Carbohydrase and Protease Enzyme Preparations Derived From Bacillus Subtilis or Bacillus Amyloliquefaciens; Affirmation of GRAS Status as Direct Food Ingredients

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Part 184

[Docket No. 84G-0257]

Carbohydrase and Protease Enzyme Preparations Derived From

Bacillus Subtilis or Bacillus Amyloliquefaciens; Affirmation of GRAS

Status as Direct Food Ingredients

AGENCY: Food and Drug Administration, HHS.

ACTION: Final rule.

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SUMMARY: The Food and Drug Administration (FDA) is affirming that

carbohydrase enzyme preparations derived from either Bacillus subtilis

or B. amyloliquefaciens and protease enzyme preparations derived from

either B. subtilis or B. amyloliquefaciens are generally recognized as

safe (GRAS) for use as direct food ingredients. This action is a

partial response to a petition filed by the Ad Hoc Enzyme Technical

Committee (now the Enzyme Technical Association).

DATES: The regulation is effective April 23, 1999. 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 certain

publications listed in 21 CFR 184.1148 and 184.1150, effective April

23, 1999.

FOR FURTHER INFORMATION CONTACT: Linda S. Kahl, Center for Food Safety

and Applied Nutrition (HFS-206), Food and Drug Administration, 200 C

St. SW., Washington, DC 20204, 202-418-3101.

SUPPLEMENTARY INFORMATION:

Table of Contents

I. Introduction

II. Standards for GRAS Affirmation

III. Background

A. Identity and Technical Effect

B. Methods of Manufacture

IV. Safety Evaluation

A. Pre-1958 History of Use in Food

1. Bacillus Subtilis

2. Bacillus Amyloliquefaciens

B. Corroborating Evidence of Safety

1. The Enzyme Components

2. Enzyme Sources, Manufacturing Methods, and Processing Aids

V. Comments

VI. Conclusions

VII. Environmental Considerations

VIII. Analysis for Executive Order 12866

IX. Regulatory Flexibility Analysis

X. Paperwork Reduction Act of 1995

XI. Effective Date

XII. References

I. Introduction

In accordance with the procedures described in Sec. 170.35 (21 CFR

170.35), the Ad Hoc Enzyme Technical Committee (now the Enzyme

Technical Association), c/o Miles Laboratories, Inc., 1127 Myrtle St.,

Elkhart, IN 46514, submitted a petition (GRASP 3G0016) requesting that

the following enzyme preparations be affirmed as GRAS for use in food:

(1) Animal-derived enzyme preparations: Catalase (bovine liver);

lipase, animal; pepsin; rennet; rennet, bovine; and trypsin; (2) plant-

derived enzyme preparations: Bromelain; malt; and papain; (3)

microbially-derived enzyme preparations: Lipase, catalase, glucose

oxidase, and carbohydrase from Aspergillus niger, var.; mixed

carbohydrase and protease from Bacillus subtilis, var.; carbohydrase

from Rhizopus oryzae; and carbohydrase from Saccharomyces species.

FDA published a notice of filing of this petition in the Federal

Register of April 12, 1973 (38 FR 9256), and gave interested persons an

opportunity to submit comments to the Dockets Management Branch (HFA-

305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061,

Rockville, MD 20852. The petition was amended by notices published in

the Federal Register of June 12, 1973 (38 FR 15471), proposing

affirmation that microbially-derived enzyme preparations (carbohydrase,

lipase, and protease) from A. oryzae are GRAS for use in food; in the

Federal Register of August 29, 1984 (49 FR 34305), proposing

affirmation that the enzyme preparations ficin, obtained from species

of the genus Ficus (fig tree), and pancreatin, obtained from bovine and

porcine pancreas, are GRAS for use in food; in the Federal Register of

June 23, 1987 (52 FR 23607), proposing affirmation that the protease

enzyme preparation from A. niger is GRAS for use in food; and in the

Federal Register of August 5, 1996 (61 FR 40648), proposing affirmation

that carbohydrase and protease enzyme preparations from B.

amyloliquefaciens are GRAS for use in food. In the June 23, 1987,

notice, FDA also noted the petitioner's assertion that pectinase enzyme

preparation from A. niger and lactase enzyme preparation from A. niger

are included under carbohydrase enzyme preparation from A. niger, and

that invertase enzyme preparation from Saccharomyces cerevisiae and

lactase enzyme preparation from Kluyveromyces marxianus are both

included under carbohydrase enzyme preparation from species of the

genus Saccharomyces. The agency further noted that, therefore,

pectinase enzyme preparation from A. niger, lactase enzyme preparation

from A. niger, invertase enzyme preparation from S. cerevisiae, and

lactase enzyme preparation from K. marxianus were to be considered part

of the petition. Interested persons were given an opportunity to submit

comments to the Dockets Management Branch (address above) on each

amendment.

