Agar

Prepared at the 49th JECFA (1997)
superseding specifications prepared at the 44th JECFA (1995),
published in FNP 52 Addendum 3 (1995)

SYNONYMS

Agar-agar; gelose; Japan agar; Bengal, Ceylon, Chinese or Japanese isinglass; Layor Carang; INS No. 406

DEFINITION

Agar is the dried hydrophilic, colloidal substance extracted from certain marine algae of the class Rhodophyceae. It is a polysaccharide, consisting primarily of D- and L-galactose units. About every tenth D-galactopyranose unit contains a sulfate ester group. Calcium, magnesium, potassium or sodium cations are also associated with the polysaccharide.

C.A.S. number

9002-18-0

Assay

Not higher than 0.25% for threshold gel concentration

DESCRIPTION

Agar is odourless or has a slight characteristic odour. Unground agar usually occurs in bundles consisting of thin, membranous, agglutinated strips, or in cut, flaked, granulated or powdered forms. It may be light yellowish orange, yellowish grey to pale yellow, or colourless. It is tough when damp, brittle when dry. Powdered agar is white to yellowish white or pale yellow.

FUNCTIONAL USES

Thickener, stabilizer, emulsifier

CHARACTERISTICS

IDENTIFICATION

Solubility

Insoluble in cold water; soluble in boiling water

Gel formation with water

Passes test
See description under TESTS

Precipitate formation with ammonium sulfate solution

Passes test
See description under TESTS

Precipitate formation with lead acetate solution

Passes test
See description under TESTS

Microscopy

Passes test
See description under TESTS

PURITY

Water absorption

Passes test
See description under TESTS

Loss on drying

Not more than 22% after drying at 105° until the difference between two weighings is less than 1 mg (about 5h). Unground agar should be cut into pieces from 2 to 5 mm² before drying.

Total ash

Not more than 6.5% on the dried basis

Acid-insoluble ash

Not more than 0.5% on the dried basis

Foreign insoluble matter

Not more than 1% See description under TESTS

Arsenic

Not more than 3 mg/kg (Method II)

Lead

Not more than 5 mg/kg Prepare a sample solution as directed for organic compounds in the Limit Test, using 5 m g of lead ion (Pb) in the control

Starch and dextrins

Not detectable
See description under TESTS

Gelatin and other proteins

Not detectable
See description under TESTS

Microbiological criteria

Total plate count: Not more than 5,000 colonies per gram.
Initially prepare a 10^-1 dilution by adding a 50 g sample to 450 ml of Butterfield's phosphate buffered dilution water and homogenizing in a high speed blender.
Yeasts and moulds: Not more than 500 colonies per gram
Coliforms: Negative by test
Salmonella: Negative by test

TESTS

IDENTIFICATION TESTS

Gel formation with water

Prepare a 1.0% solution of the sample in boiling water in a flask and place the flask in water at 30° for 15 min. A firm, resistant gel is formed. Place the flask in water at 70° for 1 h, the gel is not molten. When heating the flask at higher temperature than 95°, gel is liquefied to form a clear solution.

Precipitate formation with ammonium sulfate solution

A warm (40°) 0.5% solution of the sample gives a precipitate with half its volume of a warm (40°) 40% ammonium sulfate solution. This test distinguishes agar from alginates, gum arabic, gum ghatti, karaya gum, pectin and tragacanth.

Precipitate formation with lead acetate solution

A warm 0.5% solution of the sample gives a precipitate with one fifth its volume of basic lead acetate TS. This test distinguishes agar from methyl cellulose.

Microscopy

Place a few fragments of unground agar or some powder on a slide and add some drops of water or chloral hydrate TS. When examined under a microscope, agar in water appears granular and somewhat filamentous. In chloral hydrate TS, the powdered agar appears more transparent than in water.

PURITY TESTS

Water absorption

Place 5 g of the sample in a 100-ml graduated cylinder, fill to the mark with water, mix, and allow to stand at 25° for 24 h. Pour the contents of the cylinder through moistened glass wool, allowing the water to drain into a second 100-ml graduated cylinder. Not more than 75 ml of water should be obtained.

Foreign insoluble matter

Boil 5 g of the sample with 500 ml of water and 12 ml of sulfuric acid under a reflux condenser for 2 h. Allow to cool and filter through a tared, fine, sintered glass crucible. Wash flask and filter with 50 ml of water, dry at 105° to constant weight and weigh. Calculate as percentage.

Starch and dextrins

To a warm (40°) 0.5% solution of the sample, add 2 drops of iodine TS. Where the drops fall, a red-violet colour appears. After mixing, the solution should be golden brown and not blue or reddish.

Gelatin and other proteins

To a warm (40°) 0.5% solution of the sample add 1 volume of warm (40°) picric acid TS. No turbidity should appear within 10 min.

METHOD OF ASSAY

Threshold gel concentration: Prepare serial dilutions of the sample with known solids content (0.15%, 0.20%, 0.25%, etc.) and place in tubes, 150 mm long by 16 mm internal diameter, stoppered at both ends. Cool for 1 h at 20-25°. Allow cylinders of gel to slide from the tubes to a level surface. The lowest concentration of gel that resists gravity without rupture for 5-30 sec is the threshold concentration of the sample.

