolved in 100 ml of distilled water by heating in a water bath. After cooling, it is shaken will with 100 ml of n-butyl alcohol. It is then set aside to separate two phases. The aqueous phase is discarded and n-butyl alcohol layer is used as Test Solution. When 10 ml of the Test Solution is mixed with 10 ml of anthrone solution and heated in a water bath, the solution turns green.
(2) 0.5 g of Stevioside is dissolved in 20 ml of 5% sulfuric acid, which is heated for 2 hours in a water bath. After cooling, the solution is extracted twice with 50 ml each of ether. The extracts are added and washed with small amount of distilled water. Then ether is evaporated to dryness. The residue is crystallized by adding 1 ml of methyl alcohol. The crystallites are washed with small amount of methyl alcohol and dried. The melting point of the crystallites should be 227∼232°. |
| Purity |
(1) Arsenic : 0.5 g of Stevioside is placed in a platinum, quartz, or porcelain crucible. 10 ml of magnesium nitrate in ethyl alcohol(1→50) is added to the crucible and then alcohol is ignited. It is then reduced to ash by heating at 450∼550°. If carbonaceous substance persists, it is wetted with minute amount of nitric acid, which is further heat treated at 450∼550°. After cooling, 3 ml of hydrochloric acid is added to the residue, which is then dissolved by heating in a water bath. When test for arsenic is carried out with this test solution, it should not be more than 2ppm.
(2) Heavy Metals : 1 g of Stevioside is carbonized by heating mildly in a quartz or porcelain crucible. After cooling, add 2 ml of nitric acid and 5 drops of sulfuric acid, it is heated until white smoke disappears, which is then reduced to ash by further heating at 450∼550°. After cooling, 2 ml of hydrochloric acid is added, which is then evaporated to dryness in a water bath. 3 drops of hydrochloric acid and 10ml of hot water are added to the resulting residue, which is then heated for 2 minutes. After cooling, 1 drop of phenolphthalein indicator solution is added, then ammonia solution is added until the color of the solution becomes pale red. The resulting solution is transferred into a Nestler cylinder by rinsing with water. 50 ml of test solution is prepared by adding 2 ml of diluted acetic acid (1→20) and water. When this solution tested for heavy metals, the content should not be more than 30ppm. Color standard solution is prepared by the following procedure. 2 ml of nitric acid, 5 drops of sulfuric acid, and 2 ml of hydrochloric acid are added and evaporated to dryness in a crucible that is made of the same material used for test solution preparation. 3 drops of hydrochloric acid are added to the residue, which is then transferred into another Nestler cylinder as described above. Finally, 2 ml of lead standard solution, 2 ml of diluted acetic acid (1→20), and water are added to bring the total volume to 50 ml. |
| Loss on Drying |
When 3 g of Stevioside is dried for 2 hours at 100°, the loss should not be more than 2%. |
| Assay |
(1) Preparation of Test Solution : Approximately 50 mg of Stevioside is precisely weighed into a 30 ml Erlenmeyer flask, where 15 ml of 20% sulfuric acid added. A reflux condenser is attached to the flask, which is then heated for 2 hours in a water and cooled in running water. The solution is transferred into a separatory funnel using 10 ml of water. The flask is washed with 80 ml of ether, which is added to the separatory funnel. The mixture is well shaken and set aside to separate the phases. The aqueous phase is discarded. The ether phase is washed twice with 20 ml each of water. The ether phase is transferred into a 200 ml beaker. The separatory funnel is washed twice with 20 ml each of ether, which is added to the beaker. 10 ml of anhydrous sodium sulfate is added and set aside for 10 minutes to dehydrate the ether phase. The resulting ether phase is transferred into a 150 ml Erlenmeyer flask with a stopper. The beaker is washed three times with 10 ml each of ether, which is added to the Erlenmeyer flask. Ether is evaporated out in a 60～70° water bath. The residue is dissolved in 10 ml of chloroform. After adding 1 ml solution of diazomethane solution in ether 4(w/v%), it is plugged with a stopper and set aside for 20 minutes while stirring occasionally. 0.5 ml of glacial acetic acid is added to the solution, which is well shaken. Test Solution is prepared by adding 2 ml of internal standard solution (squalene in n-butyl alcohol 12.5 mg/ml) to the resulting solution.
(2) Standard Solution : Using 50 mg of stevioside standard, a Standard Solution is prepared by following the same procedure as the Test Solution. Test Solution and Standard Solution are tested by Gas Chromatography under the following operation conditions.
[Operation Conditions]
- Column : A glass tube with inner diameter of 3 mm x length of 2 m
- Column Filler : 2% OV-17, Chromosorb W․DMCS (80∼100 mesh)
- Detector : (Hydrogen) Flame Ionization Detector (FID)
- Temperature at injection hole : 260°
- Column Temperature : 235°
- Detector Temperature : 260°
- Carrier gas and flow rate : N2, 30～60 ml/min
A×amount of stevioside standard(mg)
Content(%) ＝ ━━━━━━━━━━━━━━━━━━━━━ × 100
As×amount of sample(mg)
A : Peak ratio of isosteviol methyl ester vs. squalene for Test Solution
As : Peak ratio of isosteviol methyl ester vs. squalene for Standard Solution |
| Permitted Use Level of Stevioside |
Should not be used for the food items listed below.
1. Loaf bread
2. Infant formula, follow-up formula
3. Formulated grain food for infants and babies, other baby foods
4. White sugar
5. Brown sugar
6. Glucose
7. Molasses
8. Candies
9. Bee honey
10. Processed milk (ice cream, powdered ice cream, ice cream mix are excluded) |
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