After the petition was filed, the agency published, as part of its

comprehensive safety review of GRAS substances, two GRAS affirmation

regulations that covered three of the enzyme preparations from animal

and plant sources included in the petition. These two regulations are:

(1) Sec. 184.1685 Rennet (animal derived) (21 CFR 184.1685), which was

published in the Federal Register of November 7, 1983 (48 FR 51151) and

includes the petitioned enzyme preparations rennet and bovine rennet;

and (2) Sec. 184.1585 Papain (21 CFR 184.1585), which was published in

the Federal Register of October 21, 1983 (48 FR 48805). Thus,

[[Page 19888]]

rennet, bovine rennet, and papain are already affirmed as GRAS and need

not be addressed further.

In a notice published in the Federal Register of September 20, 1993

(58 FR 48889), the agency announced that the petitioner had requested

that the following enzyme preparations be withdrawn from the petition

without prejudice to the filing of a future petition: (1) Pancreatin

used for its lipase activity, (2) pancreatin used for its amylase

activity, and (3) amylase derived from unmalted barley extract. In that

notice, the agency stated that, in light of the petitioner's request,

any future action by FDA on the petition would not include a

determination of the GRAS status of these three enzyme preparations.

In a final rule published in the Federal Register of June 26, 1995

(60 FR 32904), the agency affirmed as GRAS the following enzyme

preparations derived from animal sources: Catalase (bovine liver),

animal lipase, pepsin, trypsin, and pancreatin (as a source of protease

activity). In that same final rule, the agency also affirmed as GRAS

the following enzyme preparations derived from plant sources:

Bromelain, ficin, and malt.

This final rule addresses the following bacterially-derived enzyme

preparations: (1) carbohydrase enzyme preparation from B. subtilis; (2)

protease enzyme preparation from B. subtilis; (3) carbohydrase enzyme

preparation from B. amyloliquefaciens; and (4) protease enzyme

preparation from B. amyloliquefaciens. \1\ The other microbial enzyme

preparations in the petition will be dealt with separately in a future

issue of the Federal Register.

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\1\ Although the petition requested GRAS affirmation for mixed

carbohydrase and protease enzyme preparation from B. subtilis, the

petitioner subsequently agreed that this enzyme preparation be

evaluated as two separate enzyme preparations, carbohydrase enzyme

preparation from B. subtilis and protease enzyme preparation from B.

subtilis. Enzyme preparations that contain mixtures of carbohydrases

and proteases can be used either for their carbohydrase activity or

for their protease activity, and they are usually sold according to

their intended use. FDA requested the petitioner's agreement to this

change to reflect the distinct uses of mixed carbohydrase and

protease enzyme preparations in food depending on whether a

particular preparation is being used for its carbohydrase activity

or for its protease activity.

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II. Standards for GRAS Affirmation

Under Sec. 170.30 (21 CFR 170.30) and 21 U.S.C. 321(s), 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 directly or indirectly added to food. The basis of such

views may be either scientific procedures or, 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 and

ordinarily is 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, and ordinarily is based upon generally available data and

information concerning the pre-1958 use of the substance

(Sec. 170.30(c)).

For the enzyme preparations from B. subtilis and B.

amyloliquefaciens that are the subject of this document, the Enzyme

Technical Association bases its request for affirmation of GRAS status

on a history of safe food use prior to 1958. As discussed in the

preamble to the proposed rule for the most recent amendment to

Sec. 170.30, general recognition of safety through experience based on

common use in food requires a consensus on the safety of the substance

among the community of experts who are qualified to evaluate the safety

of food ingredients (50 FR 27294 at 27295, July 2, 1985).

III. Background

A. Identity and Technical Effect

Enzymes are proteins that originate from living cells and produce

chemical change by catalytic action (Random House Dictionary of the

English Language, 1987). Most enzymes are very specific in their

ability to catalyze only certain chemical reactions; this high degree

of specificity and strong catalytic activity are the most important

functional properties of enzymes (Ref. 1).

Commercial enzyme preparations such as those that are the subject

of this document usually contain several enzymes that have catalytic

activities other than those for which they are sold--i.e., other than

their characterizing enzyme activities. As discussed in more detail in

section III.B of this document, the methods of manufacture for a

specific commercial enzyme preparation are tailored to maximize the

characterizing enzyme activity. The other enzymes that are present in

the preparation generally are present at low levels.

