Glycyrrhizin, Steviosides, Thaumatin

1. Non-Nutritive Sweeteners

2. Content
Preparation, Evaluation and salient features of
• Steviosides
• Thaumatin
• Glycyrrhizin

3. Introduction
• What is non-nutritive sweeteners?
• Major non-nutritive sweeteners in use throughout the world
are mostly synthetic sweeteners such as aspartame,
saccharin, cyclamate, acesulfane-K and alitame.
• Most of the non-nutritive sweeteners now are far more
sweeter than sucrose and a very small quantity is required for
sweetening food preparation.
• Sweetness potency denotes, how many times a given
compound is more potent than sucrose on the same weight
basis.
• e.g. 0.75g/1 of aspartame, a non-nutritive sweetner matches
with the sweetness of 100g/1 of sucrose solution. Therefore ,
aspartame is considered as 133 times sweeter than sucrose.

4. Introduction
• Sweetness potency determination is a subjective matter and it
can be affected by the sensitivity and experience of the taster,
other ingredients present in the solution, texture of food, pH,
temperature of samples etc.
• The other qualities of sugar should also be present with a
good alternative sweetener.
• These include safety along with sweetness, stability with
other food ingredients, higher water solubility, dissolution
rate and easily handle able nature.
• It should be non hygroscopic, economical, should have high
degree of consumer acceptance and synergism with other
sweetening agents.
• These qualities are important for even extremely potent
sweetener in order to be developed as commercial products.

5. • The FDA has given the label “Generally Recognized
as Safe” (GRAS), to five* NNSs:
• Aspartame (NutraSweet® and Equal®)
• Acesulfame-K (Sweet One®)
• Neotame
• Saccharin (Sweet’N Low®)
• Sucralose (Splenda®)

6. Sweetness index

7. Stevioside
• Source: It is a naturally occurring sweetener. It is extracted
from a south american plant “Stevia rebaudiana” belonging
to family Compositae.
• GS: occurs in Paraguay & Brazil but commercially cultivated in
Japan. Also cultivated in Southern China, Taiwan, Thailand and
Malaysia.
• Form used: The dried leaves of the plant, the water extract of
the leaves and purified ingredients of the extract are used as
sweetening agents.
• Marketed brand: Truvia®, PureVia®, Enliten®

8. Stevioside
• Preparation:
• Stevioside can be isolated from the leaves of Stevia by
extraction with water or water ethyl alcohol mixture and
further purified by treatment with calcium and magnesium
hydroxide or carbonate.
• The ration of rebaudioside A to stevioside can be increased by
extraction with methanol.
• Two compounds isolated and purified from aqueous extract
are stevioside and rebaudioside A.
• Features: Both these compounds are diterpene glycosides.
• Stevioside is 160-170 times sweeter than sucrose.
• Its sweetness is tainted with a bitter and undesirable after
taste.

9. Stevioside
• But its wide use in Japan for over 20 years did not produce
any known deleterious side effects.
• In USA, the FDA allowed the use of stevia as a diet
supplement but not as a sweetener or an ingredient for food.
• Stevioside and Rebaudioside A give rise to its principal
metabolite, steviol which has been reported to be mutagenic.
• Stevioside, the more important product, is used as a table top
sweetener in confectionaries, soft drinks and fruit products.
• The sweetness intensity of rebaudioside A is approximately
one third higher than that of stevioside and its taste
characteristics are also superior. But its unstable and gets
decomposed to light.

10. Stevioside
• Evaluation by TLC
• Stationary phase: Silica Gel G
• Mobile phase: chloroform: methanol: water (65:25:4)
• Detection: Iodine & 10% aqueous H2SO4
• Spot color: black & light brown
• Rf value: 0.3-rebaudioside A, 0.4-Stevioside
• Reference: ijpsonline.com

11. Glycyrrhizin
• Source: it is a mixed calcium and potassium salt of glycyrrhizic
acid found in licorice root, “Glycyrrhiza glabra” belonging to
family Leguminosae.
• GS: largely cultivated in Russia, Iran, Iraq and in many other
countries of Europe & Asia.
• Features:
• It is a triterpenic glycoside of beta amyrin type which contains
2 beta-1,2-glycosidic linked glucuronic acids.
• It gives glycyrrhetic acid as an aglycone after hydrolysis.
• Licorice root extracts containing 6-14% glycyrhizinates are
available as licorice.
• Glycyrrhizin is obtained from the dried, cut licorice roots
which contains about 2-7% of this compound.