SYNONYMS	Agar-agar; gelose; Japan agar; Bengal, Ceylon, Chinese or Japanese isinglass; Layor Carang; INS No. 406
DEFINITION	Agar is the dried hydrophilic, colloidal substance extracted from certain marine algae of the class Rhodophyceae. It is a polysaccharide, consisting primarily of D- and L-galactose units. About every tenth D-galactopyranose unit contains a sulfate ester group. Calcium, magnesium, potassium or sodium cations are also associated with the polysaccharide.
C.A.S. number	9002-18-0
Assay	Not higher than 0.25% for threshold gel concentration
DESCRIPTION	Agar is odourless or has a slight characteristic odour. Unground agar usually occurs in bundles consisting of thin, membranous, agglutinated strips, or in cut, flaked, granulated or powdered forms. It may be light yellowish orange, yellowish grey to pale yellow, or colourless. It is tough when damp, brittle when dry. Powdered agar is white to yellowish white or pale yellow.
FUNCTIONAL USES	Thickener, stabilizer, emulsifier
CHARACTERISTICS
IDENTIFICATION
Solubility	Insoluble in cold water; soluble in boiling water
Gel formation with water
	Passes test See description under TESTS
Precipitate formation with ammonium sulfate solution
	Passes test See description under TESTS
Precipitate formation with lead acetate solution
	Passes test See description under TESTS
Microscopy	Passes test See description under TESTS
PURITY
Water absorption	Passes test See description under TESTS
Loss on drying	Not more than 22% after drying at 105° until the difference between two weighings is less than 1 mg (about 5h). Unground agar should be cut into pieces from 2 to 5 mm² before drying.
Total ash	Not more than 6.5% on the dried basis
Acid-insoluble ash	Not more than 0.5% on the dried basis
Foreign insoluble matter	Not more than 1% See description under TESTS
Arsenic	Not more than 3 mg/kg (Method II)
Lead	Not more than 5 mg/kg Prepare a sample solution as directed for organic compounds in the Limit Test, using 5 m g of lead ion (Pb) in the control
Starch and dextrins	Not detectable See description under TESTS
Gelatin and other proteins
	Not detectable See description under TESTS
Microbiological criteria	Total plate count: Not more than 5,000 colonies per gram. Initially prepare a 10^-1 dilution by adding a 50 g sample to 450 ml of Butterfield's phosphate buffered dilution water and homogenizing in a high speed blender. Yeasts and moulds: Not more than 500 colonies per gram Coliforms: Negative by test Salmonella: Negative by test
TESTS
IDENTIFICATION TESTS
Gel formation with water
	Prepare a 1.0% solution of the sample in boiling water in a flask and place the flask in water at 30° for 15 min. A firm, resistant gel is formed. Place the flask in water at 70° for 1 h, the gel is not molten. When heating the flask at higher temperature than 95°, gel is liquefied to form a clear solution.
Precipitate formation with ammonium sulfate solution
	A warm (40°) 0.5% solution of the sample gives a precipitate with half its volume of a warm (40°) 40% ammonium sulfate solution. This test distinguishes agar from alginates, gum arabic, gum ghatti, karaya gum, pectin and tragacanth.
Precipitate formation with lead acetate solution
	A warm 0.5% solution of the sample gives a precipitate with one fifth its volume of basic lead acetate TS. This test distinguishes agar from methyl cellulose.
Microscopy	Place a few fragments of unground agar or some powder on a slide and add some drops of water or chloral hydrate TS. When examined under a microscope, agar in water appears granular and somewhat filamentous. In chloral hydrate TS, the powdered agar appears more transparent than in water.
PURITY TESTS
Water absorption	Place 5 g of the sample in a 100-ml graduated cylinder, fill to the mark with water, mix, and allow to stand at 25° for 24 h. Pour the contents of the cylinder through moistened glass wool, allowing the water to drain into a second 100-ml graduated cylinder. Not more than 75 ml of water should be obtained.
Foreign insoluble matter	Boil 5 g of the sample with 500 ml of water and 12 ml of sulfuric acid under a reflux condenser for 2 h. Allow to cool and filter through a tared, fine, sintered glass crucible. Wash flask and filter with 50 ml of water, dry at 105° to constant weight and weigh. Calculate as percentage.
Starch and dextrins	To a warm (40°) 0.5% solution of the sample, add 2 drops of iodine TS. Where the drops fall, a red-violet colour appears. After mixing, the solution should be golden brown and not blue or reddish.
Gelatin and other proteins
	To a warm (40°) 0.5% solution of the sample add 1 volume of warm (40°) picric acid TS. No turbidity should appear within 10 min.
METHOD OF ASSAY	Threshold gel concentration: Prepare serial dilutions of the sample with known solids content (0.15%, 0.20%, 0.25%, etc.) and place in tubes, 150 mm long by 16 mm internal diameter, stoppered at both ends. Cool for 1 h at 20-25°. Allow cylinders of gel to slide from the tubes to a level surface. The lowest concentration of gel that resists gravity without rupture for 5-30 sec is the threshold concentration of the sample.