Hoshin-Sweetenersg

INTENSE SWEETENERS INTENSE SWEETENERS – – An InsightAn Insight

Dr. VISHWAS GEORGEDr. VISHWAS GEORGE

SweetnessSweetness

A fundamental component of tasteA fundamental component of taste

Due to a structural feature called glycophoreDue to a structural feature called glycophore

Sweet taste contributed by compounds like Sweet taste contributed by compounds like inorganic salts, sugars, amino acids, proteins, inorganic salts, sugars, amino acids, proteins, cyclic and aromatic organic derivatives cyclic and aromatic organic derivatives (Prodolliet, 1996)(Prodolliet, 1996)

Mechanism of sweetnessMechanism of sweetness Due to intermolecular hydrogen bonding between glycol Due to intermolecular hydrogen bonding between glycol

unit and taste bud receptor siteunit and taste bud receptor site

Glycophore comprises of AH, B coupleGlycophore comprises of AH, B couple

– A and B are electronegative atoms separated by a A and B are electronegative atoms separated by a distance of greater than 2.5 Adistance of greater than 2.5 Aoo but less than 4 A but less than 4 Aoo

– Hydrogen atom (H) is attached to one of the Hydrogen atom (H) is attached to one of the electronegative atoms by covalent bondelectronegative atoms by covalent bond

(Shallen Berger and Acree, 1967)(Shallen Berger and Acree, 1967)

A – H------------ B

B---------------H- A

RECEPTOR SITE SWEET UNIT

Classification of sweetenersClassification of sweeteners Sugars (refined sugars, sucrose, fructose, glucose, dextrose, maltose, etc.) Bulk

(nutritive) Sugar replacements, polyols or sugar alcohols (sorbitol, mannitol, xylitol, isomalt, etc.)

Sweeteners

Natural /plant origin (glycyrrhizin, steviosides, thaumatin, neohesperidine dihydrochalcone)

Intense (non-nutritive)

Synthetic (aspartame, acesulfame-K, saccharin,

sucralose, cyclamate, etc.)

Artificial sweetenersArtificial sweeteners Intensely sweet substances other than sucrose with no or Intensely sweet substances other than sucrose with no or

low caloric valueslow caloric values

Result of specific chemical reaction, with diverse structuresResult of specific chemical reaction, with diverse structures

Different physical and chemical characteristicsDifferent physical and chemical characteristics

Advantages – no effect on blood sugar, non-caloricAdvantages – no effect on blood sugar, non-caloric

Disadvantages – unpleasant aftertaste, cannot replace sugar Disadvantages – unpleasant aftertaste, cannot replace sugar entirely in baked productsentirely in baked products

An ideal sweetenerAn ideal sweetener Tastes like sugar Tastes like sugar

As sweet as or sweeter than As sweet as or sweeter than sucrosesucrose

Pleasant taste with no Pleasant taste with no aftertaste aftertaste

Colorless and odorlessColorless and odorless

Readily solubleReadily soluble

Stable in all processing Stable in all processing environments environments

Cost competitive Cost competitive

Low / no calories Low / no calories

Doesn't promote tooth Doesn't promote tooth decay decay

Weight maintenance/ Weight maintenance/ reductionreduction

Diabetes managementDiabetes management

Non toxic to all persons Non toxic to all persons

Easily availableEasily available

ApplicationsApplications Tabletop sweetenersTabletop sweeteners

– Saccharin, aspartame, sucralose, acesulfame potassium, steviosidesSaccharin, aspartame, sucralose, acesulfame potassium, steviosides

Processed foodsProcessed foods– Sucralose, acesulfame potassium, saccharin, cyclamateSucralose, acesulfame potassium, saccharin, cyclamate

BeveragesBeverages– Sucralose, aspartame, acesulfame-k, saccharin, stevia, Neotame, Sucralose, aspartame, acesulfame-k, saccharin, stevia, Neotame,

Neohesperidine dihydrochalcone (NHDC)Neohesperidine dihydrochalcone (NHDC)

Confectionaries, candy and chewing gumsConfectionaries, candy and chewing gums– Sucralose, aspartame, acesulfame-k, saccharinSucralose, aspartame, acesulfame-k, saccharin

Dairy productsDairy products– Sucralose, acesulfame potassium, saccharin, aspartame, cyclamateSucralose, acesulfame potassium, saccharin, aspartame, cyclamate

AlitameAlitame– Aspartic acid containing dipeptideAspartic acid containing dipeptide– 2000 times sweeter than sucrose 2000 times sweeter than sucrose – No aftertaste, no phenylalanineNo aftertaste, no phenylalanine– Mexico, Australia, NZ, ChinaMexico, Australia, NZ, China

NeotameNeotame– 8000-13000 times sweeter than sucrose 8000-13000 times sweeter than sucrose – Moderately heat stableModerately heat stable

SteviosideStevioside– 300 times sweeter than sucrose 300 times sweeter than sucrose – Heat stableHeat stable– SA, Japan, China, KoreaSA, Japan, China, Korea

CyclamateCyclamate– 30-50 times sweeter than sucrose30-50 times sweeter than sucrose– Heat stableHeat stable– US, Canada, SwitzerlandUS, Canada, Switzerland

Neohesperidine dihydrochalconeNeohesperidine dihydrochalcone– 1500-1800 times sweeter than sucrose 1500-1800 times sweeter than sucrose – Heat stableHeat stable– Synergistic & Flavour enhancerSynergistic & Flavour enhancer

Intense sweeteners (Outside India)Intense sweeteners (Outside India)

National Regulatory Standards PFA Act, 1954; PFA Rules, 1955 (2007 Ed.)

