[Federal Register Volume 60, Number 203 (Friday, October 20, 1995)]
[Rules and Regulations]
[Pages 54190-54193]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-26054]
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DEPARTMENT OF HEALTH AND HUMAN SERVICES
21 CFR Part 184
[Docket No. 87G-0406]
Direct Food Substances Affirmed as Generally Recognized as Safe;
Aminopeptidase Enzyme Preparation Derived From Lactococcus Lactis
AGENCY: Food and Drug Administration, HHS.
ACTION: Final rule.
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SUMMARY: The Food and Drug Administration (FDA) is amending its
regulations to affirm that the use of an aminopeptidase enzyme
preparation derived from Lactococcus lactis (formerly known as
Streptococcus lactis) in the manufacturing of cheddar cheese and in the
preparation of protein hydrolysates is generally recognized as safe
(GRAS). This action is in response to a petition filed by Imperial
Biotechnology, Ltd.
DATES: Effective October 20, 1995. The Director of the Office of the
Federal Register approves the incorporation by reference in accordance
with 5 U.S.C. 552(a) and 1 CFR part 51 of a publication listed in new
Sec. 184.1985, effective October 20, 1995.
FOR FURTHER INFORMATION CONTACT: Aydin Orstan, Center for Food Safety
and Applied Nutrition (HFS-217), Food and Drug Administration, 200 C
St. SW., Washington, DC 20204, 202-418-3076.
SUPPLEMENTARY INFORMATION:
I. Background
In accordance with the procedures described in 21 CFR 170.35,
Imperial Biotechnology, Ltd., Imperial College Rd., South Kensington,
London, SW7 2BT, United Kingdom, submitted a petition (GRASP 8G0335)
proposing that aminopeptidase from L. lactis be affirmed as GRAS as a
direct human food ingredient.
FDA published a notice of filing of this petition in the Federal
Register of February 23, 1988 (53 FR 5319), and gave interested parties
an opportunity to submit comments to the Dockets Management Branch
(HFA-305), Food and Drug Administration, rm. 1-23, 12420 Parklawn Dr.,
Rockville, MD 20857. FDA received no comments in response to that
notice.
II. Standards for GRAS Affirmation
Under Sec. 170.30 (21 CFR 170.30), general recognition of safety
may be based only on the views of experts qualified by scientific
training and experience to evaluate the safety of substances added to
food. The basis of such views may be either: (1) Scientific procedures,
or (2) in the case of a substance used in food prior to January 1,
1958, experience based on common use in food (Sec. 170.30(a)). General
recognition of safety based upon scientific procedures requires the
same quantity and quality of scientific evidence as is required to
obtain approval of a food additive regulation and ordinarily is to be
based upon published studies, which may be corroborated by unpublished
studies and other data and information (Sec. 170.30(b)). In its
petition, Imperial Biotechnology, Ltd., relies on scientific
procedures, primarily published scientific papers and books,
corroborated by unpublished information, to demonstrate the safety of
aminopeptidase enzyme preparation produced from L. lactis for use in
the manufacturing of cheddar cheese and in the preparation of protein
hydrolysates.
III. Identity, Technical Effect, and Production
A. Identity
Aminopeptidase enzyme preparation is a mixture of intracellular
peptidases derived from the bacterium L. lactis. Peptidases are enzymes
that cleave peptide bonds to liberate free amino acids or dipeptides
(Ref. 1). The natural occurrence of peptidases in the cellular extracts
of L. lactis and in extracts of cheese made with this organism is
documented in the scientific literature (Ref. 2).
For simplicity, the trivial name aminopeptidase is used to describe
the enzyme preparation. The Chemical Abstracts Service (CAS) Registry
Number for aminopeptidase is 9031-94-1. The Enzyme Commission (EC)
numbers of the enzymes present in aminopeptidase enzyme preparation are
as follows: aminopeptidase, EC 3.4.11.1; tripeptide aminopeptidase, EC
3.4.11.4; dipeptidase, EC 3.4.13.11; proline dipeptidase, EC 3.4.13.9;
dipeptidylpeptide hydrolases (EC 3.4.14.1-3) (Ref. 1). The agency finds
that the petitioned preparation meets the requirements for enzyme
preparations found in the Food Chemicals Codex, 3d ed. (1981), which is
incorporated by reference in new Sec. 184.1985.