Carbohydrases, which are also known as glycosidases, are enzymes

whose catalytic activity is the hydrolysis (i.e., splitting) of O-

glycosyl bonds in carbohydrates. The carbohydrase enzyme preparations

that are the subject of this document each contain two or more

carbohydrases, including: (1) -amylase, which hydrolyzes

-1,4-glucan bonds in polysaccharides (e.g., starch) yielding

monosaccharides, linear oligosaccharides and branched oligosaccharides

(dextrins), and (2) -glucanase, which hydrolyzes 1,3 and some

1,4 linkages in -D-glucans (polysaccharides that are common in

cereals such as oats, barley, and rye), yielding oligosaccharides and

glucose (Refs. 2 and 3). Because the major carbohydrase in the

carbohydrase enzyme preparations derived from B. subtilis or B.

amyloliquefaciens is -amylase, the primary use of these enzyme

preparations is the hydrolysis of starch in processes such as the

preparation of starch syrups and the fermentation of beer (Refs. 3

through 5).

Proteases are enzymes whose catalytic activity is the hydrolysis of

peptide bonds in proteins, yielding peptides and amino acids. The

protease enzyme preparations that are the subject of this document each

contain two or more proteases, including subtilisin and neutral

proteinase (Refs. 2 and 3). The primary use of the protease enzyme

preparations derived from B. subtilis or B. amyloliquefaciens is in the

preparation of protein hydrolysates and the tenderizing of meat (Refs.

3 through 5).

Table 1 lists the characterizing enzyme activities and associated

International Union of Biochemistry Enzyme Commission (EC) numbers of

the carbohydrase and protease enzyme preparations derived from B.

subtilis or B. amyloliquefaciens.

#Table 1.--Enzyme Activities and EC Numbers Associated With Enzyme

Preparations Derived From B. Subtilis or B. Amyloliquefaciens

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Characterizing Enzyme

Enzyme Preparation Activity EC Number

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Carbohydrase -Amylase 3.2.1.1

-Glucanase 3.2.1.6

Protease Subtilisin 3.4.21.62

Neutral Proteinase 3.4.24.28

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B. Methods of Manufacture

All microbial strains, including bacterial strains, used to

manufacture enzyme preparations are started from a

[[Page 19889]]

pure laboratory culture and grown, or ``fermented,'' in a sterile

liquid nutrient medium or sterile moistened semisolid medium. Accepted

microbiological techniques are used to exclude contaminating organisms

and to avoid development of substrains from within the culture itself

(Ref. 6). Although specific conditions of fermentation vary from

manufacturer to manufacturer, common fermentation procedures are: (1)

The submerged culture method, which uses closed fermenters equipped

with agitators, aeration devices, and jackets or coils for temperature

control; and (2) the semisolid culture method, which uses horizontal

rotating drums or large chambers fitted with trays (Refs. 5 and 6).

During fermentation by either method, the pH, temperature, appearance

or disappearance of certain ingredients, purity of culture, and level

of enzyme activity must be carefully controlled. The fermentation is

harvested at the point where laboratory tests indicate that maximum

production of enzyme activity has been attained.

In practice, the processes by which microbial-derived enzyme

preparations are produced vary widely. Each single strain of

microorganism produces a large number of enzymes (Ref. 5). The absolute

and relative amounts of various individual enzymes produced vary

markedly among species and even among strains of the same species. They

also vary depending upon the composition of medium on which the

microorganism grows, and upon the fermentation conditions. The

petitioner states that for a specific enzyme preparation the production

strain, medium composition, and fermentation conditions are optimized

to maximize the desired enzyme activity (Refs. 7 and 8).

The carbohydrase and protease enzymes from B. subtilis and B.

amyloliquefaciens are excreted into the fermentation medium (Refs. 9

through 11). In the semisolid culture method, an enzyme that is present

in the fermentation medium is extracted either directly from the moist

material, or later after the culture mass has been dried. In the

submerged culture method, the microorganisms and other insolubles are

removed from the fermentation medium by decanting, filtering, or

centrifuging, and therefore an extraction step is not required. In

either method, further processing steps may involve clarification,

evaporation, precipitation, drying, and grinding (Refs. 6 and 9 through

12).

IV. Safety Evaluation

A. Pre-1958 History of Use in Food

Enzyme preparations have been safely used for many years in the

production and processing of food, for example, in the baking, dairy,

and brewing industries (e.g., see Refs. 1, 4, and 13).

1. Bacillus Subtilis

The petitioner has provided generally available information,

including published reviews, showing that carbohydrase and protease

enzyme preparations derived from B. subtilis were commonly used in food

prior to 1958 (Refs. 4 and 5). This information is summarized in Table

2.