Artificial sweetener Article of food Maximum limit (ppm)

SODIUM SACCHARIN

Carbonated Water 100Soft drink concentrate 100

Supari 4000Pan Masala 8000

Pan flavouring material 8 percentChocolate (White, Milk, Plain,

Composite and Filled)500

Sugar based/Sugar free confectionery 3000Chewing gum/Bubble gum 3000

Sweets (carbohydrates based and milk products based):-Halwa, Mysore Pak,

Boondi ladoo, Jalebi, Khoya burfi, Peda, Gulab Jamun, Rasogolla and

similar product based sweets

500


ASPARTAME


Biscuits, Bread, Cakes & Pastries 2200Jam, Jellies, Marmalades, Custard

powder 1000

Chocolate (White, Milk, Plain, Composite and Filled)

2000

Sugar based/Sugar free confectionery 10000Chewing gum/Bubble gum 10000Vegetarian jelly crystals 3000




200


ACESULFAME POTASSIUM


Biscuits, Bread, Cakes & Pastries 1000RTS tea & coffee based beverages 600

Chocolate (White, Milk, Plain, Composite and Filled) and cereal

based beverages

500

Sugar based/Sugar free confectionery 3500Chewing gum/Bubble gum 5000

Ice lollies/Ice candy 800Sweets (carbohydrates based and milk products based):-Halwa, Mysore Pak,



500


SUCRALOSE

Carbonated Water, Soft drink concentrate

300

Yoghurt, Sweetened Butter milk 300Biscuits, Bread, Cakes & Pastries 750

RTS tea & coffee based beverages 600Ice-cream 400

Sugar based/Sugar free confectionery 1500Chewing gum/Bubble gum 1250Jam, Jellies & Marmalades 450




750


SUCRALOSE

Ice lollies/Ice candy, Doughnuts, Chutney

800

Concentrates for vegetable juice and vegetable nectar

1250

Vegetable juice & vegetable nectar 250Cake mixes 700

Cookies 750Frozen fruit 150

Sweeteners- tradenamesSweeteners- tradenames

AspartameAspartame Equal, NutrasweetEqual, Nutrasweet

SaccharinSaccharin SucarylSucaryl

Acesulfame-KAcesulfame-K Sunett, Sweet OneSunett, Sweet One

SucraloseSucralose Splenda, ZeroSplenda, Zero

CyclamateCyclamate Sweet & LowSweet & Low

SteviosideStevioside Rebiana, StugarRebiana, Stugar

ThaumatinThaumatin TalinTalin

http://www.sweetone.com/box.html

Structural features of intense Structural features of intense sweetenerssweeteners

Methyl ester of a dipeptideMethyl ester of a dipeptide Derivative of oxathiazinDerivative of oxathiazin

Derivative of isothiazolDerivative of isothiazol Chlorinated disaccharideChlorinated disaccharide

Comparative properties of intense sweetenersComparative properties of intense sweetenersPROPERTY ASPARTAME ACESULFAME-K SACCHARIN SUCRALOSE

Chemical nature/Structure

Methyl ester of a dipeptide

Derivative of oxathiazin Derivative of isothiazol Chlorinated dissacharide

Relative sweetness (w.r.t.

sucrose)

200 -300 200 400-500 600 - 800

After taste No Little bitter Bitter, metal like No

Stability Stable to low acidic and alkaline

conditions (pH 3-6), heat unstable

Stable to heat, acid and alkali

Stable to heat, acid and alkali

Stable to heat, acid, alkali and light

Solublility Sparingly soluble in water and slightly

soluble in ethanol (10 g/l at 20C)

Freely soluble in water, very slightly soluble in ethanol (270 g/l at

20C)

Slightly soluble in water, sparingly soluble in alcohol (2 g/l – acid saccharin, 100 g/l – Na

saccharin)

Freely soluble in water, methanol and

alcohol, slightly soluble in ethyl

acetate (283 g/l)

pH for max. stability

4.5 – 6.0 6.5 – 7.5 7.0 – 8.5 5.0 – 6.0

Melting point (C) 246 200 (decomposes before reaching this temp.)