B. Technical Effect
The progressive breakdown of milk proteins to peptides and amino
acids during the ripening of cheese leads to the development of typical
cheese texture and flavors. This process is catalyzed by aminopeptidase
and other peptidases produced by the bacteria added to milk as starter
cultures (Refs. 3 through 6). Also, these enzymes may be extracted from
bacterial cultures and used in improving flavor and eliminating the
bitterness of protein hydrolysates (Ref. 7), which are used in many
foods for a variety of functions, including as formulation aids,
leavening agents, stabilizers, thickening agents, nutrient supplements,
protein sources, flavorings, and flavor enhancers. The petitioner
intends to use the aminopeptidase enzyme preparation to accelerate
flavor development during cheddar cheese ripening and to improve the
flavor of protein hydrolysates used in various foods.
The petitioner has presented published information demonstrating
that peptidase enzymes from L. lactis perform their intended technical
effect in cheese manufacturing (Ref. 8). Furthermore, the petitioner
provided a European patent office publication containing an approved
patent application that demonstrates that the aminopeptidase enzyme
preparation performs its intended technical effect in the manufacture
of protein hydrolysates (Ref. 7). The petitioner also presented
unpublished, corroborative studies demonstrating that the
aminopeptidase enzyme preparation performs its intended technical
effects in the manufacture of cheddar cheese and protein hydrolysates.
C. Production and Purification
The production process for aminopeptidase enzyme preparation,
described in detail in GRASP 8G0335, may be summarized as follows: L.
lactis, started from a pure culture, is aseptically grown at 30 deg.C
in stainless steel fermenters in a medium containing lactose, casein
hydrolysate, yeast extract, ascorbic acid, disodium hydrogen phosphate,
magnesium sulfate, and polypropylene glycol P-2,000 as a defoaming
agent. Samples of the medium are removed aseptically at various stages
of fermentation and examined microscopically for typical morphology of
the production organism and for the presence of contaminating
organisms. During fermentation, the pH of the culture is maintained
within a range of 6.4-6.6 with sodium hydroxide. Once the maximum cell
density of the production organism, as measured by
[[Page 54191]]
optical density, has been reached, the fermentation is terminated by
cooling the contents of the fermenter down to 5-10 deg.C. The
bacterial cells are collected by centrifugation, resuspended in
phosphate buffer, and the intracellular enzymes are released by
physical disruption. The fraction containing aminopeptidase and other
enzymes is separated from unwanted material by ultrafiltration or
diafiltration. The enzyme fraction is dried, mixed, and packaged.
IV. Safety Evaluation
In evaluating the safety of aminopeptidase enzyme preparation as a
food ingredient, the agency considered the following issues: (1) The
safety of the producing organism; (2) the safety of the enzyme
component; and (3) exposure levels of the enzyme preparation in food.
A. The Producing Organism
The producing organism L. lactis was formerly named S. lactis.
However, genetic studies have demonstrated that this organism and
several of its relatives are not as closely related to the other
streptococci as was once thought, and the new information prompted
their transfer to the newly created genus Lactococcus in 1985 (Ref. 9).
Thus, in the older literature and various Federal regulations L. lactis
is referred to as S. lactis.
L. lactis and its related organisms belong to a group of bacteria
commonly known as the ``lactic acid bacteria'' (Ref. 10). All of the
cheese standards FDA lists in part 133 (21 CFR part 133) provide for
the use of lactic acid bacteria in the manufacture of cheese (for
example, Sec. 133.113 Cheddar cheese). Published information
demonstrates that L. lactis and several of its subspecies are commonly
used in cheese manufacturing (Refs. 2, 3, 4, 10, 11, and 12). The
Catalogue of Strains of the National Collection of Food Bacteria in the
United Kingdom lists several strains of L. lactis as cheese starter
cultures (Ref. 13), and the Catalogue of Bacteria of the American Type
Culture Collection cites various food uses for the same organism (Ref.
14).
Furthermore, certain strains of S. lactis are used to prepare two
substances that FDA has affirmed as GRAS, nisin (21 CFR 184.1538) and
starter distillate (21 CFR 184.1848). Additionally, the standards of
identity for acidified sour cream (21 CFR 131.162); sour half-and-half
(21 CFR 131.185); acidified sour half-and-half (21 CFR 131.187); and
bread, rolls, and buns (21 CFR 136.110) provide for the use of lactic
acid bacteria in the manufacture of these foods. S. lactis has been
used to manufacture cheese, buttermilk, and other fermented foods for
decades (Ref. 15). Lactic acid bacteria are the subject of a prior
sanction by the United States Department of Agriculture (Ref. 16) and
are listed as approved substances for use in several meat products in 9
CFR 318.7.
The information in the petition indicates that viable cells of the
producing organism L. lactis may remain in the final product. The
agency concludes that the presence of the viable cells of L. lactis in
aminopeptidase enzyme preparation is not a safety concern, however,
because: (1) The published information summarized above demonstrates
the widespread food uses of this organism without any safety concerns;
and (2) ``Bergey's Manual of Systematic Bacteriology,'' which describes
the pathogenicity of Streptococcus species, contains no reference to
pathogenicity of S. lactis (Ref. 17).
B. The Enzyme Component and Processing Aids
Published data demonstrate that aminopeptidase and other peptidases
are naturally present in cheese prepared using S. lactis as a starter
culture. In a study using a modified electrophoretic starch gel
technique on cheddar cheese, researchers detected aminopeptidase
activity in fractions of the cheese extracts (Ref. 2).
The petitioner also provided unpublished animal feeding studies as
corroborative evidence of the safety of the aminopeptidase enzyme
preparation. During a dietary range-finding study, rats were fed up to
2,000 milligrams (mg) aminopeptidase enzyme preparation per kilogram
(kg) body weight (bw) per day (d) for 28 days. There were no reported
deaths, clinical signs or group differences in liver and kidney weights
that could be ascribed to treatment. Also, weight gains and food intake
for all treatment groups were similar to those for controls.
During a second study, rats were fed aminopeptidase enzyme
preparation for 13 weeks at doses up to 2,000 mg/kg bw/d. There were no
deaths and no treatment-related clinical signs. Weight gains and food
intake for all treatment groups were similar to those for controls.
There were no macroscopic, pathologic, or histopathologic changes that
could be ascribed to treatment with aminopeptidase enzyme preparation.
Statistical analyses of organ weights showed no dose-related
differences between treated and control groups.
The agency concludes from the evidence summarized above that the
enzyme component of the aminopeptidase preparation does not raise
safety concerns; therefore, the relevant safety issue becomes whether
the enzyme preparations contain toxic contaminants. Enzyme preparations
used in food processing are usually not chemically pure but contain, in
addition to the enzyme component, materials that derive from the enzyme
source, as well as from the manufacturing methods used to generate the
finished enzyme preparation.
In accordance with Sec. 170.30(h)(1), the enzyme preparations
affirmed as GRAS in this document must comply with the general
requirements and additional requirements for enzyme preparations in the
Food Chemicals Codex, 3d ed., pp. 107-110. These include the
requirement that aminopeptidase enzyme preparation from L. lactis be
produced by methods and under culture conditions that ensure a
controlled fermentation, thus preventing the introduction of bacterial
cells that could be the source of toxic materials and other undesirable
substances. Moreover, any compounds that become or are intended to
become functional components of aminopeptidase enzyme preparation, such
as water, salts, preservatives, or stabilizers, must be either GRAS
ingredients or food additives approved as safe for this purpose.
Therefore, the agency concludes that the presence of added substances
and impurities derived from the enzyme source or introduced by
manufacturing does not present a basis for concern about the safety of
the enzyme preparation.
Additionally, the petitioner presented results of tests showing
that aminopeptidase enzyme preparation derived from the strain of L.
lactis used by the petitioner contains no detectable antibiotics that
might promote the development of antibiotic resistance.
C. Estimated Exposure Levels
For exposure estimates, the agency has considered the proposed uses
of aminopeptidase enzyme preparation in the manufacturing of cheddar
cheese and in the preparation of protein hydrolysates. Estimates of
enzyme use level and intake are usually based on the total organic
solids (TOS) content of the enzyme preparation. The petitioner provided
data indicating that the average TOS content of aminopeptidase enzyme
preparation is 85 percent by weight. Based on information on
consumption of cheese and processed foods containing protein
hydrolysates and on the amount of aminopeptidase enzyme preparation
needed to produce foods under conditions of current good
[[Page 54192]]
manufacturing practice (CGMP), the estimated daily intake (EDI) of
aminopeptidase enzyme preparation, expressed as TOS, is 33 mg/person/d
at the 90th percentile level of consumption of these products. As
discussed above, aminopeptidase and other peptidases are naturally
present in cheese made by using S. lactis as a starter culture (Ref.
2). The EDI at the 90th percentile level of consumption of
aminopeptidase and other peptidases naturally present in cheese
prepared with L. lactis as the starter culture expressed as TOS is 77
mg/person/d, which exceeds the EDI calculated above for added
aminopeptidase enzyme preparation.
Moreover, the data obtained in the corroborative unpublished 13-
week rat feeding study showed no adverse effects at the highest dose of
2,000 mg aminopeptidase enzyme preparation/kg bw/d. Correction of this
value for TOS and application of a 1,000-fold safety factor produces,
for a 60 kg person, an acceptable daily intake (ADI) of 102 mg TOS of
aminopeptidase enzyme preparation/person/d, which exceeds the EDI
reported above (33 mg TOS/person/d).
V. Conclusion
FDA has evaluated the published information in the petition, along
with other corroborative information, and finds that the use of
aminopeptidase enzyme preparation from L. lactis in the manufacturing
of cheddar cheese and preparation of protein hydrolysates is GRAS.
Furthermore, these data show no potential risk from any foreseeable
use of the aminopeptidase enzyme preparation. Therefore, in accordance
with 21 CFR 184.1(b)(1), the agency is affirming that the use of
aminopeptidase enzyme preparation from L. lactis is GRAS with no limits
on its conditions of use other than CGMP.
VI. Environmental Impact
The agency has carefully considered the potential environmental
effects of this action. FDA has concluded that the action will not have
a significant impact on the human environment, and that an
environmental impact statement is not required. The agency's finding of
no significant impact and the evidence supporting that finding,
contained in an environmental assessment, may be seen in the Dockets
Management Branch (address above) between 9 a.m. and 4 p.m., Monday
through Friday.
VII. Analysis of Impacts
FDA has examined the economic implications of this final rule
affirming the GRAS status of the use of aminopeptidase enzyme
preparation from L. lactis in the manufacturing of cheddar cheese and
preparation of protein hydrolysates under Executive Order 12866 (Pub.
L. 96-354). Executive Order 12866 directs agencies to assess all costs
and benefits of available regulatory alternatives and, when regulation
is necessary, to select regulatory approaches that maximize net
benefits (including potential economic, environmental, public health,
and safety effects; distributive impacts; and equity). The agency
believes that this final rule is consistent with the regulatory
philosophy and principles identified in the Executive Order. In
addition, the final rule is not a significant regulatory action as
defined by the Executive Order and so is not subject to review under
the Executive Order.
The Regulatory Flexibility Act requires agencies to analyze
regulatory options that would minimize any significant impact of a rule
on small entities. Because no current activity is prohibited by this
final rule, the compliance cost to firms is zero. Since no increase in
the health risks faced by consumers will result from this final rule,
total costs are also zero. Potential benefits include the wider use of
this substance to achieve its intended technical effects, and any
resources saved by eliminating the need to prepare further petitions to
affirm the GRAS status of this substance. Affirming that the use of
aminopeptidase enzyme preparation from L. lactis in the manufacturing
of cheddar cheese and preparation of protein hydrolysates under
conditions of CGMP is GRAS will expand product formulation
possibilities for food manufacturers, including small entities.
Therefore, under the Regulatory Flexibility Act, FDA has also
determined that this rule will have a positive impact on small
entities.
VIII. Effective Date
As this rule recognizes an exemption from the food additive
definition in the Federal Food, Drug, and Cosmetic Act, and from the
approval requirements applicable to food additives, no delay in
effective date is required by the Administrative Procedure Act, 5
U.S.C. 553(d). The rule will therefore be effective immediately (5
U.S.C. 553(d)(1)).
IX. References
The following references have been placed on display in the Dockets
Management Branch (address above) and may be seen by interested persons
between 9 a.m. and 4 p.m., Monday through Friday.
1. ``Enzyme Nomenclature 1978,'' pp. 300-309, Academic Press,
NY, 1979.
2. Cliffe, A. J., and B. A. Law, ``An Electrophoretic Study of
Peptidases in Starter Streptococci and in Cheddar Cheese,'' Journal
of Applied Bacteriology, 47:65-73, 1979.
3. Perry, K. D., ``A Comparison of the Influence of
Streptococcus lactis and Str. cremoris Starters on the Flavour of
Cheddar Cheese,'' Journal of Dairy Research, 28:221-229, 1961.
4. Dawson, D. J., and J. T. Feagan, ``Bacteriology of Cheddar
Cheese,'' Journal of Dairy Research, 24:210-224, 1957.
5. Law, B. A., M. E. Sharpe, and B. Reiter, ``The Release of
Intracellular Dipeptidase from Starter Streptococci During Cheddar
Cheese Ripening,'' Journal of Dairy Research, 41:137-146, 1974.
6. Reiter, B., Y. Sorokin, A. Pickering, and A. J. Hall,
``Hydrolysis of Fat and Protein in Small Cheeses Made Under Aseptic
Conditions,'' Journal of Dairy Research, 36:65-76, 1969.
7. Parker, D. M., and D. Pawlett, ``Flavour Control of Protein
Hydrolysates,'' European Patent Office Publication No. 0 223 560,
Bulletin 87/22, May 5, 1987.
8. Law, B. A., and A. S. Wigmore, ``Accelerated Ripening of
Cheddar Cheese with a Commercial Proteinase and Intracellular
Enzymes from Starter Streptococci,'' Journal of Dairy Research,
50:519-525, 1983.
9. Schleifer, K. H., J. Kraus, C. Dvorak, R. Kilpper-Balz, M. D.
Collins, and W. Fischer, ``Transfer of Streptococcus Lactis and
Related Streptococci to the Genus Lactococcus gen. nov.,''
Systematic Applied Microbiology, 6:183-195, 1985.
10. Jay, J. M., ``Modern Food Microbiology,'' 2d ed., pp. 255
and 265-266, D. Van Nostrand, NY, 1978.
11. Potter, N. N., ``Food Science,'' 4th ed., pp. 374-376, Van
Nostrand Reinhold, NY, 1986.
12. Reiter, B., T. F. Fryer, A. Pickering, H. R. Chapman, R. C.
Lawrence, and M. E. Sharpe, ``The Effect of the Microbial Flora on
the Flavour and Free Fatty Acid Composition of Cheddar Cheese,''
Journal of Dairy Research, 34:257-272, 1967.
13. National Collection of Food Bacteria, Catalog of Strains, 3d
ed., Reading, United Kingdom, pp. 116-126, 1986.
14. American Type Culture Collection, Catalogue of Bacteria and
Phages, 8th ed., Rockville, MD, pp. 176-177, 1992.
15. Frazier, W. C., ``Food Microbiology,'' pp. 49 and 215,
McGraw-Hill, NY, 1958.
16. Food Drug Cosmetic Law Journal, pp. 834-840, December, 1958.
17. ``Bergey's Manual of Systematic Bacteriology,'' vol. 2,
edited by P. H. A. Sneath, pp. 1002 and 1065-1066, Williams and
Wilkins, Baltimore, 1986.
List of Subjects in 21 CFR Part 184
Food ingredients, Incorporation by reference.
Therefore, under the Federal Food, Drug, and Cosmetic Act and under
[[Page 54193]]
authority delegated to the Commissioner of Food and Drug and
redelegated to the Director, Center for Food Safety and Applied
Nutrition, 21 CFR part 184 is amended as follows:
PART 184--DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED
AS SAFE
1. The authority citation for 21 CFR part 184 continues to read as
follows:
Authority: Secs. 201, 402, 409, 701 of the Federal Food, Drug,
and Cosmetic Act (21 U.S.C. 321, 342, 348, 371).
2. New Sec. 184.1985 is added to read as follows:
Sec. 184.1985 Aminopeptidase enzyme preparation derived from
lactococcus lactis.
(a) Aminopeptidase enzyme preparation is derived from the
nonpathogenic and nontoxicogenic bacterium Lactococcus lactis
(previously named Streptococcus lactis). The preparation contains the
enzyme aminopeptidase (CAS Reg. No. 9031-94-1; EC 3.4.11.1) and other
peptidases that hydrolyze milk proteins. The preparation is produced by
pure culture fermentation.
(b) The ingredient meets the specifications for enzyme preparations
in the Food Chemicals Codex, 3d ed. (1981), pp. 107-110, which are
incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR
part 51. Copies are available from the National Academy Press, 2101
Constitution Ave. NW., Washington, DC 20418, or may be examined at the
Division of Petition Control (HFS-215), Center for Food Safety and
Applied Nutrition, Food and Drug Administration, 1110 Vermont Ave. NW.,
suite 1200, Washington, DC, or at the Office of the Federal Register,
800 North Capitol St. NW., suite 700, Washington, DC.
(c) In accordance with Sec. 184.1(b)(1), the ingredient is used in
food with no limitations other than current good manufacturing
practice. The affirmation of this ingredient as generally recognized as
safe as a direct human food ingredient is based upon the following
current good manufacturing practice conditions of use:
(1) The ingredient is used as an enzyme, as defined in
Sec. 170.3(o)(9) of this chapter, as an optional ingredient for flavor
development in the manufacture of cheddar cheese, in accordance with
Sec. 133.113 of this chapter, and in the preparation of protein
hydrolysates.
(2) The ingredient is used at levels not to exceed current good
manufacturing practice.
Dated: September 29, 1995.
Fred R. Shank,
Director, Center for Food Safety and Applied Nutrition.
[FR Doc. 95-26054 Filed 10-19-95; 8:45 am]
BILLING CODE 4160-01-F