12. Glycyrrhizin
• Preparation:
• A crude licorice extract is obtained by counter current
extraction with water.
• After removal of removal of polysaccharides, glycyrrhizic acid
can be precipitated from the crude extract with sulphuric acid.
• Glycyrrhizic acid is then converted to ammonium
glycyrrhizinate by treatment with aqueous ammonia and
subsequently dried or precipitated with ethanol.
• Repeated crystallization yields almost colorless
monoammonium glycyrrhizinate.
• Ammonium salt improves the solubility of glycyrrhizic acid in
water.

13. Glycyrrhizin
• Features:
• Glycyrrhizin is claimed to be about 50-100 times sweeter than
sucrose in dilute aqueous solution where as ammonium
glycyrrhizinate is about 50 times sweeter as compared to
sugar.
• Glycyrrrhizin and its salts are characterized by a delayed
sweetness onset, a lingering sweetness with a characteristic
liquorice taste and therefore considered as a flavouring agent
rather than sweetener.
• Glycyrrhizinates are used as flavouring agents in confectionary
and pharmaceuticals.
• In addition to their flavouring properties, glycyrrhizinates
show flavour enhancing effect in certain products.

14. Glycyrrhizin
• Features:
• It is also used in soft drinks, tobacco products and oral
hygiene products.
• Glycyrrhizin is characterized by certain physiological activities
such as oedema and hypertension, which discourage its use in
large quantities for sweetening purpose.
• It is not an approved sweetener in many countries.
• However, glycyrrhiza root extracts containing at least 90%
glycyrrhizin are widely used in Japan, second only to Stevia
sweeteners.
• Due to its other pharmacological effects, its consumption
should be less than 200 mg/day in drug formulation.

15. Glycyrrhizin
• Evaluation by TLC
• Stationary phase: Silica Gel G
• Mobile phase: Chloroform: GAA: Methanol: Water
(60:32:12:8)
• Detection: Anisaldehyde H2SO4 acid reagent
• Spot color: violey zone
• Rf value: 0.35-0.4
• Reference: Wagner book

16. Thaumatin
• Source: It is mixture of proteins isolated from the fruit of an
African plant “Thaumatococcus danielli” belonging to family
Marantaceae.
• GS: marketed in USA and Japan (Talin)
• Composition:
• The frozen fruit undergoes an aqueous extraction and the
protein fraction is resolved by physical techniques
(ultrafiltration).
• The extract contains 2 chief proteins (Thaumatin I & II) which
are each composed of 207 amino acids and differ from one
another only in 5 positions, their structure includes 8
disulphide bridges.

17. Thaumatin
• Thaumatin is readily soluble in water and soluble in dilute
alcohols.
• Its stability is maximum at pH 2.7-3 (the sweet taste turns
acidic at pH <2) and the sweetening power does not
disappear upon heating.
• Solutions maybe pasteurized, but prolonged treatment
(sterilization) destroys the sweet taste.
• Properties:
• It is a potent sweetener: its activity can be detected at a
concentration of 10(-8) M

18. Thaumatin
• The sweet sensation induced by thaumatin is slightly delayed,
it persists for 15-20 minutes (with licorice aftertaste), hence
its application in products such as chewing gums or breath
fresheners.
• It is not toxic, not carcinogenic and it enhances aromas and
flavours at low doses.
• At higher doses, it is an intense sweetener.
• Research efforts to come up with production methods that do
not require using the plant (transgenic organism) have not yet
succeeded in achieving sufficient yields.
• Commercial product is sweeter between 750 to 1600 times
than sucrose.

19. References
• Pharmacognosy & Phytochemistry by Vinod
Rangari Vinod, Page no. 125-131
• Pharmacognosy by Bruneton, Page no. 215-
216.
• https://www.fda.gov/media/124271/downloa
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