228.8 – 229.7 (acid saccharin),

> 300 (Na saccharin)

125

Degradation products

Diketo piperazine (DKP), -, -aspartyl phenylalanine (AP), methanol, aspartic

acid, phenylalanine, phenylalanine methyl ester, - aspartame

Acetoamide, acetoamide-N-sulfonic acid,

acetoacetic acid, acetoacetamide N-sulfonic

acid, acetone, CO2, ammonium hydrogen

sulphate, amido sulfate

2–sulfobenzoic acid, 2-sulphamoyl

benzoic acid

1,6- dichloro fructose, 4-chloro-4-deoxy galactose, 3,6-anhydro--D-

fructofuranosyl-4-chloro-4-deoxy--D-galactopyranoside

Decomposition products of Decomposition products of Aspartame Aspartame

α – Aspartame (α – APM)

Basic pH

Acidic pH

Diketopiperazine (DKP)

α – Aspartylphenylalanine (α – AP)

Aspartic acid (ASP) (Phenylalanine methyl ester (PM)

β – Aspartame (β – APM)

Phenylalanine aspartic acid (PA)

Aspartic acid (ASP) Phenylalanine (PHE)

β – Aspartylphenylalanine (β – AP)

Decomposition products of aspartame (Mazur, 1972)

acetone carbondioxide ammonium hydrogen sulphate

amidosulphate acetoamide acetoamide N-sulfonic acid

acetoacetic acid acetoacetamide N-sulfonic acid

Acidic pH(2.5)

Alkaline pH (3-10.5)

O

CH3-C-CH3 + CO2 + (NH4)2 HSO4

CONH2SO4 + CH3CONH2 + CH3CONHSO3H

CH3COCH2COOH + CH3COCH2CONHSO3H

(Arpe, 1978)

Decomposition products of acesulfame-K

Decomposition products of saccharin

(De Garmo, 1952)

Saccharin 2-sulfamoyl-benzoic acid

2-sulfobenzoic acid

Decomposition of sucraloseDecomposition of sucralose

High temperature, low pH

At high pH

3,6-anydro--D-fructofuranosyl-4-chloro-4-deoxy--D-galacto pyranoside

(Goldsmith and Merkel, 2004)

Sample preparationSample preparation

Filtration

Sample

Dilution with water

Dilution with water

Sample

Homogenization

Filtration

Carrez clarification

Centrifugation

Sample

Filtration

Dilution with water

Homogenization

Solid phase extraction

Ultrasonification


Centrifugation

Ultrasonification

Sample

Homogenization

Filtration


Centrifugation

Dilution with water

Clear liquid products Clear liquid products Cloudy liquid productsCloudy liquid products Low fat productsLow fat products Semi-solid and solid Semi-solid and solid productsproducts

(Wood, 2004)(Wood, 2004)

Analysis Methods

Safety AspectsSafety Aspects

SWEETENER

ACCEPTABLE DAILY INTAKE (ADI)

(mg/Kg body wt/day)

Aspartame 50

Acesulfame-K 15

Saccharin 5

Sucralose 5

AspartameAspartame Degrades to: Aspartic acid (40 %), Phenylalanine (50 %) and Degrades to: Aspartic acid (40 %), Phenylalanine (50 %) and

methanol (10 %) and some amounts of diketopiperazine (DKP)methanol (10 %) and some amounts of diketopiperazine (DKP) Headache, seizures, weight gain, depression, nausea, etc.Headache, seizures, weight gain, depression, nausea, etc.

Extensive studies by FDA in animals and humans show that its Extensive studies by FDA in animals and humans show that its safe except for phenylketonuricssafe except for phenylketonurics Inability to produce enzyme to metabolize phenylalanine in Inability to produce enzyme to metabolize phenylalanine in

bodybody

Approved by Joint Expert Committee on Food Additives (JECFA) Approved by Joint Expert Committee on Food Additives (JECFA) and Scientific Committee on Food of the European Union (SCF)and Scientific Committee on Food of the European Union (SCF)

Normally 2-10 mg/kg body wt. normally consumedNormally 2-10 mg/kg body wt. normally consumed

Acesulfame-KAcesulfame-K Based on pharmacokinetic studies, long term feeding in rats and Based on pharmacokinetic studies, long term feeding in rats and

dogs show no mutagenicity or carcinogenicitydogs show no mutagenicity or carcinogenicity

Approved by JECFA, SCF and USFDAApproved by JECFA, SCF and USFDA

Approved safe for use Approved safe for use

SaccharinSaccharin Human studies conductedHuman studies conducted

Bladder tumors in some male rats fed on high dosesBladder tumors in some male rats fed on high doses

Declared safe by JECFA, SCF and National Toxicology Program (NTP)JECFA, SCF and National Toxicology Program (NTP)

Studies limited to clinical trials Studies limited to clinical trials

SucraloseSucralose Reviews by FDA in humans and animals for toxic Reviews by FDA in humans and animals for toxic

effects showed that sucralose is safe for human effects showed that sucralose is safe for human consumptionconsumption

Does not cause tooth decay, cancer, genetic Does not cause tooth decay, cancer, genetic changes, birth defectschanges, birth defects

