[Federal Register Volume 59, Number 232 (Monday, December 5, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-29731]
[[Page Unknown]]
[Federal Register: December 5, 1994]
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DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration
21 CFR Part 184
[Docket No. 83G-0277]
]-Amylase Enzyme Preparation; Affirmation of GRAS Status as
Direct Human Food Ingredient
AGENCY: Food and Drug Administration, HHS.
ACTION: Tentative final rule.
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SUMMARY: The Food and Drug Administration (FDA) is tentatively
affirming that -amylase enzyme preparation derived from
Bacillus stearothermophilus is generally recognized as safe (GRAS) for
use in the processing of starch to make maltodextrins and nutritive
carbohydrate sweeteners.
DATES: Written comments by February 3, 1995.
ADDRESSES: Submit written comments to the Dockets Management Branch
(HFA-305), Food and Drug Administration, rm. 1-23, 12420 Parklawn Dr.,
Rockville, MD 20857.
FOR FURTHER INFORMATION CONTACT: Vincent E. Zenger, Center for Food
Safety and Applied Nutrition (HFS-206), Food and Drug Administration,
200 C St. SW., Washington, DC 20204, 202-418-3105.
SUPPLEMENTARY INFORMATION:
I. Background
In accordance with the procedures described in Sec. 170.35 (21 CFR
170.35), CPC International Inc., International Plaza, Englewood Cliffs,
NJ 07632, submitted a petition (GRASP 3G0284) requesting that
-amylase enzyme from B. stearothermophilus used in the
production of nutritive saccharides from starch be affirmed as GRAS as
a direct human food ingredient. The petition includes information about
the identity of, and manufacturing processes for, -amylase
enzyme preparations derived from B. stearothermophilus; information
about the history of human food use of -amylase derived from
B. stearothermophilus; final reports and published articles of safety
studies with -amylase enzyme preparation derived from B.
stearothermophilus; and published literature with respect to -
amylase and bacterial -amylase preparations.FDA published a
notice of the filing of this petition in the Federal Register of
September 21, 1983 (48 FR 43096). FDA gave interested persons an
opportunity to submit comments to the Dockets Management Branch
(address above). FDA did not receive any comments in response to that
notice.
In the filing notice the agency gave notice that the petition had
requested that -amylase enzyme derived from B.
stearothermophilus be affirmed as GRAS for use in production of
sweeteners from starch. However, the petition requested, and the agency
evaluated, the use of this enzyme preparation in the production of
nutritive saccharides (which includes maltodextrins as well as
nutritive carbohydrate sweeteners). The end products of the -
amylase hydrolysis of starch are maltodextrins, which are not sweet and
are not used as sweeteners in food, as well as nutritive carbohydrate
sweeteners. Maltodextrins may be used as a food ingredient or used as a
raw material in the manufacture of nutritive carbohydrate sweeteners,
for example, glucose syrups. Therefore, FDA finds that the phrase
``production of maltodextrins and nutritive carbohydrate sweeteners
from starch'' is a more accurate description of the petitioned food use
of -amylase enzyme preparation. FDA is publishing this
document as a tentative final rule to afford interested persons the
opportunity to comment on this change. To avoid confusion between
-amylase, the enzyme, and -amylase, the enzyme
preparation (in which -amylase is the principal active
component, but which also contains other components derived from the
production organism or the fermentation media), this document will use
the term ``-amylase'' to refer to the former and ``-
amylase enzyme preparation'' to refer to the latter.
II. Standards for GRAS Affirmation
Pursuant to 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. 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. 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)). General
recognition of safety through experience based on common use in food
prior to January 1, 1958, may be determined without the quantity or
quality of scientific procedures required for approval of a food
additive regulation but ordinarily is to be based upon generally
available data and information concerning its pre-1958 use
(Sec. 170.30(c)).
III. Safety Evaluation
A. IntroductionStarch produced in plants exists in two main forms. The
linear form is composed of -D-glucose sugar residues bonded
together with a type of linkage termed -1,4 (Ref. 1). This
linear form is commonly termed amylose. The other form of starch,
termed amylopectin, is composed of amylose molecules linked together at
branch points. In this form, resembling a tree-like structure, the
branch points are formed by a different kind of linkage termed
-1,6. An -amylase enzyme (1,4--D glucan
glucanohydrolase (International Union of Biochemistry Enzyme Commission
(E.C.) 3.2.1.1)) can hydrolyze, i.e., break, the -1,4 linkages
found in amylose and amylopectin (Ref. 2). Treatment with -
amylase enzyme lowers the molecular weight of the starch molecules to
form molecules collectively called maltodextrins.
