CFR - Code of Federal Regulations Title 21

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[Code of Federal Regulations]

[Title 21, Volume 3]

[CITE: 21CFR173]

TITLE 21--FOOD AND DRUGS
CHAPTER I--FOOD AND DRUG ADMINISTRATION
DEPARTMENT OF HEALTH AND HUMAN SERVICES
					
SUBCHAPTER B - FOOD FOR HUMAN CONSUMPTION (CONTINUED)
					
							PART 173SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION

Subpart B - Enzyme Preparations and Microorganisms

Sec. 173.110 Amyloglucosidase derived from Rhizopus niveus.

Amyloglucosidase enzyme product, consisting of enzyme derived from Rhizopus niveus, and diatomaceous silica as a carrier, may be safely used in food in accordance with the following conditions:

(a) Rhizopus niveus is classified as follows: Class, Phycomycetes; order, Mucorales; family, Mucoraceae; genus, Rhizopus; species, niveus.

(b) The strain of Rhizopus niveus is nonpathogenic and nontoxic in man or other animals.

(c) The enzyme is produced by a process which completely removes the organism Rhizopus niveus from the amyloglucosidase.

(d) The additive is used or intended for use for degrading gelatinized starch into constituent sugars, in the production of distilled spirits and vinegar.

(e) The additive is used at a level not to exceed 0.1 percent by weight of the gelatinized starch.

Sec. 173.115 Alpha-acetolactate decarboxylase ([alpha]-ALDC) enzyme preparation derived from a recombinant Bacillus subtilis.

The food additive alpha-acetolactate decarboxylase ([alpha]-ALDC) enzyme preparation, may be safely used in accordance with the following conditions:

(a) The food additive is the enzyme preparation derived from a modified Bacillus subtilis strain that contains the gene coding for [alpha]-ALDC from Bacillus brevis.

(b)(1) The manufacturer produces the additive from a pure culture fermentation of a strain of Bacillus subtilis that is nonpathogenic and nontoxigenic in man or other animals.

(2) The manufacturer may stabilize the enzyme preparation with glutaraldehyde or with other suitable approved food additives or generally recognized as safe substances.

(3) The enzyme preparation must meet the general and additional requirements for enzyme preparations in the Food Chemicals Codex, 4th ed., 1996, pp. 133-134, which is incorporated by reference. The Director of the Office of the Federal Register approves this incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from the National Academy Press, 2101 Constitution Ave. NW., Washington, DC 20055, or may be examined at the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 20852, 240-402-7500, between 9 a.m. and 4 p.m., Monday through Friday, or 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 in an amount not in excess of the minimum required to produce its intended effect as a processing aid in the production of alcoholic malt beverages and distilled liquors.

[66 FR 27022, May 16, 2001, as amended at 88 FR 17722, Mar. 24, 2023]

Sec. 173.120 Carbohydrase and cellulase derived from Aspergillus niger.

Carbohydrase and cellulase enzyme preparation derived from Aspergillus niger may be safely used in food in accordance with the following prescribed conditions:

(a) Aspergillus niger is classified as follows: Class, Deuteromycetes; order, Moniliales; family, Moniliaceae; genus, Aspergillus; species, niger.

(b) The strain of Aspergillus niger is nonpathogenic and nontoxic in man or other animals.

(c) The additive is produced by a process that completely removes the organism Aspergillus niger from the carbohydrase and cellulase enzyme product.

(d) The additive is used or intended for use as follows:

(1) For removal of visceral mass (bellies) in clam processing.

(2) As an aid in the removal of the shell from the edible tissue in shrimp processing.

(e) The additive is used in an amount not in excess of the minimum required to produce its intended effect.

Sec. 173.130 Carbohydrase derived from Rhizopus oryzae.

Carbohydrase from Rhizopus oryzae may be safely used in the production of dextrose from starch in accordance with the following prescribed conditions:

(a) Rhizopus oryzae is classified as follows: Class, Phycomycetes; order, Mucorales; family, Mucoraceae; genus, Rhizopus; species, Rhizopus oryzae.

(b) The strain of Rhizopus oryzae is nonpathogenic and nontoxic.

(c) The carbohydrase is produced under controlled conditions to maintain nonpathogenicity and nontoxicity, including the absence of aflatoxin.