No effect on carbohydrate metabolism, blood No effect on carbohydrate metabolism, blood glucose control, insulin secretion, immune glucose control, insulin secretion, immune systemsystem

FOOD SAMPLE PREPARATION

COLUMN MOBILE PHASE DETECTION (nm)

SWEETENERS ANALYZED

REFERENCES

1 Orange drink

Diabetic chocolate

Solid phase extraction (SPE)

Defatting, SPE

Partisil 10 ODS3, 10 m

Methanol - tetrabutyl ammonium

hydrogen sulfate in phosphate buffer (32.5: 67.5), pH 4.0

227 Acesulfame-K (AK), Saccharin

(SA)

Slack (1985)

Slack (1989)

2 Beverage None Bondapak C18, 10 m

Methanol - acetic acid - water

(20:5:75)

254 Acesulfame-K, Saccharin

Veerabhadrarao et al.,

(1987)3 Beverage,

JamDilution with

water Recoveries (%): 96.5 – 103.6 (AK)

100.4 – 103.2 (SA)

Spherisorbis ODS-1, 5 m

Methanol - 0.02 M KH2PO4 (8:92),

pH 6.7


Detection limit (DL): <

1mg/l)

Hannisdal (1992)

4 Diet soft drinks,

tabletop sweeteners

Diet puddings &

dessert toppings

Dilution with water, filtration

Ethanol extraction,

filtration, dilution with mobile phase

Bondapak C18, 10 m

Acetonitrile - 0.02 M KH2PO4 (3:97), pH 5.0

+ Acetonitrile - 0.02 M KH2PO4 (20:80), pH 3.5

200 Acesulfame-K, Saccharin, Aspartame, Sucralose

DL (ng): 20 (AK), 5 (SA), 25 (AS), 1600

(SU)

Lawrence and Charbonneau

(1988)

5 Lemonade, cola drink, tabletop

sweetener

Dilution with water

Superspher RP –

select B, 4 m

Acetonitrile - 0.02 M KH2PO4 (10:90), pH

4.2 – 4.4

220 Acesulfame-K, Saccharin, Aspartame

Hagenauer –Hener et al.,

(1990)

6 Cola beverage

Degassing, dilution with

water

Bondapak C18

Acetonitrile – triethylammoniu

m phosphate (15:85), pH 4.3

214 Saccharin, Aspartame

Tyler (1984)

HPLC methods for the determination of sweeteners in foodsHPLC methods for the determination of sweeteners in foods


COLUMN MOBILE PHASE

DETECTION (nm)

SWEETENERS ANALYZED

REFERENCES

7 Tabletop sweetener,

candy

Liquid beverage

Other foods

Dilution with water

Dilution with water,

degassing, clean up on C18 cartridge

Water extraction, carrez

clarification Recoveries

(%): 95.2 – 106.8

(AK)

Bondapak

C18

Acetonitrile - 0.0125 M KH2PO4

(10:90), pH 3.5

220 Acesulfame-K, Saccharin,

Aspartame and its degradation products

DL: 100 pg/injection

(AK)

Prodolliet and Bruelhart

(1993)

8 Cherry, nectar, brine

Yoghurt

Chocolate

Mayonnaise

None

Carrez

clarification

Defatting, Carrez clarification

Defatting, Water extraction

Lichrospher N –

select B, 5m


(10:90)


Lehr and Schmid (1993)

9 Candy

Gum

Dissolution in water

Extraction with glacial acetic acid

– CHCl3 (50:25)

AS4, anion exchange

2.8 mM Na2CO3


Biemer (1989)

10 Tabletop sweetener,

candy, powdered beverage

Liquid beverage

Cream, yoghurt

Water extraction

Water dilution,

degassing, cleaning on C18

cartridgeWater extraction,

centrifugation

Bondapak C18, 10 m

Acetonitrile - 0.0125 M

KH2PO4 (2:98), (10:90) or

(15:85), pH 3.5

214, 220 Acesulfame-K, Saccharin, Aspartame,

Diketopiperazine (DKP),

Phenylalanine methyl ester

(PM)

Prodolliet and Bruelhart

(1993)



SWEETENERS ANALYZED

REFERENCES

11 Soft drink

Soft drink

Degassing, dilution with acetonitrile

Degassing, cleanup on NH4+

and C18 cartridges

Recoveries (%): > 95

Bondapak C18 radial-pak

Lichrosorb C18, 10 m

Acetonitrile - 0.5 %

ammonium acetate in 2 % glacial acetic

acid (15:85)

Acetonitrile – 0.1 M NaH2PO4 (7:93), pH 4.5

254

215 (AS), 230 (SA)

Saccharin, Aspartame


Delaney et al., (1985)

Moors et al., (1991)

12 Beverages

Degassing, water dilution

Spherisorb ODS-2, 3 m

Methanol - 0.03 M KH2PO4

(28:72), pH 3.2

214 Saccharin, Aspartame, PHE, DKP

Nordic committee on food analysis,

no. 142 (1992)

