Gellan gum
Prepared at the 49th JECFA (1997)
superseding specifications prepared at the 46th JECFA (1996),
published in FNP 52 Addendum 4 (1996)
SYNONYM INS No. 418
DEFINITION Gellan gum is a high molecular weight polysaccharide gum produced by
a pure culture fermentation of a carbohydrate by Pseudomonas elodea,
purified by recovery with isopropyl alcohol, dried, and milled. The high
molecular weight polysaccharide is principally composed of a
tetrasaccharide repeating unit of one rhamnose, one glucuronic acid,
and two glucose units, and is substituted with acyl (glyceryl and acetyl)
groups as the O-glycosidically-linked esters. The glucuronic acid is
neutralized to a mixed potassium, sodium, calcium, and magnesium salt.
It usually contains a small amount of nitrogen containing compounds
resulting from the fermentation procedures..
C.A.S. number 71010-52-1
Formula weight Approximately 500,000
Assay Yields, on the dried basis, not less than 3.3% and not more than 6.8% of
carbon dioxide (CO2).
DESCRIPTION Off-white powder
FUNCTIONAL USES Thickening agent, gelling agent, stabilizer
CHARACTERISTICS
IDENTIFICATION
Solubility Soluble in water, forming a viscous solution; insoluble in ethanol
Gel test with calcium ion
Passes test
See description under TESTS
Gel test with sodium ion
Passes test
See description under TESTS
PURITY
Loss on drying Not more than 15% (105°, 2½h)
Lead Not more than 2 mg/kg
Prepare a sample solution as directed for organic compounds in the Limit
Test and determine the lead content by atomic absorption
Nitrogen Not more than 3%
Isopropyl alcohol Not more than 750 mg/kg
See description under TESTS
Microbiological Total plate count: Not more than 10,000 colonies per gram
criteria E. coli Negative by test
Salmonella: Negative by test
Yeasts and moulds: Not more than 400 colonies per gram
See description under TESTS
TESTS
IDENTIFICATION TESTS
Gel test with calcium ion
Add 1.0 g of the sample to 99 ml of water, and stir for about 2 h, using a
motorized stirrer having a propeller-type stirring blade. Draw a small
amount of this solution into a wide bore pipet and transfer into a 10%
solution of calcium chloride. A tough worm-like gel will be formed
immediately.
Gel test with sodium ion
To the 1% solution of the sample prepared for the previous test, add 0.50
g of sodium chloride, heat to 80° with stirring, and hold at 80° for 1
min. Allow the solution to cool to room temperature. A firm gel is formed.
PURITY TESTS
Isopropyl alcohol Isopropyl alcohol (IPA) Standard Solution:
Transfer 500.0 mg of chromatographic quality isopropyl 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.
Tertiary butyl alcohol (TBA) Standard Solution:
Transfer 500.0 mg of chromatographic quality tert-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 IPA standard 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 40g each of
isopropyl alcohol and of tert-butyl 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
round-bottom distilling flask. Add about 5 g of the sample, accurately
weighed, and shake the flask for 1 h, on a wrist-action mechanical
shaker. Connect the flask to a fractionating column and distil about 100
ml; adjust 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.
Procedure:
Inject about 5 l of the Mixed Standard Solution into a suitable gas
chromatograph equipped with a flame-ionization detector and a 1.8-m x
2.3-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 200°, the column temperature 165°, and the
detector temperature 200°. The retention time of isopropyl alcohol is
about 2 min, and that of tert-butyl alcohol about 3 min.
Determine the areas of the IPA and TBA peaks, and calculate the
response factor, f, by the formula AIPA/ATBA, in which AIPA is the area of
the isopropyl alcohol peak, and ATBA is the area of the tert-butyl alcohol
Peak.
Similarly, inject about 5 ul of the Sample Preparation, and determine the
peak areas, recording the area of the isopropyl alcohol peak as aIPA, and
that of the tert-butyl alcohol peak as aTBA. Calculate the isopropyl alcohol
content, in mg/kg, in the sample taken by the formula:
(aIPA x 4000)(f x aTBA x W)
in which W is the weight of the sample taken, in g.
