Prepared at the 51st JECFA (1998), published in FNP 52 Add 6 (1998) superseding specifications prepared at the 44th JECFA (1995), published in FNP 52 Add 3 (1995). Group ADI "not specified" (temporary) for carrageenan and PES, established at the 51 st JECFA in 1998.


PES, PNG-carrageenan, semi-refined carrageenan; INS No. 407a


A substance with hydrocolloid properties obtained from either Eucheuma cottonii or E. spinosum (from the Rhodophyceae class of red seaweeds). In addition to carrageenan polysaccharides, processed euchema seaweed may contain up to 15% of insoluble algal cellulose and minor amounts of other insoluble matter. Articles of commerce may include sugars for standardization purposes or salts to obtain specific gelling or thickening characteristics. It is distinguished from carrageenan (INS 407) by its higher content of cellulosic matter and by the fact that it is not solubilized and precipitated during processing.

The functional component of the product obtained from E. cottonii is kappa-carrageenan (a copolymer of D-galactose-4-sulfate and 3,6-anhydro-D-galactose). From E. spinosum it is iota-carrageenan (a copolymer of D-galactose-4-sulfate and 3,6-anhydro-D-galactose-2-sulfate).

Processing consists of soaking the cleaned seaweed in alkali for a short time at elevated temperatures. The material is then thoroughly washed with water to remove residual salts followed by purification, drying, and milling to a powder. Alcohols that may be used during purification are restricted to methanol, ethanol, and isopropanol.


Light tan to white coarse to fine powder


Thickener, gelling agent, stabilizer, emulsifier




Forms cloudy viscous suspensions in water; insoluble in ethanol

A 1 g sample disperses and partially dissolves in 100 ml of water at 80o giving a cloudy opalescent solution. (The sample disperses in water more readily if first moistened with alcohol, glycerol, or a saturated solution of glucose or sucrose in water).

Test for sulfate

Dissolve a 100 mg sample in 20 ml of water. Heat to boiling, cool to room temperature, and add 3 ml of barium chloride TS and 5 ml of hydrochloric acid, dilute TS. Filter the mixture. Boil the filtrate for 5 min. A white, crystalline precipitate appears.

Test for galactose and anhydrogalactose

Proceed as directed under Gum Constituents Identification, using the following as reference standards: galactose, rhamnose, galacturonic acid, 3,6-anhydrogalactose, mannose, arabinose and xylose. Galactose and 3,6-anhydrogalactose should be present.

Identification of hydrocolloid and predominant type of copolymer

Add 4 g of sample to 200 ml of water, and heat the mixture in water bath at 80o, with constant stirring, until dissolved. Replace any water lost by evaporation, and allow the solution to cool to room temperature. It becomes viscous and may form a gel. To 50 ml of the solution or gel, add 200 mg of potassium chloride, then reheat, mix well, and cool. A short-textured ("brittle") gel indicates a carrageenan of a predominantly kappa-type. A compliant ("elastic") gel indicates a predominantly iota-type.

Infrared absorption

Passes test

See description under TESTS


Loss on drying

Not more than 12% (105o to constant weight)


Between 8 and 11 (1 in 100 suspension)


Not less than 5 cp at 75o (1.5% solution)

See description under TESTS


Not less than 15% and not more than 40% (as SO42-) on a dry weight basis

See description under TESTS

Total ash

Not less than 15% and not more than 30% on a dry weight basis

See description under TESTS

Acid-insoluble ash

Not more than 1%

Acid-insoluble matter

Not less than 8% and not more than 15% on a dry weight basis

Use 2 g of sample obtained from part (a) of the procedure for sulfate determination

Residual solvents

Not more than 0.1% of ethanol, isopropanol, or methanol, singly or in combination

See description under TESTS

Microbiological criteria

Initially prepare a 10-1 dilution by adding a 50 g sample to 450 ml of Butterfield's phosphate-buffered dilution water and homogenizing the mixture in a high speed blender.

Total (aerobic) plate count: Not more than 5000 cfu/g

Salmonella spp.: Negative per test

E. coli: Negative in 1 g


Not more than 3 mg/kg (Method II)

Determine by atomic absorption hydride technique using a 3 g sample


Not more than 2 mg/kg

Prepare a sample solution as directed for organic compounds in the Limit Test and determine by atomic absorption spectroscopy


Not more than 1 mg/kg

Determine by atomic absorption spectroscopy (Method I)


Not more than 1 mg/kg

Determine by atomic absorption cold vapour technique



Infrared absorption

Prepare a 0.2% aqueous solution of the sample, cast films of 0.5 mm thickness (when dry) on a suitable non-sticking surface such as Teflon, and obtain the infrared absorption spectrum of each film. (Alternatively, the spectra may be obtained of films cast on potassium bromide plates if care is taken to avoid moisture).

Iota- and kappa-carrageenan have strong, broad absorption bands, typical of all polysaccharides, in the 1000 to 1100 cm-1 region. Other characteristic absorption bands and their intensities relative to the absorbance at 1050 cm-1 are as follows:

Wave number


Molecular Assignment

Absorbance Relative to 1050 cm-1

Kappa Iota




ester sulfate














Hydrolysed sulfate groups are precipitated as barium sulfate.


(a) Disperse an accurately weighed 8 g sample of commercial product into 400 ml of 60% w/w isopropanol/water at room temperature. Stir gently for 4 h. Filter through ash-free filter paper. Discard the filtrate. Wash the material remaining on the filter paper with two 10 ml portions of 60% isopropanol/water. Dry the material at 105° to constant weight.

Approximately 1 g of the dried matter is to be used for part (b). The remainder should be retained for determination of Total ashand Acid-insoluble matter.