Table 2.--Applications of Bacterial Carbohydrase and Protease Enzyme

Preparations in Food Prior to 1958

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Technical

Enzyme effect or

preparation Food categories industry References

application

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Carbohydrase Beer Mashing1 4 and 5

Syrup for cocoa Reduction of 4 and 5

and chocolate viscosity 4 and 5

Sugar Recovery from 4 and 5

Distilled scrap candy 4

beverages Mashing

Precooked Modification of

cereals cereal starches

to improve

characteristics

Protease Beer Chillproofing 4

Condiments Not reported 5

Milk Protein 5

hydrolysis

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\1\ Mashing is the conversion of starch to sugars.

In the published article by Underkofler et al. (Ref. 5), the

authors use the general terms ``bacterial amylase'' and ``bacterial

protease'' to refer to bacterially-derived carbohydrase and protease

enzyme preparations used in food at the time of the article. However,

the article also includes a table in which the source bacterium for

bacterially-derived enzyme preparations is identified as B. subtilis.

In the published article by Underkofler and Ferracone (Ref. 4), the

authors use the general terms ``bacterial carbohydrase'' and

``bacterial protease'' to refer to bacterially-derived carbohydrase and

protease enzyme preparations used in food at the time of the article.

Unlike the Underkofler et al. article, however, the Underkofler and

Ferracone article does not identify the source bacterium for these

enzyme preparations. Although it is not possible to determine

conclusively whether the descriptor ``bacterial'' in the Underkofler

and Ferracone article refers to B. subtilis, the use of this term by

the same principal author in two scientific articles published in

consecutive years to describe the source of protease and carbohydrase

enzyme preparations used in the food industry, coupled with the

identification of the source bacterium for these enzyme preparations as

B. subtilis in the Underkofler et al. article, makes it likely that the

source bacterium referred to by Underkofler and Ferracone was in fact

B. subtilis.

The food uses shown in Table 2, using terminology from the cited

reference(s), were documented in articles that were published before or

during 1958; the cited references demonstrate that the use of these

enzyme preparations in a variety of foods was widely recognized by

1958. Therefore, the agency concludes that carbohydrase and protease

enzyme preparations derived from B. subtilis were in common use in food

prior to January 1, 1958.

2. Bacillus Amyloliquefaciens

According to the petitioner (Refs. 8 and 14 through 16), the

species B. amyloliquefaciens was not classified under the name B.

amyloliquefaciens until it was taxonomically separated from the species

B. subtilis in the late 1980's (Refs. 17 and 18). Therefore, the

petitioner asserts, references in contemporaneous scientific literature

to pre-1958 food use of enzyme preparations from B. amyloliquefaciens

occur under the name B. subtilis.

With respect to carbohydrase components of the petitioned enzyme

[[Page 19890]]

preparations, the petitioner cites scientific literature describing a

distinctive group of bacteria, within the group originally considered

to be B. subtilis, that are known to possess a high level of -

amylase activity and are currently designated as B. amyloliquefaciens

(Refs. 19 through 22). The petitioner also cites a scientific review

article (Ref. 23) that states that the source organism for commercial

preparations of -amylase from B. amyloliquefaciens was called

B. subtilis prior to its current designation as B. amyloliquefaciens.

With respect to the protease components of the petitioned enzyme

preparations, the petitioner cites a statement in the same scientific

review article (Ref. 23) that most bacterial protease preparations

produced before 1960 were derived from B. amyloliquefaciens.

As FDA noted in the preamble to another final rule affirming an

enzyme preparation as GRAS (58 FR 27197 at 27199, May 7, 1993), the

taxonomic placement and name of an organism may change as a result of

scientific advances. If internationally accepted rules of nomenclature

are observed, references to a particular organism can be followed

historically in the scientific literature. Thus, changes in the

taxonomic placement of an organism should not affect the ability to

identify scientific references to the organism, including scientific

references to its toxigenicity, pathogenicity, or use in the production

of food or enzymes.In reviewing the petition, FDA has evaluated whether

the scientific information documenting pre-1958 food use of

bacterially-derived carbohydrase and protease enzyme preparations

pertains to carbohydrase and protease enzyme preparations from B.

amyloliquefaciens. Although it is not possible to determine

conclusively whether any one reference to B. subtilis in the scientific

literature refers to the species now referred to as B.

amyloliquefaciens, the totality of the scientific evidence supports a

determination that some carbohydrase and some protease enzyme

preparations that were described in scientific literature documenting

their common use in food before 1958 as derived from B. subtilis were

in fact derived from B. amyloliquefaciens. Therefore, the agency

concludes that carbohydrase and protease enzyme preparations derived

from B. amyloliquefaciens were in common use in food prior to January

1, 1958.

