[Code of Federal Regulations]
[Title 21, Volume 3]
[Revised as of April 1, 2006]
From the U.S. Government Printing Office via GPO Access
TITLE 21--FOOD AND DRUGS
CHAPTER I--FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN
PART 173_SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN
CONSUMPTION--Table of Contents
Subpart B_Enzyme Preparations and Microorganisms
Sec. 173.165 Candida lipolytica.
The food additive Candida lipolytica may be safely used as the
organism for fermentation production of citric acid in accordance with
the following conditions:
(a) The food additive is the enzyme system of the organism Candida
lipolytica and its concimitant metabolites produced during the
(b)(1) The nonpathogenic organism is classified as follows:
(2) The taxonomic characteristics of the culture agree in essential
with the standard description for Candida lipolytica variety lipolytica
listed in ``The Yeasts--A Toxonomic Study,'' 2d Ed. (1970), by Jacomina
Lodder, which is incorporated by reference. Copies are available from
the Center for Food Safety and Applied Nutrition (HFS-200), Food and
Drug Administration, 5100 Paint Branch Pkwy., College Park, MD 20740, or
available for inspection at the National Archives and Records
Administration (NARA). For information on the availability of this
material at NARA, call 202-741-6030, or go to: http://www.archives.gov/
(c) The additive is used or intended for use as a pure culture in
the fermentation process for the production of citric acid from purified
(d) The additive is so used that the citric acid produced conforms
to the specifications of the ``Food Chemicals Codex,'' 3d Ed. (1981),
pp. 86-87, which is incorporated by reference. Copies may be obtained
from the National Academy Press, 2101 Constitution Ave. NW., Washington,
DC 20418, or may be examined at the National Archives and Records
Administration (NARA). For
information on the availability of this material at NARA, call 202-741-
6030, or go to: http://www.archives.gov/federal--register/code--of--
federal--regulations/ibr--locations.html. The additive meets the
following ultraviolet absorbance limits when subjected to the analytical
procedure described in this paragraph:
Ultraviolet absorbance per centimeter path length Maximum
280 to 289 millimicrons..................................... 0.25
290 to 299 millimicrons..................................... 0.20
300 to 359 millimicrons..................................... 0.13
360 to 400 millimicrons..................................... 0.03
Analytical Procedure for Citric Acid
Because of the sensitivity of the test, the possibility of errors
arising from contamination is great. It is of the greatest importance
that all glassware be scrupulously cleaned to remove all organic matter
such as oil, grease, detergent residues, etc. Examine all glassware
including stoppers and stopcocks, under ultraviolet light to detect any
residual fluorescent contamination. As a precautionary measure it is
recommended practice to rinse all glassware with purified isooctane
immediately before use. No grease is to be used on stopcocks or joints.
Great care to avoid contamination of citric acid samples in handling is
essential to assure absence of any extraneous material arising from
inadequate packaging. Because some of the polynuclear hydrocarbons
sought in this test are very susceptible to photo-oxidation, the entire
procedure is to be carried out under subdued light.
1. Aluminum foil, oil free.
2. Separatory funnels, 500-milliliter capacity, equipped with
tetrafluoroethylene polymer stopcocks.
3. Chromatographic tubes: (a) 80-millimeter ID x 900-millimeter
length equipped with tetrafluoroethylene polymer stopcock and course
fritted disk; (b) 18-millimeter ID x 300-millimeter length equipped with
tetrafluoroethylene polymer stopcock.
4. Rotary vacuum evaporator, Buchi or equivalent.
5. Spectrophotometer--Spectral range 250-400 nanometers with
spectral slit width of 2 nanometers or less; under instrument operating
conditions for these absorbance measurements, the spectrophotometer
shall also meet the following performance requirements:
Absorbance repeatability, 0.01 at 0.4
Wavelength repeatability, 0.2 nanometer.
Wavelength accuracy, 1.0 nanometer.
The spectrophotometer is equipped with matched 1 centimeter path
length quartz microcuvettes with 0.5-milliliter volume capacity.
