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173.165 Candida lipolytica.

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[Code of Federal Regulations]
[Title 21, Volume 3]
[Revised as of April 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 21CFR173.165]

[Page 132-135]
 
                        TITLE 21--FOOD AND DRUGS
 
CHAPTER I--FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN 
                          SERVICES (CONTINUED)
 
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 
fermentation process.
    (b)(1) The nonpathogenic organism is classified as follows:

Class: Deuteromycetes.
Order: Moniliales.
Family: Cryptococcaceae.
Genus: Candida.
Species: lipolytica.

    (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/
federal--register/code--of--federal--regulations/ibr--locations.html.
    (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 
normal alkanes.
    (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

[[Page 133]]

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

                          general instructions

    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.

                                apparatus

    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 
absorbance.
    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 
deep.

                         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 
test:
    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 
nanometers.
    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 
otherwise.

                            eluting mixtures

    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

[[Page 134]]

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 
following test:
    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.

                                procedure

    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 
isooctane extract.
    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.

[[Page 135]]

    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:


Source: U.S. Code of Federal Regulations - CFR Title 21, Part 173, Section 165


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