13 Coffee containing

drink

Degassing RP 18, 10 m Methanol - 0.0125 M KH2PO4

(30:70), pH 4.5

217 Saccharin, Aspartame,

DKP

Bestimmung Des

Aspartamgehaltes In

Coffeinhaltigen Brausen

(1989)14 Food stuffs Dilution with

water, SPE, Carrez clarification

Bondapak C18

Phosphate buffer-

Acetonitrile (90:10, 80:20, 85:15, 95:5,

98:2)


Wood (2004)

(BS EN 12856: 1999)

15 Soft drinks

Soft

drinks

Soft drinks

Degassing

Degassing

Degassing, dilution with

water

Bondapak C18

Spherisorb ODS-5

Fractogel TSK

HW-40 (S), 25-40 m

Methanol – 1 M acetic acid

(20:80), pH 4.2

Methanol – 0.05 M

NaH2PO4 (10:90) to

(60:40), pH 3.6

0.067 M KH2PO4, pH 4.3; 0.015 M

KH2PO4 – 0.015 M Na2HPO4,

pH 6.9

254

214

214

Saccharin, Aspartame,

DKP


Saccharin, Aspartame,

DKP

Bidlingmeyer. (1991)

Hann and Gilkison (1987)

Muller and Jork (1990)


COLUMN MOBILE PHASE

DETECTION (nm)

SWEETENERS ANALYZED

REFERENCES

16 Beverages Degassing, dilution with water

Bondapak C18, 37-50

m

2-propanol – 10 % acetic

acid (3:97), pH 3.0

254 Saccharin, Aspartame DL: 0.1 g /

injection

Webb and Beckmann

(1984)

17 Soft drinks Degassing Bondapak C18


(10:90), pH 3.5


Tsang et al., (1985)

18 Dried soft drinks

Dilution with water MCH - 5 Methanol - 0.01 M KH2PO4 (10:90),

pH 2.5


Cross and Cunico (1984)

19 Pharmaceutical and dietary

formulations

Extraction with mobile phase

Hypersil RP – 18, 10 m

Methanol – THF – 0.08 M

triethyl ammonium phosphate

(5.5:90), pH 3.0

270 Acesulfame-K, Saccharin, Aspartame,

DKP

Di Pietra et al., (1990)

20 Soy sauce, pickles, miso,

powdered instant

puddings

Dialyze with 1% H3PO4, cleanup on Bond Elut SCX for AS and on Bond

Elut C18 for AK and SA

Recoveries (%): 90 – 104 (AK)

92-102 (SA)

90-103 (AS)

Fine pak C18 S

Tetrapropyl-ammonium

hydroxide in methanol –

water (20:80), pH 4.0


Aspartame DL (g/g): 10(AK), 5(SA),

10(AS)

Morriyasu et al., (1991)

21 Beverages None Bondapak C18, 10 m

Acetonitrile - 0.02 M KH2PO4 (3:97), pH 5.0


DL: 1-2 g/ml

Lawrence (1987)

22 Beverages Dilution with water Bondapak C18, 10 m

Acetone – 20 mM KH2PO4

(2:98)


Lawrence (1990)



SWEETENERS ANALYZED

REFERENCES

23 Vanilla yoghurt with

cherries, multivitamin

juice

Methanol extraction, filtration

Recoveries (%): 63.0 – 81.1

Li Chrospher 60 RP – Select B, 5

m

20 mM tetra-n-butyl

ammonium hydrogen

sulfate (75:15 to 5:95), 20

mM phosphate (pH 4.3) – acetonitrile

(90:10 to 5:95)

217, 285 Acesulfame-K, Saccharin, Aspartame

Haush (1996)

24 Fishery products, creams,

mayonnaise, ice cream

Water extraction, SPE Recoveries (%): 84 - 102

Nucleosil 100– 5 C18, 5

m

20 mM KH2PO4–

acetonitrile–water, 20 mM

phosphate buffer (pH

6.7)-acetonitrile

232 (AK) 214 (SA,

AS)

Acesulfame-K, Saccharin, Aspartame

Ostermeyer (1995)

25 Soy sauce, sugared fruit,

dried roast beef

Acetone extraction,

cleanuo on silica cartridge Recoveries (%):

76 - 95

Shoko monomeric /

Shoko polymeric; C18, 5 m

Acetonitrile – 50 mM - hydroxy

isobutyric acid (pH 4.5;

2.2:3.4), 2.5 mM

hexadecyltrimethyl

ammonium bromide


Chen and Fu (1996)

26 Tabletop sweetener

Fruit juice drinks

Carbonated drinks

Water dilution

Water dilution

Degassing, Water dilution Recoveries (%):

93 - 107

Ionpoc AS4A - SCA

Na2CO3

(1 to 12.5 mM)


Aspartame DL (g/ml): 0.044 (AK), 0.019 (SA), 0.035 (AS)

Chen et al., (1997)

METHOD FOOD SAMPLE PREPARATIO

N

STATIONARY PHASE/

COLUMN/ FILM

MOBILE PHASE/ BUFFER/

CONDITIONS

DETECTION (nm)

SWEETENERS ANALYZED

REF.

