LECITHIN

Prepared at the 41st JECFA (1993), published in FNP 52 Add 2 (1993)

superseding specifications prepared at the 30th JECFA (1986), published
in FNP 37 (1986) and FNP 52 (1992). Metals and arsenic specifications
revised at the 61st JECFA (2003). An ADI not limited' was established at
the 17th JECFA (1973)

SYNONYMS Phosphatides, phospholipids; INS No. 322(i)

DEFINITION Usually prepared from oil-bearing seeds used for food, especially
soybeans; may also be prepared from animal sources; a complex mixture
of acetone-insoluble phosphatides which consists chiefly of phosphatidyl-
choline, phosphatidyl- ethanolamine, and phosphatidyl-inositol, combined
with various amounts of other substances such as triglycerides, fatty acids,
and carbohydrates; refined grades may contain any of these components in
varying proportions and combinations depending on the type of
fractionation used; its oil-free forms, the preponderance of triglycerides and
fatty acids is removed and the product contains 90% or more of
phosphatides representing all or certain fractions of the total phosphatide
complex.

C.A.S. number 8002-43-5

Assay Not less than 60% of acetone-insoluble matter (phosphatides)

DESCRIPTION Consistency of both natural grades and refined grades may vary from
plastic to fluid, depending upon free fatty acid and oil content, and upon the
presence or absence of other diluents; from light yellow to brown,
depending on the source, on crop variations, and on whether it is bleached
or unbleached; odourless or has a characteristic, slight nut-like odour.
Edible diluents, such as cocoa butter and vegetable oils, often replace
soybean oil to improve functional and flavour characteristics.

FUNCTIONAL USES Emulsifier, antioxidant

CHARACTERISTICS

IDENTIFICATION

Solubility (Vol. 4) Only partially soluble in water; readily hydrates to form emulsions; oil-free
phosphatides are soluble in fatty acids, but practically insoluble in fixed oils

Test for phosphorus Ignite 1 g of the sample with 2 g of anhydrous sodium carbonate. Cool and
dissolve the residue in 5 ml of water and 5 ml of nitric acid. Add 5 ml of
ammonium molybdate TS and heat to boiling. A yellow precipitate is
obtained.

Test for choline To 0.5 g of the sample, add 5 ml of diluted hydrochloric acid (1+1), heat in a
water bath for 2 h, and filter. Use this solution as the test solution. Perform
 Paper Chromatography with 10 µl of the test solution, using choline chloride
solution (1+200) as the control solution and n-butanol-water-acetic acid
mixture (4:2:1) as the developing solvent. A red-orange spot corresponding
to the spot obtained from the control solution is observed. For the filter
paper, use a No. 2 filter paper for chromatography. Stop the development
when the developing solvent rises about 25 cm, air-dry, spray with
Dragendorff TS to develop a colour, and observe in daylight.

Test for fatty acids Reflux 1 g of the sample for 1 h with 25 ml of 0.5 N ethanolic potassium
hydroxide. When cooled to 0o, a precipitate of potassium soap is obtained.

Test for hydrolysis To a 800 ml beaker add 500 ml of water (30-35o). Then slowly add 50 ml of
the sample with constant stirring. Hydrolyzed lecithin will form a
homogeneous emulsion. Non-hydrolyzed lecithin will form a distinct mass
of about 50 g.

PURITY

Loss on drying (Vol. 4) Not more than 2% (105º, 1 h)

Acid value Not more than 36

See description under TESTS

Peroxide value Not more than 10

See description under TESTS

Toluene-insoluble matter Not more than 0.3%

See description under TESTS

Lead (Vol. 4) Not more than 2 mg/kg

Determine using an atomic absorption technique appropriate to the

specified level. The selection of sample size and method of sample
preparation may be based on the principles of the method described in
Volume 4, “Instrumental Methods.”

TESTS

Acid value Weigh accurately about 2 g of the well-mixed sample into a 250-ml
Erlenmeyer flask. Dissolve in 50 ml of petroleum ether by shaking gently.
Then add 50 ml of ethanol, previously neutralized to phenolphthalein with
0.1 N sodium hydroxide, and shake to mix. Add 4 drops of phenolphthalein
TS and titrate while shaking with 0.1 N sodium hydroxide until the pink
colour persists for 5 sec.

Peroxide value Reagents

- Acetic acid-chloroform solution: Mix 3 volumes of acetic acid with 2
 volumes of chloroform.
- Potassium iodide solution, saturated: Dissolve excess potassium iodide in
freshly boiled water. Excess solid must remain. Store in the dark. Test daily
by adding 0.5 ml to 30 ml of the acetic acid-chloroform solution, then add 2
drops of starch TS. If the solution turns blue, requiring more than 1 drop of
0.1 N sodium thiosulfate to discharge the colour, prepare a fresh solution.