Certain maltodextrins may be subjected to subsequent processing.
For instance, corn maltodextrins may be further hydrolyzed by another
enzyme, glucoamylase, to produce glucose (also known as dextrose) which
may in turn be isomerized to form high fructose corn syrups. These corn
sweeteners are refined with ion exchange resins to remove impurities
and are then concentrated. The processed corn sweeteners are then used
in a wide variety of products in the food industry. Current technology
sometimes requires the -amylase enzyme to function at high
temperatures, up to 110 deg. C (Refs. 1 and 3). Therefore, much effort
has gone into research on -amylases from thermophilic
microorganisms such as B. stearothermophilus (Ref. 3).
In evaluating this petition to affirm as GRAS the use of -
amylase enzyme preparation from B. stearothermophilus as a food
ingredient, the agency considered six aspects of its manufacture and
use: (1) The identity of the -amylase enzyme component; (2)
the identity and safety of the source (production) organism for the
-amylase enzyme preparation; (3) the manufacturing process of
the -amylase enzyme preparation; (4) the intended uses for the
-amylase enzyme preparation in food and exposure to residual
levels of the -amylase enzyme preparation; (5) the
specifications for the formulation of the enzyme preparation; and (6)
toxicological studies of the enzyme preparation.
B. The Enzyme Component
The -amylase enzyme from B. stearothermophilus is
extracellular (Ref. 2). That is, the enzyme is secreted by the bacteria
into the surrounding media. Data and published information in the
petition confirm that the petitioner's enzyme preparation from B.
stearothermophilus functions in the hydrolysis of starch as an
-amylase (1,4--D glucan glucanohydrolase (E.C.
3.2.1.1)) (Refs. 4 through 7).
Published data show that the -amylase enzyme functions at
an optimum temperature of 80 deg. C and at pH values below 6 (Ref. 7),
which is consistent with previous published reports characterizing
-amylase from B. stearothermophilus and other thermophilic
Bacillus species (Refs. 2 and 3).
The published data further show that the petitioner's enzyme has a
molecular weight of 58 kilodaltons (kd) (Ref. 7) which is consistent
with the 58 kd mass reported by Sen (Ref. 8) and within experimental
error of the predicted 61 kd mass based on deoxyribonucleic acid (DNA)
sequence analysis (Ref. 9).
The -amylases are functionally divided into two
categories, saccharifying -amylases, which break approximately
40 to 60 percent of the -1,4 linkages in a starch, and
liquefying -amylases, which break only 30 to 40 percent of the
linkages in the starch (Ref. 3). The -amylase from B.
stearothermophilus is of the liquefying type and is very similar in
protein sequence to liquefying -amylases from other Bacillus
species that have been commonly used in food processing (Refs. 1, 3,
and 9 through 12), for example, Bacillus amyloliquefaciens (Ref. 1) and
Bacillus licheniformis (see 21 CFR 184.1027).
C. The Production Organism
The source organism for this enzyme preparation is the bacterium B.
stearothermophilus. The petition includes data to show that the strain
used by the petitioner, B. stearothermophilus (AS-154), conforms to the
description of B. stearothermophilus in ``Bergey's Manual of
Determinative Bacteriology,'' 8th ed. (Ref. 13), which is a standard
compendium for the taxonomy of bacteria. The petition also contains
data to show that this strain of B. stearothermophilus is an
asporogenic variant and does not produce antibiotics or toxins.
Published scientific literature as well as standard textbooks on
food microbiology demonstrate that B. stearothermophilus and its spores
are widely distributed in nature and they are commonly found in fresh
foods (Refs. 13 and 14). B. stearothermophilus is also reported to be
the typical organism causing nontoxic sour spoilage in low acid foods
(Ref. 14).
The petition contains one published pathogenicity study that
demonstrated that B. stearothermophilus is not pathogenic (Ref. 15).
The petition also contains an extensive search of the published
literature from 1917 to 1992 involving over 1,700 references and
citations relating to B. stearothermophilus concerning pathogenicity,
pathogen formation, toxicology and toxins, and disease or infection.
The search failed to disclose a single report that implicated B.
stearothermophilus as the etiologic agent of a disease state in man or
animals. There were no reports of any toxicity or pathogenicity
associated with the presence of this organism in food.
D. The Manufacturing Process
The -amylase enzymes of Bacillus are extracellular enzymes
(Ref. 10). Therefore, the manufacturing procedures follow those
generally used in the enzyme industry to separate and concentrate
extracellular enzymes (Ref. 16). Under the method of manufacture of
-amylase enzyme preparation described in the petition, B.
stearothermophilus is maintained as a pure culture under conditions
that minimize any genetic changes and is grown in a pure culture
fermentation. When fermentation is complete, the broth is clarified by
treating it with calcium hydroxide, and cells are removed from the
broth by filtration using a diatomaceous earth filter aid (Ref. 17).