1 Thin layer chromatograp

hy

Cosmetics

Carbonated

beverages

Dietetic candy, jellies, canned fruits

None

Acidify with HCl, ethyl

ether extraction

Blend with water,

acidify with HCl, extract with ethyl

ether

Polyamide

Silica gel G

Silica gel G

Xylene – propanol – formic acid

(5:5:1)

Ethyl acetate – isopropanol

– acetone – methanol –

water (50:15:15:4:

16)

0.2 % Dichlorofluor

es-cein in methanol

Br2 vapour + 0.05 %

fluorescein

Acesulfame-K, Saccharin DL (g): 2.0

(AK)

Saccharin, Sucrose

Von Rymon Lipinski (1979)

Das et al., (1970)

2 Paper

chromatography

Carbonated water

None Cellulose n-butanol – acetic acid –

water (40:10:22)

0.93 % Phthalic acid + 1.63 % aniline in butanol

Saccharin, Sucrose

Komoda et al., (1962)

3

Flow-through sensor/

Electrochemical biosensor/ Bilayer lipid membranes

Tabletop sweeteners, diet soft

drinks, wines,

yoghurts

None Recoveries (%): 96 – 106

Filter-supported bilayer lipid

membranes of egg

phosphatidylchol-ine

0.1 M KCl, Temperature

: 25 1C

- Saccharin, Acesulfame-K DL (M):

0.5 (AK), 0.2 (SA)

Nikolelis et al., (2001)

Other methods used for the determination of multiple sweeteners in foodsOther methods used for the determination of multiple sweeteners in foods

METHOD FOOD SAMPLE PREPARATIO

N

STATIONARY PHASE/

COLUMN/ FILM


CONDITIONS

DETECTION (nm)

SWEETENERS ANALYZED

REF.

4 Capillary electrophores

is

Low-joule soft

drinks, cordials, tabletop

sweeteners

Cola beverages

Soft drinks

Standards

Preserved fruits

Dilute with water

Filtration

Degassing

None

Water extraction,

SPERecoveries

(%): 90

Micellar electrokinetic

capillary (MEKC)

MEKC, hydrodynamic injection, 3 s

Capillary zone

electrophoresis (CZE)

electro kinetic injection at 5

KV, 7 s

MEKC

10 mM borate, 10 mM phosphate,

50 mM deoxycholate

(DOC) [pH 8.6, 20 KV, 50 cm x 75

m, 20 kPaS vaccum]

20 mM borate, 15 mM sodium

dodecylsulphate (SDS), 35 mM

sodium cholate (SC), 10%

methanol [pH 9.3, 20 KV, 52 cm

x 75 m]

20 mM carbonate, 62

mM SDS [pH 9.5, 20 KV, 40 cm x

50 m]

1.5 mM tetra borate

20 mM borate, 20 mM phosphate, 50 mM DOC, 5% acetonitrile (pH

5.6)

220

214

200

Potentiometr

ic coated wire ion selective electrode

(ISE)

214




Acesulfame-K,

SaccharinDL(µM) : 11


DL (µg/g): 10-25

Thompson et al., (1995)

Boyce (1999)

Frazier

et al., 2000)

Schnierie et al., (1998)

Lin et al., (2000)

METHOD FOOD SAMPLE PREPARATION

STATIONARY PHASE/

COLUMN/ FILM


CONDITIONS

DETECTION (nm)

SWEETENERS ANALYZED

REF.

5 UV – Visible Spectrophotomet

ry

Beverages, ice candy,

syrups

Ice cream

HCl addition, extraction with diethyl ether (SA) or ethyl acetate (AK), evaporate, dissolve in water

HCl addition, extraction as above, re-extract with 1% Na2CO3, adjust to pH 7.0

Recoveries (%): 98.5 – 99.4 (SA) 97.8 – 99.5 (AK)

Sevron Blue 5G reagent

CHCl3/ Phosphate buffer, pH

7.0


Sastry et al., (1995)

Isolation and analysis of sweeteners Isolation and analysis of sweeteners (aspartame, acesulfame-k and saccharin) (aspartame, acesulfame-k and saccharin)

Dilution with water

Product sample

Blending


Ultrasonification


Eluant

HPLC analysis using UV detector

Confirmation

By scanning over photodiode array detector (200-380 nm) using similarity index (0.85 – 1.00)

Isolation and analysis of sucraloseIsolation and analysis of sucraloseProduct sample

Blending

Dilution with water

Ultrasonification


Eluent

Quantification

By using Biorad software (Quantity one)


Filtration

HPTLC analysis

Derivatization on amino based plate

(Spangenberg et.al, 2003)

HPLC analysis using UV detector

Charring on silica gel plates

Carrez solution No. 1: 3.6 g of potassium ferrocyanide dissolved in 100 ml water.