Procedure
Weigh accurately about 5 g of the sample into a 250-ml Erlenmeyer flask.
Add 30 ml of the acetic acid-chloroform solution and swirl to dissolve. Add
0.5 ml of the saturated potassium iodide solution, allow to stand with
occasional shaking for 1 min, and add 30 ml of water. Slowly titrate with
0.01 N sodium thiosulfate with vigorous shaking until the yellow colour is
almost gone. Add about 0.5 ml of starch TS, and continue the titration,
shaking vigorously to release all the iodine from the chloroform layer, until
the blue colour disappears.

Perform a blank determination and make any necessary correction.

where
S = ml of N sodium thiosulfate
N = normality of sodium thiosulfate
W = weight of the sample (g)

Toluene-insoluble matter Weigh 10 g of the well-mixed sample into a 250-ml flask. Add 100 ml of
toluene and shake until dissolved. Filter through a tared filter funnel G3 or
equivalent with a porosity of 16-40 µm. Wash the flask with 25-ml portions
of toluene and pour the washings through the funnel. Place the funnel in a
forced-draft oven and dry at 105o for 1 h. Weigh dried funnel and subtract
tare to determine weight of toluene insoluble residue:

METHOD OF Purification of phosphatides

ASSAY Wash about 10 g of the sample 3 times well with each 100 ml of acetone.
The insoluble residue (phosphatides) is used. Residues (phosphatides)
obtained from assays carried out previously can also be used. Dissolve 5 g
of these phosphatides in 10 ml of petroleum ether, and add 25 ml of
acetone to the solution. Transfer approximately equal portions of the
precipitate to each of two 40-ml centrifuge tubes using additional portions of
acetone to facilitate the transfer. Stir thoroughly, dilute to 40 ml with
acetone, stir again, chill for 15 min in an ice bath, stir again, and then
centrifuge for 5 min. Decant the acetone, stir, chill, centrifuge, and decant
as before. The solids after the second centrifugation require no further
purification and may be used for preparing the phosphatide-acetone
solution. To saturate about 16 litres of acetone, 5 g of the purified
phosphatides are required.
Phosphatide acetone solution
Add a quantity of purified phosphatides to sufficient acetone, previously
cooled to a temperature of about 5o, to form a saturated solution, and
maintain the mixture at this temperature for 2 h., shaking it vigorously at 15-
min. intervals. Decant the solution through a rapid filter paper, avoiding the
transfer of any undissolved solids to the paper and conducting the filtration
under refrigerated conditions (not above 5º).

Procedure
If lecithin is plastic or semisolid, soften a portion of the sample by warming
it in a water bath at a temperature not exceeding 60o and then mixing it
thoroughly. Transfer about 2 g of a well-mixed sample, accurately weighed,
into a previously tared 40-ml centrifuge tube, containing a glass stirring rod,
and add 15 ml of Phosphatide-Acetone Solution from a buret. Warm the
mixture in a water bath until the lecithin melts, but avoid evaporation of the
acetone. Stir until the sample is completely disintegrated and dispersed,
and then transfer the tube into an ice bath, chill for 5 min, remove from the
ice bath, and add about one half of the required volume of Phosphatide-
Acetone Solution, previously chilled for 5 min in an ice bath. Stir the mixture
to complete dispersion of the sample, dilute to 40 ml with chilled
Phosphatide-Acetone Solution (5o), again stir, and return the tube and
contents to the ice bath for 15 min. At the end of the 15-min chilling period,
stir again while still in the ice bath, remove the stirring rod, temporarily
supporting it in a vertical upside-down position, and centrifuge the mixture
immediately at about 2000 rpm for 5 min. Decant the supernatant liquid
from the centrifuge tube, crush the centrifuged solids with the same stirring
rod previously used, and refill the tube to the 40-ml mark with chilled (5o)
Phosphatide-Acetone Solution and repeat the chilling, stirring,
centrifugation, and decantation procedure previously followed. After the
second centrifugation and decantation of the supernatant acetone, again
crush the solids with the assigned stirring rod, and place the tube and its
contents in a horizontal position at room temperature until the excess
acetone has evaporated. Mix the residue again, dry the centrifuge tube and
its contents at 105o for 45 min in a forced-draft oven, cool, and weigh.

Calculate the percentage of acetone-insoluble matter by the formula

(100R/S) - B, in which R is the weight of residue, S is the weight of the
sample, and B is the percentage of toluene-insoluble matter (see TESTS).