The filtered, clarified broth containing the soluble enzyme is then
ultrafiltered to remove all particulate matter. The filtrate,
containing the -amylase enzyme, is then evaporated to a
concentrate of the desired enzyme potency, usually about a three-fold
concentration. Sodium chloride is added to the concentrate so that the
final salt concentration is 20 percent by weight of the enzyme
preparation. Data submitted in the petition show that the enzyme
preparation produced by this method of manufacture does not contain any
viable bacterial cells.
FDA finds that the manufacturing method does not require the use of
any processing materials that are not GRAS or approved food additives.
Therefore, the agency concludes that the manufacturing steps will not
introduce impurities into the enzyme preparation that will adversely
affect the safety of the preparation.
E. Estimated Exposure Levels
The amount of the enzyme preparation used will vary based on the
catalytic activity of the enzyme in any particular batch of enzyme
preparation. Estimates of enzyme use level and intake are usually based
on the total organic solids (TOS) content of the enzyme preparation
(Ref. 18). TOS is the sum of all organic compounds present in the final
enzyme preparation, excluding diluents or carriers, if added. TOS is
calculated as follows: TOS percent=100-(A+W+D) where A is the percent
of ash, W is the percent of water, and D is the percent of diluents or
carriers.
FDA's estimate of exposure to -amylase enzyme preparation
from B. stearothermophilus is based on the food use of maltodextrins
and nutritive carbohydrate sweeteners, data for general usage of
-amylase preparations, and the relative enzymatic potency of
this particular enzyme preparation compared to typical preparations.
FDA calculates that the intake of typical -amylase enzyme
preparations reported as TOS is 25 milligrams (mg) TOS per person per
day (TOS/person/day). The subject preparation has an enzymatic potency
about six-fold higher than typical preparations; therefore, the
estimated daily intake (EDI) is one-sixth of 25 mg or about 4 mg/TOS/
person/day, or 67 micrograms (g)/kilograms (kg) body weight/
day for a 60 kg person.
F. Enzyme Preparation Specifications
The petition contains data showing that the -amylase
enzyme preparation from B. stearothermophilus produced in this manner
meets the general and additional requirements for enzyme preparations
in the ``Food Chemicals Codex,'' 3d ed. (Ref. 19).
G. Safety of Enzyme Preparation
The petition contains published animal feeding studies to support
the safety of the enzyme preparation. These include a 90-day subchronic
oral toxicity study in dogs and a 90-day subchronic oral toxicity study
in F1 rats exposed in utero. No adverse treatment-related effects were
identified in the 90-day studies (Ref. 20).
The petition also contained several unpublished, corroborative
safety studies. These animal feeding studies of the -amylase
enzyme preparation included an acute oral toxicity study in rats and
14-day palatability studies in both rats and dogs. None of these
studies demonstrated any adverse treatment-related effects.
Based upon the 90-day dog study, FDA estimated an acceptable daily
intake (ADI) of 377 g/kg body weight, which is 1/1000 of the
highest no-effect level (377 mg/kg body weight, which was the highest
dose tested). These studies show that the ADI for the enzyme
preparation (377 g/kg body weight/day) exceeds the EDI for
uses of this enzyme preparation (67 g/kg body weight/day).
IV. Conclusions
The petition requested affirmation of GRAS status of -
amylase preparation from B. stearothermophilus based on its similarity
to other -amylase enzyme preparations that have a history of
common use in food prior to 1958. The petition cites data that report
that -amylase enzyme preparation from B. subtilis has been
used commercially since 1929, when it was used in the manufacture of
chocolate syrup to reduce its viscosity (Ref. 21). The petition stated
that bacterial -amylase enzyme preparations were first
described in the preparation of corn sweeteners in 1962, but that
common use of these enzymes by major food processors did not occur
until some time later. The petition also stated that today, corn
sweeteners prepared with bacterial amylase enzyme preparations are used
in nearly all commercially prepared foods.
The agency evaluated the petition using the criteria of
Sec. 170.30(c) and concluded that although -amylase enzyme
preparations have had a long history of use before 1958, the data
provided no evidence for history of use of -amylase enzyme
preparation from B. stearothermophilus, and that based on the data in
the petition, this preparation is not eligible for GRAS affirmation
based on history of common use in food. However, the agency has also
evaluated the petition using the criteria of Sec. 170.30(b) and
concludes that -amylase enzyme preparation from B.
stearothermophilus is eligible for GRAS affirmation based on scientific
procedures.