Carrez solution No. 2: 7.2 g of zinc sulphate dissolved in 100 ml water.

Mobile phase A: 0.02M KH2PO4 (pH 5.0) – Acetonitrile (97+3).

Mobile phase B: 0.02M KH2PO4 (pH 3.5) adjusted with 5% H3PO4 – Acetonitrile (80+20).

Solid Phase Extraction (SPE) ASSEMBLY

Column stationary phase : Shimpak C18, S-5µm, 120A, 250x4.6mm IDPhase : Reverse phaseUV detector wavelength : 200 nm, 220 nmMobile Phase (A) : 0.02 M phosphate buffer, pH 5.0: acetonitrile (97:3)Mobile Phase (B) : 0.02 M phosphate buffer, pH 3.5: acetonitrile (80:20)Flow rate : 1 ml/minRun time : 30 min.Maximum pressure : 400 kgmf

Solvent filters : 0.45 µm durapore (Millipore) (47 mm)

Concentration of standards : 200 ng/20 uL [100 µL of standards(1µg/µl) dissolved in 10ml of mobile phase mix of A and B (1:1)]

Binary gradient programming:

Time (min) B concentration (%) Actual pressure observed (Kgmf)0.1 0 808 0 80

13 100 16025 100 16027 0 8030 STOP 80

HPLC Analytical Conditions

HPLC chromatogram of sweeteners and degradation product standards

1 6

4

5

6

(1) Diketopiperazine [2.953 min] (2) Acetoacetamide [3.192 min] (3) 2-sulfobenzoic acid [3.420 min]

(4) Acesulfame-K [3.789 min] (5) L-phenylalanine [3.990 min] (6) Saccharin [4.440 min] (7) Aspartame [19.190 min]

1

2 3 4

5

6 7

1

23

4

56

7

HPTLC Analysis of Sucralose

Development of isolation procedure for sucralose.

Qualitative detection over amino HPTLC plates.

Development of quantitative procedure over silica gel HPTLC plates.

Development of isolation procedure for sucralose

Product Sample

Ultra -Sonification (40°C/20 min)

Carrez clarification (2 ml Carrez solution No.1 + 2 ml Carrez solution No.2)

Filtration (Whatman No. 1 filter paper)

HPTLC Analysis

Derivitization on Charring on Amino base plates Silica gel plates

Semi-quantification Quantification(Spangenberg et.al, 2003)

By using Biorad software (Quantity one)

Qualitative detection over Amino HPTLC Plates

Plate : Amino F254s HPTLC Plates

Activation time : 30 min at 1000C

Solvent system : Acetonitrile : Water (4:1)

Reagent free derivatization by heating : 1900C for 20 min

Development of Quantitative method over Silica Gel HPTLC Plates

Plates : Silica gel 60

Activation time : 30 min at 1000C

Solvent system : Dichloromethane:Methanol (4:1)

Heating time : 10 min at 1200C

Derivatization : Sulphuric acid 15% (v/v)

Quantification : Bio-rad Quantity one software

HPTLC of standard sucralose

Stability of sweetenersStability of sweeteners Acesulfame-KAcesulfame-K

Prolonged continuous exposure to 30Prolonged continuous exposure to 30C did not cause C did not cause losses exceeding 10%, the threshold for recognition of losses exceeding 10%, the threshold for recognition of sweetener’s difference (Hoppe and Gassmann, 1980) sweetener’s difference (Hoppe and Gassmann, 1980)

Threshold for detection of sweetness differences was Threshold for detection of sweetness differences was exceeded after several months, for products having pH exceeded after several months, for products having pH 3.0 or less, even at temperatures of 403.0 or less, even at temperatures of 40C (Lipinski and C (Lipinski and Hanger, 2001) Hanger, 2001)

Acesulfame-K containing beverages can be pasteurized Acesulfame-K containing beverages can be pasteurized and sterilized under normal conditions, without loss of and sterilized under normal conditions, without loss of sweetness sweetness (Lipinski, 2004)

Stability of acesulfame-K in buffered aqueous solutionStability of acesulfame-K in buffered aqueous solution

2020CC

Storage time (weeks)Storage time (weeks) pH 3.0 recovery (%)pH 3.0 recovery (%) pH 3.5 recovery (%)pH 3.5 recovery (%)

1616 9898 9898

3030 9898 9999

5050 9898 9999

100100 9595 9898

3030CC

Storage time (weeks)Storage time (weeks) pH 3.0 recovery (%)pH 3.0 recovery (%) pH 3.5 recovery (%)pH 3.5 recovery (%)

1616 9797 100100

3030 9595 9797

5050 9191 9696

No change in stability after long periods of storage at pH No change in stability after long periods of storage at pH common for beveragescommon for beverages

SaccharinSaccharin

In its bulk form, saccharin and its salts show no In its bulk form, saccharin and its salts show no detectable decomposition over long periods and detectable decomposition over long periods and are highly stable in aqueous solutions over a are highly stable in aqueous solutions over a wide pH range wide pH range