(d) The carbohydrase is produced by a process which completely removes the organism Rhizopus oryzae from the carbohydrase product.

(e) The carbohydrase is maintained under refrigeration from production to use and is labeled to include the necessity of refrigerated storage.

Sec. 173.135 Catalase derived from Micrococcus lysodeikticus.

Bacterial catalase derived from Micrococcus lysodeikticus by a pure culture fermentation process may be safely used in destroying and removing hydrogen peroxide used in the manufacture of cheese, in accordance with the following conditions.

(a) The organism Micrococcus lysodeikticus from which the bacterial catalase is to be derived is demonstrated to be nontoxic and nonpathogenic.

(b) The organism Micrococcus lysodeikticus is removed from the bacterial catalase prior to use of the bacterial catalase.

Sec. 173.140 Esterase-lipase derived from Mucor miehei.

Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by a pure culture fermentation process, with maltodextrin or sweet whey as a carrier, may be safely used in food in accordance with the following conditions:

(a) Mucor miehei var. Cooney et Emerson is classified as follows: Class, Phycomycetes; subclass, Zygomycetes; order, Mucorales; family, Mucoraceae; genus, Mucor; species, miehei; variety Cooney et Emerson.

(b) The strain of Mucor miehei var. Cooney et Emerson is nonpathogenic and nontoxic in man or other animals.

(c) The enzyme is produced by a process which completely removes the organism Mucor miehei var. Cooney et Emerson from the esterase-lipase.

(d) The enzyme is used as a flavor enhancer as defined in § 170.3(o)(12).

(e) The enzyme is used at levels not to exceed current good manufacturing practice in the following food categories: cheeses as defined in § 170.3(n)(5) of this chapter; fat and oils as defined in § 170.(3)(n)(12) of this chapter; and milk products as defined in § 170.(3)(n)(31) of this chapter. Use of this food ingredient is limited to nonstandarized foods and those foods for which the relevant standards of identity permit such use.

(f) The enzyme is used in the minimum amount required to produce its limited technical effect.

[47 FR 28090, June 29, 1982; 48 FR 2748, Jan. 21, 1983]

Sec. 173.145 Alpha-Galactosidase derived from Mortierella vinaceae var. raffinoseutilizer.

The food additive alpha-galactosidase and parent mycelial microorganism Mortierella vinaceae var. raffinoseutilizer may be safely used in food in accordance with the following conditions:

(a) The food additive is the enzyme alpha-galactosidase and the mycelia of the microorganism Mortierella vinaceae var. raffinoseutilizer which produces the enzyme.

(b) The nonpathogenic microorganism matches American Type Culture Collection (ATCC) No. 20034, ¹ and is classified as follows:

Class: Phycomycetes.

Order: Mucorales.

Family: Mortierellaceae.

Genus: Mortierella.

Species: vinaceae.

Variety: raffinoseutilizer.

(c) The additive is used or intended for use in the production of sugar (sucrose) from sugar beets by addition as mycelial pellets to the molasses to increase the yield of sucrose, followed by removal of the spent mycelial pellets by filtration.

(d) The enzyme removal is such that there are no enzyme or mycelial residues remaining in the finished sucrose.

¹ Available from: American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.

[42 FR 14526, Mar. 15, 1977, as amended at 54 FR 24897, June 12, 1989]

Sec. 173.150 Milk-clotting enzymes, microbial.

Milk-clotting enzyme produced by pure-culture fermentation process may be safely used in the production of cheese in accordance with the following prescribed conditions:

(a) Milk-clotting enzyme is derived from one of the following organisms by a pure-culture fermentation process:

(1) Endothia parasitica classified as follows: Class, Ascomycetes; order, Sphaeriales; family, Diaporthacesae; genus, Endothia; species, parasitica.

(2) Bacillus cereus classified as follows: Class, Schizomycetes; order, Eubacteriales; family, Bacillaceae; genus, Bacillus; species, cereus (Frankland and Frankland).

(3) Mucor pusillus Lindt classified as follows: Class, Phycomycetes; subclass, Zygomycetes; order, Mucorales; family, Mucoraceae; genus, Mucor; species, pusillus; variety, Lindt.

(4) Mucor miehei Cooney et Emerson classified as follows: Class, Phycomycetes; subclass, Zygomycetes; order, Mucorales; family, Mucoraceae; genus, Mucor; species, miehei; variety, Cooney et Emerson.