The agency has evaluated the information in the petition along with
other available information and concludes, based on evaluation of
published information, corroborated by unpublished data and
information, that use of the -amylase enzyme preparation
derived by fermentation from B. stearothermophilus to hydrolyze starch
to produce maltodextrins and nutritive carbohydrate sweeteners is GRAS.
Furthermore, the data show no basis for a potential risk from any use
of this -amylase preparation that can be anticipated.
Therefore, the agency is tentatively affirming that the use of the
enzyme is GRAS with no limits other than current good manufacturing
conditions in accordance with 21 CFR 184.1(b)(1).
The agency further finds that because the principal active
ingredient of the -amylase enzyme preparation is safe and
because expected impurities in the -amylase enzyme preparation
do not provide any basis for a safety concern that the general and
additional requirements given for enzyme preparations in the ``Food
Chemicals Codex,'' 3d ed. (1981), pp. 107-110, are adequate for
defining minimum criteria for a food-grade -amylase enzyme
preparation derived from B. stearothermophilus.
V. Environmental Effects
The agency has determined under 21 CFR 25.24(b)(7) that this action
is of a type that does not individually or cumulatively have a
significant effect on the human environment. Therefore, neither an
environmental assessment nor an environmental impact statement is
required.
VI. Analysis of Impacts
FDA has examined the impacts of the tentative final rule under
Executive Order 12866, and the Regulatory Flexibility Act (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,
and other advantages; distributive impacts; and equity). The agency
believes that this tentative final rule is consistent with the
regulatory philosophy and principles identified in the Executive Order.
In addition, the tentative 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
tentative final rule, the compliance cost to firms is zero. Because no
increase in the health risks faced by consumers will result from this
tentative final rule, total costs are also zero. Potential benefits
include the wider use of this enzyme because of reduced uncertainty
concerning its GRAS status, and any resources saved by eliminating the
need to prepare further petitions to affirm the GRAS status of this
enzyme for this use. The agency certifies, therefore, that the
tentative final rule will not have a significant economic impact on a
substantial number of small entities. Therefore, under the Regulatory
Flexibility Act, no further analysis is required.
VII. 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. MacAllister, R. V., ``Manufacture of High Fructose Corn Syrup
Using Immobilized Glucose Isomerase,'' in ``Immobilized Enzymes for
Food Processing,'' W. H. Pitcher, Jr., editor, CRC Press, Inc., Boca
Raton, FL, pp. 81-111, 1980.
2. Vihinen, M. and P. Mantsala, ``Microbial Amylolytic
Enzymes,'' CRC Critical Reviews in Biochemistry and Molecular
Biology, 24:329-418, 1989.
3. Tomazic, S. J. and A. M. Klibanov, ``Mechanisms of
Irreversible Thermal Inactivation of Bacillus -Amylases,''
Journal of Biological Chemistry, 263:3086-3091, 1988.
4. Tamuri, M. et al., ``Heat and Acid Stable -Amylase
Enzymes and Processes for Producing the Same,'' U.S. Patent No.
4,284,722, 1981.
5. Brumm, P. J. and W. M. Teague, ``Effect of Additives on the
Thermostability of Bacillus stearothermophilus -Amylase,''
Biotechnology Letters, 11:541-544, 1989.
6. Henderson, W. E. and W. M. Teague, ``A Kinetic Model of
Bacillus stearothermophilus -Amylase under Process
Conditions,'' Starch/Starke, 40:412-418, 1988.
7. Brumm, P. J. et al., ``Purification and Properties of a New
Commercial, Thermostable Bacillus stearothermophilus -
Amylase,'' Food Biotechnology, 2:67-80, 1988.
8. Sen, S. and P. Oriel, ``Multiple Amylase Genes in Two Strains
of Bacillus stearothermophilus,'' Gene, 76:137-144, 1989.
9. Ihara, H. et al., ``Complete Nucleotide Sequence of a
Thermophilic -Amylase Gene: Homology between Prokaryotic
and Eukaryotic -Amylases at the Active Sites,'' Journal of
Biochemistry, 98:95-103, 1985.
10. Suominen, I. et al., ``Extracellular Production of Cloned
-Amylase by Escherichia coli,'' Gene, 61:165-176, 1987.
11. Tsukamoto, A. et al., ``Nucleotide Sequence of the
Maltohexaose-Producing Amylase Gene from an Alkalophilic Bacillus
sp. No. 707 and Structural Similarity to Liquefying Type -
Amylases,'' Biochemical and Biophysical Research Communications,
151:25-31, 1988.