Microbiological test
Total plate count: Using aseptic technique, disperse 1 g of sample into 99
ml of phosphate buffer and use a Stomacher, shaker or stirrer to fully
dissolve. Limit dissolving time to about 10 min and then pipette 1 ml of
the solution into separate, duplicate, appropriately marked petri dishes.
Pour over the aliquot of sample in each petri dish 12-15 ml of Plate Count
Agar previously tempered to 44-46°. Mix well by alternate rotation and
back and forth motion of the plates, allow the agar to solidify. Invert the
plates and incubate for 48±2 h at 35±1°.
After incubation count the growing colonies visible on each plate and
record the number of colonies. Take the average of both plates, and
multiply by the sample dilution factor, 100. Where no colonies are visible,
express the result as less than 100 cfu/g.
E. coli determination: Using aseptic technique, disperse 1 g of sample in
99 ml of Lactose broth using either a Stomacher, shaker or stirrer to fully
dissolve the sample. Limit the dissolving time to about 15 min and then
lightly seal the container and incubate the broth for 18-24 h at 35±1°.
Using a sterile pipette, inoculate 1 ml of the incubate into a tube
containing 10 ml GN broth. Incubate for 18-24 h and then streak any GN
broths showing positive growth or gas production onto duplicate plates of
Levine EMB agar. Incubate the plates for 24±2 h at 35±1° and then
examine for colonies typical of E. coli i.e. showing strong purple growth
with dark centre and a green metallic sheen sometimes spreading onto
the agar. Record any typical E. coli colonies as presumptive positive,
otherwise negative.
Streak any well isolated suspect colonies onto a plate of PCA and
incubate for 18-24 h at 35±1°. Perform a Gram stain on any growth to
confirm it is Gram negative. If so, disperse any colony growth into a small
volume of 0.85% saline and perform chemical tests to confirm the
identity of the bacterial growth. This can most conveniently be done by
using API 20E or Micro ID strips or equivalent systems.
After completion of the tests, identify the organism from the Identification
manual of the system used and record the final result.
Media
GN Broth (Gram Negative Broth)
Peptone 20.0 g
Dextrose 1.0 g
Mannitol 2.0 g
Sodium citrate 5.0 g
Sodium deoxycholate 0.5 g
Potassium phosphate (dibasic) 4.0 g
Potassium phosphate (monobasic) 1.5 g
Sodium chloride 5.0 g
Make up to 1 litre with distilled or de-ionised water, pH 7.0±0.2 at 25°
.
Salmonella determination: Using aseptic technique, disperse 5 g of
sample into 200 ml of sterile lactose broth using either a Stomacher,
shaker or stirrer to maximise dissolution over a 15 min period. Loosely
seal the container and incubate at 35±1° for 24±2h.
Continue as per method on page 221 of FNP 5/Rev. 2 (1991).
Identification can be more conveniently done using API or Micro ID
systems or equivalent.
Yeasts and moulds: Using aseptic technique, disperse 1 g of sample into
99 ml of phosphate buffer and use a Stomacher, shaker or stirrer to fully
dissolve. Limit dissolving time to about 10 min and then pipette 1 ml of
the solution into separate, duplicate, appropriately marked petri dishes.
Pour over the aliquot of sample in each petri dish 15-20 ml of Potato
dextrose Agar (either acidified or containing antibiotic) previously
tempered to 44-46°. Mix well by alternate rotation and back and forth
motion of the plates, and allow the agar to solidify. Invert the plates and
incubate for 5 days at 20-25°.
After incubation, count the growing colonies visible on each plate using a
colony counter and record the number of colonies. Separate the yeasts
from the moulds according to their morphology and count them
separately. Take the average of both plates and multiply by the sample
dilution factor, 100. Where no colonies are visible, express the result as
less than 100 cfu/g.
METHOD OF ASSAY
Processed as directed in the test for Carbon Dioxide Determination by
Decarboxylation in the General Methods, using about 1.2 g of the sample
weighed accurately.