6. Vacuum oven, minimum inside dimensions: 200 mm x 200 mm x 300 mm
reagents and materials
Organic solvents. All solvents used throughout the procedure shall
meet the specifications and tests described in this specification. The
methyl alcohol, isooctane, benzene, hexane and 1,2-dichloroethane
designated in the list following this paragraph shall pass the following
The specified quantity of solvent is added to a 250-milliliter round
bottom flask containing 0.5 milliliter of purified n-hexadecane and
evaporated on the rotary evaporator at 45 [deg]C to constant volume. Six
milliliters of purified isooctane are added to this residue and
evaporated under the same conditions as above for 5 minutes. Determine
the absorbance of the residue compared to purified n-hexadecane as
reference. The absorbance of the solution of the solvent residue shall
not exceed 0.03 per centimeter path length between 280 and 299
nanometers and 0.01 per centimeter path length between 300 and 400
Methyl alcohol, A.C.S. reagent grade. Use 100 milliliters for the
test described in the preceding paragraph. If necessary, methyl alcohol
may be purified by distillation through a Virgreaux column discarding
the first and last ten percent of the distillate or otherwise.
Benzene, spectrograde (Burdick and Jackson Laboratories, Inc.,
Muskegon, Mich., or equivalent). Use 80 milliliters for the test. If
necessary, benzene may be purified by distillation or otherwise.
Isooctane (2,2,4-trimethylpentane). Use 100 milliliters for the
test. If necessary, isooctane may be purified by passage through a
column of activated silica gel, distillation or otherwise.
Hexane, spectrograde (Burdick and Jackson Laboratories, Inc.,
Muskegon, Mich., or equivalent). Use 100 milliliters for the test. If
necessary, hexane may be purified by distillation or otherwise.
1,2-Dichloroethane, spectrograde (Matheson, Coleman and Bell, East
Rutherford, N.J., or equivalent). Use 100 milliliters for the test. If
necessary, 1,2-dichloroethane may be purified by distillation or
1. 10 percent 1,2-dichloroethane in hexane. Prepare by mixing the
purified solvents in the volume ratio of 1 part of 1,2-dichloroethane to
9 parts of hexane.
2. 40 percent benzene in hexane. Prepare by mixing the purified
solvents in the volume
ratio of 4 parts of benzene to 6 parts of hexane.
n-Hexadecane, 99 percent olefin-free. Determine the absorbance
compared to isooctane as reference. The absorbance per centimeter path
length shall not exceed 0.00 in the range of 280-400 nanometers. If
necessary, n-hexadecane may be purified by percolation through activated
silica gel, distillation or otherwise.
Silica gel, 28-200 mesh (Grade 12, Davison Chemical Co., Baltimore,
MD, or equivalent). Activate as follows: Slurry 900 grams of silica gel
reagent with 2 liters of purified water in a 3-liter beaker. Cool the
mixture and pour into a 80 x 900 chromatographic column with coarse
fritted disc. Drain the water, wash with an additional 6 liters of
purified water and wash with 3,600 milliliters of purified methyl
alcohol at a relatively slow rate. Drain all of the solvents and
transfer the silica gel to an aluminum foil-lined drying dish. Place
foil over the top of the dish. Activate in a vacuum oven at low vacuum
(approximately 750 millimeters Mercury or 27 inches of Mercury below
atmospheric pressure) at 173[deg] to 177 [deg]C for at least 20 hours.
Cool under vacuum and store in an amber bottle.
Sodium sulfate, anhydrous, A.C.S. reagent grade. This reagent should
be washed with purified isooctane. Check the purity of this reagent as
described in Sec. 172.886 of this chapter.
Water, purified. All water used must meet the specifications of the
Extract 600 milliliters of water with 50 milliliters of purified
isooctane. Add 1 milliliter of purified n-hexadecane to the isooctane
extract and evaporate the resulting solution to 1 milliliter. The
absorbance of this residue shall not exceed 0.02 per centimeter path
length between 300-400 nanometers and 0.03 per centimeter path length
between 280-299 nanometers. If necessary, water may be purified by
distillation, extraction with purified organic solvents, treatment with
an absorbent (e.g., activated carbon) followed by filtration of the
absorbent or otherwise.