12. Satoh, H. et al., ``Evidence for Movement of the -
Amylase Gene into Two Phylogenetically Distant Bacillus
stearothermophilus Strains,'' Journal of Bacteriology, 170:1034-
1040, 1988.
13. ``Bergey's Manual of Determinative Bacteriology,'' 8th ed.,
Williams and Wilkins Co., Baltimore, p. 1135, 1975.
14. Ito, K. A., ``Thermophilic Organisms in Food Spoilage: Flat-
Sour Aerobes,'' Journal of Food Protection, 44:157-163, 1981.
15. Sattar, S. A. et al., ``Hazard Inherent in Microbial
Tracers: Reduction of Risk by the Use of Bacillus stearothermophilus
Spores in Aerobiology,'' Applied Microbiology, 23:1053-1059, 1972.
16. Frost, G. M. and D. A. Moss, ``Production of Enzymes by
Fermentation,'' in ``Biotechnology, Vol. 7A, Enzyme Technology,'' H.
J. Rehm and G. Reed, editors, J. F. Kennedy, Vol. editor, VCH, New
York, pp.72-76, 1987.
17. Brummer, W. and G. Gunzer, ``Laboratory Techniques of Enzyme
Recovery,'' in ``Biotechnology, Vol. 7A, Enzyme Technology,'' H. J.
Rehm and G. Reed, editors, J. F. Kennedy, Vol. editor, VCH, New
York, pp. 217-219 and 273, 1987.
18.``The 1978 Enzyme Survey Summarized Data,'' National Research
Council/National Academy of Sciences, Washington, DC; U.S.
Department of Commerce, National Technical Information Service PB81-
216897, 1981, pp. i-iii.
19. Monograph on Enzyme Preparations, in ``Food Chemicals
Codex,'' 3d ed., National Academy Press, Washington, DC, pp. 107-
110, 1981.
20. MacKenzie, K. M. and S. R. W. Petsel, ``Subchronic Toxicity
Studies in Dogs and In Utero Rats Fed Diets Containing Bacillus
stearothermophilus -Amylase from a Natural or Recombinant
DNA Host,'' Food and Chemical Toxicology, 27:599-606, 1989.
21. Reed, T., ``Enzymes in Food Processing,'' Academic Press,
New York, p. 406, 1966.
VIII. Comments
FDA is publishing this document as a tentative final rule to afford
interested persons the opportunity to comment on the use of the enzyme
preparations in the production of maltodextrins, which was not
discussed in the filing notice.
Interested persons may, on or before February 3, 1995, submit to
the Dockets Management Branch (address above) written comments
regarding this tentative final rule. Two copies of any comments are to
be submitted, except that individuals may submit one copy. Comments are
to be identified with the docket number found in brackets in the
heading of this document. Received comments may be seen in the office
above between 9 a.m. and 4 p.m., Monday through Friday.
List of Subjects in 21 CFR Part 184
Food ingredients, Incorporation by reference.
Therefore, under the Federal Food, Drug, and Cosmetic Act and under
authority delegated to the Commissioner of Food and Drugs and
redelegated to the Director, Center for Food Safety and Applied
Nutrition, it is proposed that 21 CFR part 184 be amended as follows:
PART 184--DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED
AS SAFE1.
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. 184.1012 is added to subpart B to read as follows:
Sec. 184.1012 -Amylase enzyme preparation from Bacillus
stearothermophilus.
(a) -Amylase enzyme preparation is obtained from the
culture filtrate that results from a pure culture fermentation of a
nonpathogenic and nontoxicogenic strain of Bacillus stearothermophilus.
Its characterizing enzyme activity is -amylase (1,4--
D glucan glucanohydrolase (E. C. 3.2.1.1)).
(b) The ingredient meets the general and additional requirements
for enzyme preparations in the ``Food Chemicals Codex,'' 3d ed. (1981),
pp. 107-110, which is incorporated by reference in accordance with 5
U.S.C. 552(a). Copies are available from the National Academy Press,
2101 Constitution Ave. NW., Washington, DC 20418, or available for
inspection 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 limitation other than current good manufacturing
practices. The affirmation of this ingredient as GRAS 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, in the hydrolysis of edible starch to
produce maltodextrins and nutritive carbohydrate sweeteners.
(2) The ingredient is used at levels not to exceed current good
manufacturing practices.
Dated: November 22, 1994.
Fred R. Shank,
Director, Center for Food Safety and Applied Nutrition.
[FR Doc. 94-29731 Filed 12-2-94; 8:45 am]
BILLING CODE 4160-01-F
