§ 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 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 × 900-millimeter length equipped with tetrafluoroethylene polymer stopcock and course fritted disk; （b） 18-millimeter ID × 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 × 200 mm × 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 °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 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 × 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° to 177 °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 §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 °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 × 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 °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 °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]
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