(5) Aspergillus oryzae modified by recombinant deoxyribonucleic (DNA) techniques to contain the gene coding for aspartic proteinase from Rhizomucor miehei var. Cooney et Emerson as defined in paragraph (a)(4) of this section, and classified as follows: Class, Blastodeuteromycetes (Hyphomycetes); order, Phialidales (Moniliales); genus, Aspergillus ; species oryzae.

(b) The strains of organism identified in paragraph (a) of this section are nonpathogenic and nontoxic in man or other animals.

(c) The additive is produced by a process that completely removes the generating organism from the milk-clotting enzyme product.

(d) The additive is used in an amount not in excess of the minimum required to produce its intended effect in the production of those cheeses for which it is permitted by standards of identity established pursuant to section 401 of the Act.

[42 FR 14526, Mar. 15, 1977; 42 FR 56728, Oct. 28, 1977, as amended at 62 FR 59284, Nov. 3, 1997]

Sec. 173.160 Candida guilliermondii.

The food additive Candida guilliermondii 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 viable organism Candida guilliermondii and its concomitant metabolites produced during the fermentation process.

(b)(1) The nonpathogenic and nontoxicogenic organism descending from strain, American Type Culture Collection (ATCC) No. 20474, ¹ is classified as follows:

Class: Deuteromycetes.

Order: Moniliales.

Family: Cryptococcaceae.

Genus: Candida.

Species: guilliermondii.

Variety: guilliermondii.

(2) The toxonomic characteristics of the reference culture strain ATCC No. 20474 agree in the essentials with the standard description for Candida guilliermondii variety guilliermondii listed in "The Yeasts - A Toxonomic Study;" 2d Ed. (1970), by Jacomina Lodder, which is incorporated by reference. Copies are available from the Office of Food Additive Safety (HFS-200), Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Dr., College Park, MD 20740, 240-402-1200, 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)(1) The additive is used or intended for use as a pure culture in the fermentation process for the production of citric acid using an acceptable aqueous carbohydrate substrate.

(2) The organism Candida quilliermondii is made nonviable and is completely removed from the citric acid during the recovery and purification process.

(d) The additive is so used that the citric acid produced conforms to the specifications of the Food Chemicals Codex, 7th ed. (2010), pp. 226-227, which is incorporated by reference. The Director of the Office of the Federal Register approves this incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You may obtain copies from the United States Pharmacopeial Convention, 12601 Twinbrook Pkwy., Rockville, MD 20852 (Internet address http://www.usp.org ). Copies may be examined at the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 20852, 240-402-7500, between 9 a.m. and 4 p.m., Monday through Friday, or 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/cfr/ibr-locations.html.

¹ Available from: American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.

[42 FR 14526, Mar. 15, 1977, as amended at 47 FR 11838, Mar. 19, 1982; 49 FR 10106, Mar. 19, 1984; 54 FR 24897, June 12, 1989; 78 FR 71466, Nov. 29, 2013; 88 FR 17722, Mar. 24, 2023]

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 Office of Food Additive Safety (HFS-200), Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5001 Campus Dr., College Park, MD 20740, 240-402-1200, 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.

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 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 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 173deg. 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 § 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 * 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; 78 FR 71466, Nov. 29, 2013; 88 FR 17722, Mar. 24, 2023]

Sec. 173.170 Aminoglycoside 3'-phosphotransferase II.

The food additive aminoglycoside 3'-phosphotransferase II may be safely used in the development of genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions:

(a) The food additive is the enzyme aminoglycoside 3'-phosphotransferase II (CAS Reg. No. 58943-39-8) which catalyzes the phosphorylation of certain aminoglycoside antibiotics, including kanamycin, neomycin, and gentamicin.

(b) Aminoglycoside 3'-phosphotransferase II is encoded by the kan ^r gene originally isolated from transposon Tn5 of the bacterium Escherichia coli.

(c) The level of the additive does not exceed the amount reasonably required for selection of plant cells carrying the kan ^r gene along with the genetic material of interest.

[59 FR 26711, May 23, 1994]

Authority: 21 U.S.C. 321, 342, 348.
Source: 42 FR 14526, Mar. 15, 1977, unless otherwise noted.