(b) Transfer a 1 g sample (W1 ) from (a), accurately weighed, to a 100-ml long-neck round-bottom flask. Add 50 ml of 0.2 N hydrochloric acid. Fit a condenser, preferably one with at least 5 condensing bulbs, to the flask and reflux for 1 h. Add 25 ml of a 10% (by volume) hydrogen peroxide solution and resume refluxing for about 5 h or until the solution becomes completely clear. Transfer the solution to a 600-ml beaker, bring to a boil, and add dropwise 10 ml of a 10% barium chloride solution. Heat the reaction mixture for 2 h on a boiling water bath. Filter the mixture through ash-free slow-filtration filter paper (e.g., Blue Ribbon). Wash with boiling distilled water until the filtrate is free from chloride. Dry the filter paper and contents in a drying oven. Gently burn and ash the paper at 800° in a tared porcelain or silica crucible until the ash is white. Cool in a desiccator.

Weigh the crucible containing the ash. Calculate the percentage sulfate from the weight in g (W2) of the ash (barium sulfate) using the formula: (W2/W1) x 100 x 0.4116.

Total ash

Accurately weigh 2 g of the dried sample (W1) obtained from part (a) under the procedure for Sulfate determination. Transfer to a previously ignited, tared, silica or platinum crucible. Heat the sample with a suitable infrared lamp, increasing the intensity gradually, until the sample is completely charred; continue heating for an additional 30 min. Transfer the crucible with charred sample into a muffle furnace and ignite at about 550o for 1 h. Cool in a desiccator and weigh. Repeat the ignition in the muffle furnace until a constant weight (W2) is obtained. If a carbon-free ash is not obtained after the first ignition, moisten the charred spot with a 1 in 10 solution of ammonium nitrate and dry under an infrared lamp. Repeat the ignition step. Calculate the percentage of total ash of the sample: (W2/W1) x 100.

Retain the ash for the Acid-insoluble ash test.


Transfer 7.5 g of the sample into a tared, 600-ml tall-form (Berzelius) beaker, and disperse with agitation for 10 to 20 min in 450 ml of deionized water. Add sufficient water to bring the final weight to 500 g and heat in a water bath, with continuous agitation, until a temperature of 80o is reached (20-30 min). Add 7.5 g of diatomaceous earth or perlite filter aid.

Stir for two minutes. Add water to adjust for loss by evaporation. Filter the solution through a Büchner funnel (pre-heated with hot water to 80o) equipped with a coarse filter paper. Place the filter assembly in a vacuum receiver bottle.

Filter 200 ml of solution. Cool to 76-77o, and heat in a constant temperature bath at 75o. Pre-heat the bob and guard of a Brookfield LVF viscometer to approximately 75o in water, then dry the bob and guard, and attach them to the viscometer, which should be equipped with a No. 1 spindle (19 mm in diameter, approximately 65 mm in length) and capable of rotating at 30 rpm. Adjust the height of the bob in the sample solution, start the viscometer rotating at 30 rpm and, after six complete revolutions of the viscometer, take the viscometer reading on the 0-100 scale.

If the viscosity is very low, increased precision may be obtained by using the Brookfield UL (ultra low) adapter or equivalent.

Record the results in centipoises, obtained by multiplying the reading on the scale by the factor given by the Brookfield manufacturer.

Residual solvents

Standard Alcohol Solution:
Transfer 500 mg each of chromatographic quality methanol, ethanol, and isopropanol into a 50 ml volumetric flask. Dilute to volume with water, and mix. Pipet 10 ml of this solution into a 100-ml volumetric flask. Dilute to volume with water and mix.

TBA Standard Solution:
Transfer 500 mg of chromatographic quality tertiary-butyl alcohol into a 50-ml volumetric flask. Dilute to volume with water, and mix. Pipet 10 ml of this solution into a 100-ml volumetric flask. Dilute to volume with water and mix.

Mixed Standard Solution:
Pipet 4 ml each of the Standard Alcohol Solution and of the TBA Standard Solution into a 125-ml graduated Erlenmeyer flask. Dilute to about 100 ml with water and mix. This solution contains approximately 40 µg of each alcohol per ml.

Sample Preparation:
Disperse 1 ml of a suitable antifoam emulsion, such as Dow-Corning G-10 or equivalent, in 200 ml of water contained in a 1000-ml 24/40 round-bottom distilling flask. Add about 5 g of the sample, accurately weighed, and shake for 1 h on a wrist-action mechanical shaker. Connect the flask to a fractionating column, and distil about 100 ml, adjusting the heat so that foam does not enter the column. Add 4.0 ml of TBA Standard Solution to the distillate to obtain the Sample Preparation.

Inject 5 µl of the Mixed Standard Solution into a suitable gas chromatograph equipped with a flame-ionization detector and a 1.8-m x 3.2-mm stainless steel column packed with 80-100-mesh Porapak QS or equivalent. The carrier is helium flowing at 80 ml per min. The injection port temperature is 200o; the column temperature is 165o; and the detector temperature is 200o. The retention time of isopropanol is about 2 min, and that of tertiary-butyl alcohol about 3 min.

Measure the areas of the methanol, ethanol, isopropanol, and TBA peaks. Calculate each response factor, f, by the formula A/ATBA, in which A is the area of each alcohol peak.

Similarly, inject 5 µl of the Sample Preparation, and measure the peak areas, recording the area of each alcohol peak as a, and that of the tertiary-butyl alcohol peak as aTBA. Calculate the concentration of each alcohol (mg/kg) in the sample taken, by the formula

a · 4000 / f · aTBA · W

where W is the weight of the sample taken (grams)

Source: Joint FAO/WHO Expert Committee on Food Additives (JECFA)

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