Saccharin solutions buffered at pH’s ranging Saccharin solutions buffered at pH’s ranging from 3.3 – 8.0, were essentially unchanged after from 3.3 – 8.0, were essentially unchanged after heating for 1 hour at 150heating for 1 hour at 150C (DeGarmo C (DeGarmo et al.et al., , 1952)1952)

Saccharin hydrolyzes to a measurable extent only Saccharin hydrolyzes to a measurable extent only under severe conditions of high temperature and low under severe conditions of high temperature and low pH, over an extended period (PMC Specialties Group, pH, over an extended period (PMC Specialties Group, 2004)2004)

Saccharin hydrolysis at 20C Saccharin hydrolysis at 40C

Saccharin hydrolysis at 80C

SucraloseSucralose

Sucralose is freely soluble in water and ethanol, and its Sucralose is freely soluble in water and ethanol, and its solubility increases with temperature (Jenner and Smithson, solubility increases with temperature (Jenner and Smithson, 1989)1989)

Sucralose solubility in water and ethanol

Aqueous stability of sucralose, effect of pH at 30C

Sucralose solutions stored at 30Sucralose solutions stored at 30C for up to 1 year at pH 3.0, C for up to 1 year at pH 3.0, 4.0, 6.0 and 7.5 showed no measurable loss at pH 4.0, 6.0 and 4.0, 6.0 and 7.5 showed no measurable loss at pH 4.0, 6.0 and 7.5, while there was less than 4% loss at pH 3.0 (Mulligan 7.5, while there was less than 4% loss at pH 3.0 (Mulligan et alet al, , 1988)1988)

Aqueous stability of sucralose, effect of pH at 100C

Sucralose solutions stored at 100Sucralose solutions stored at 100C for 2 hours, showed 2% C for 2 hours, showed 2% loss after 1 hour and 2%, 3% and 4% loss at pH 5.0, 7.0 and 3.0 loss after 1 hour and 2%, 3% and 4% loss at pH 5.0, 7.0 and 3.0 after 2 hours respectively (Anonymous, 1987) after 2 hours respectively (Anonymous, 1987)

AspartameAspartame

Stability of aspartame in solutions at 25Stability of aspartame in solutions at 25C was C was maximum at pH 4.3 (Beck, 1978) maximum at pH 4.3 (Beck, 1978)

60% of the original aspartame remained after 6 60% of the original aspartame remained after 6 months at 21.1months at 21.1C (Fellows C (Fellows et al., et al., 1991) 1991)

Kinetics of aspartame degradation in low to Kinetics of aspartame degradation in low to intermediate moisture systems, showed intermediate moisture systems, showed maximum stability at pH 5.0 and it decreased, as maximum stability at pH 5.0 and it decreased, as pH decreased or increased (Leonard and pH decreased or increased (Leonard and Theodore, 1993) Theodore, 1993)

Higher levels of initial microbial population and its Higher levels of initial microbial population and its subsequent increase over time resulted in higher subsequent increase over time resulted in higher rates of aspartame degradation.(Keller rates of aspartame degradation.(Keller et al., et al., 1990)1990)

Aspartame degradation is minimal in the pH range Aspartame degradation is minimal in the pH range of 4-5, which is below the pH of protein stabilization of 4-5, which is below the pH of protein stabilization (Bell and Labuza, 1991)(Bell and Labuza, 1991)

Multiple sweetener approachMultiple sweetener approach

An ideal sweetener does not existAn ideal sweetener does not exist

Differences exhibited like sweetness lag, Differences exhibited like sweetness lag, lingering aftertaste or bitterness, lack of lingering aftertaste or bitterness, lack of bulking properties, stability during storage and bulking properties, stability during storage and competitive pricescompetitive prices

Multi-sweetener blendsMulti-sweetener blends

Overcomes limitations of single sweetenersOvercomes limitations of single sweeteners

Development of new productsDevelopment of new products

Special featuresSpecial features

Improves the quality of sweet tasteImproves the quality of sweet taste

Masks the shortcoming of the individual sweetenerMasks the shortcoming of the individual sweetener

Taste profile similar to sugarTaste profile similar to sugar

Adds versatility to productsAdds versatility to products

Lengthens sweetness shelf-lifeLengthens sweetness shelf-life

Cost effectiveCost effective

SynergySynergy

Blends commonly used:Blends commonly used:

Acesulfame-K + SucraloseAcesulfame-K + SucraloseAspartame + Acesulfame-KAspartame + Acesulfame-KAspartame + SaccharinAspartame + SaccharinAcesulfame-K + Aspartame + SaccharinAcesulfame-K + Aspartame + Saccharin

CONCLUSIONCONCLUSION Intense sweeteners: low to non-caloric, Intense sweeteners: low to non-caloric,

sweeter than sucrosesweeter than sucrose

Diabetic and calorie conscious friendlyDiabetic and calorie conscious friendly

Wide applications in industryWide applications in industry

Future beckoning Future beckoning

Hoshin-Sweetenersg

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