Separate portions of 200 milliliters of purified water are taken
through the procedure for use as control blanks. Each citric acid sample
is processed as follows: Weigh 200 grams of anhydrous citric acid into a
500 milliliter flask and dissolve in 200 milliliters of pure water. Heat
the solution to 60 [deg]C and transfer to a 500 milliliter separatory
funnel. Rinse the flask with 50 milliliters of isooctane and add the
isooctane to the separatory funnel. Gently shake the mixture 90 times
(caution: vigorous shaking will cause emulsions) with periodic release
of the pressure caused by shaking.
Allow the phases to separate for at least 5 minutes. Draw off the
lower aqueous layer into a second 500-milliliter separatory funnel and
repeat the extraction with a second aliquot of 50 milliliters of
isooctane. After separation of the layers, draw off and discard the
water layer. Combine both isooctane extracts in the funnel containing
the first extract. Rinse the funnel which contained the second extract
with 10 milliliters of isooctane and add this portion to the combined
A chromatographic column containing 5.5 grams of silica gel and 3
grams of anhydrous sodium sulfate is prepared for each citric acid
sample as follows: Fit 18 x 300 column with a small glass wool plug.
Rinse the inside of the column with 10 milliliters of purified
isooctane. Drain the isooctane from the column. Pour 5.5 grams of
activated silica gel into the column. Tap the column approximately 20
times on a semisoft, clean surface to settle the silica gel. Carefully
pour 3 grams of anhydrous sodium sulfate onto the top of the silica gel
in the column.
Carefully drain the isooctane extract of the citric acid solution
into the column in a series of additions while the isooctane is draining
from the column at an elution rate of approximately 3 milliliters per
minute. Rinse the separatory funnel with 10 milliliters of isooctane
after the last portion of the extract has been applied to the column and
add this rinse to the column. After all of the extract has been applied
to the column and the solvent layer reaches the top of the sulfate bed,
rinse the column with 25 milliliters of isooctane followed by 10
milliliters of a 10-percent dichloroethane in hexane solution. For each
rinse solution, drain the column until the solvent layer reaches the top
of the sodium sulfate bed. Discard the rinse solvents. Place a 250-
milliliter round bottom flask containing 0.5 milliliter of purified n-
hexadecane under the column. Elute the polynuclear aromatic hydrocarbons
from the column with 30 milliliters of 40-percent benzene in hexane
solution. Drain the eluate until the 40-percent benzene in the hexane
solvent reaches the top of the sodium sulfate bed.
Evaporate the 40-percent benzene in hexane eluate on the rotary
vacuum evaporator at 45 [deg]C until only the n-hexadecane residue of
0.5 milliliter remains. Treat the n-hexadecane residue twice with the
following wash step: Add 6 milliliters of purified isooctane and remove
the solvents by vacuum evaporation at 45 [deg]C to constant volume,
i.e., 0.5 milliliter. Cool the n-hexadecane residue and transfer the
solution to an 0.5-milliliter microcuvette. Determine the absorbance of
this solution compared to purified n-hexadecane as reference. Correct
the absorbance values for any absorbance derived from the control
reagent blank. If the corrected absorbance does not exceed the limits
prescribed, the samples meet the ultraviolet absorbance specifications.
The reagent blank is prepared by using 200 milliliters of purified
water in place of the citric acid solution and carrying the water sample
through the procedure. The typical control reagent blank should not
exceed 0.03 absorbance per centimeter path length between 280 and 299
nanometers, 0.02 absorbance per centimeter path length between 300 and
359 nanometers, and 0.01 absorbance per centimeter path length between
360 and 400 nanometers.
[42 FR 14491, Mar. 15, 1977, as amended at 47 FR 11838, Mar. 19, 1982;
49 FR 10106, Mar. 19, 1984; 54 FR 24897, June 12, 1989]
Additives that reference this regulation: