[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