B. Corroborating Evidence of Safety

Because enzymes are highly efficient catalysts, they are needed in

only minute quantities to perform their function. When used in

accordance with current good manufacturing practice (CGMP), the amounts

added to food represent only a minute fraction of the total food mass.

FDA estimates dietary exposure to enzyme preparations derived from B.

subtilis or B. amyloliquefaciens at 200 mg/person/day (Ref. 24). This

estimate is exaggerated because the agency used the total consumption

of microbially-derived enzyme preparations in food as an approximation

for the consumption of enzyme preparations derived from B. subtilis or

B. amyloliquefaciens. Thus, the estimate relies on the worst-case

assumption that all microbially-derived enzyme preparations that are

consumed in food are derived from B. subtilis or B. amyloliquefaciens.

This assumption is extremely conservative because there are numerous

microbially-derived enzyme preparations that are GRAS for use in food

(see, e.g., 21 CFR 184.1012, 184.1027, 184.1387, 184.1388, 184.1924,

and 184.1985).

1. The Enzyme Components

Enzymes, including carbohydrase and protease enzymes in the enzyme

preparations that are the subject of this document, are naturally

occurring proteins that are ubiquitous in living organisms. A wide

variety of enzymes has always been present in human food. Many

naturally occurring enzymes remain active in unprocessed food and

therefore are consumed as active enzymes. For example, active enzymes

are present in fresh fruits and vegetables and are not inactivated

unless the fruits or vegetables are cooked (Refs. 1 and 25).

Enzymes derived from microorganisms have been used as components of

foods that have been safely consumed as part of the diet throughout

human history (Ref. 26). For example, such common foods as bread and

yogurt are produced using enzymes derived from microorganisms (Refs. 26

and 27).

The carbohydrase and protease enzymes in the enzyme preparations

that are the subject of this document are substantially equivalent \2\

to carbohydrase and protease enzymes from other microorganisms that FDA

has evaluated and found to be safe and that are routinely consumed as

part of a normal diet in the United States. For example, FDA has

affirmed the use of a mixed carbohydrase and protease enzyme

preparation derived from Bacillus licheniformis is GRAS (see 21 CFR

184.1027). In addition, carbohydrases derived from various fungi (e.g.,

Rhizopus niveus, Rhizopus oryzae, and A. niger) are approved for use as

secondary direct food additives (see 21 CFR 173.110, 173.130, and

173.120, respectively).

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\2\ A 1996 report of the joint Food and Agriculture Organization

and World Health Organization (FAO/WHO) consultation group (Ref. 28)

stated that ``[s]ubstantial equivalence embodies the concept that if

a new food or food component is found to be substantially equivalent

to an existing food or food component, it can be treated in the same

manner with respect to safety (i.e. the food or food component can

be concluded to be as safe as the conventional food or food

component). Account should be taken of any processing that the food

or food component may undergo as well as the intended use and the

intake by the population.'' As discussed more fully in FDA's

proposal to amend the agency's regulations pertaining to substances

that are generally recognized as safe (62 FR 18938 at 18944, April

17, 1997), international expert groups such as the FAO/WHO

consultation group and the Organization for Economic Co-operation

and Development (OECD) consultation group have recommended that the

concept of ``substantial equivalence'' be applied to the safety

assessment of foods and substances intentionally added to food.

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In general, issues relevant to a safety evaluation of proteins such

as the enzyme component of an enzyme preparation are potential toxicity

and allergenicity. Pariza and Foster (Ref. 1) note that very few toxic

agents have enzymatic properties, and those that do (e.g., diphtheria

toxin and certain enzymes in the venom of poisonous snakes) catalyze

unusual reactions that are not related to the types of catalysis that

are common in food processing and that are the subject of this

document. Further, as the agency has noted in the context of guidance

to industry regarding the safety assessment of new plant varieties,

enzymes do not generally raise safety concerns (57 FR 22984 at 23000,

May 29, 1992). Exceptions include enzymes that catalyze the formation

of toxic substances or substances that are not ordinarily digested and

metabolized. The catalytic activities of the enzymes that are the

subject of this document are well known; they split proteins or

carbohydrates into smaller subunits that are readily metabolized by the

human body and that do not have toxic properties.

According to Pariza and Foster (Ref. 1), there have been no

confirmed reports of allergies or primary irritations in consumers

caused by enzymes used in food processing. There have been, however,

some reports of allergies and primary irritations from skin contact

with enzymes or inhalation of dust from concentrated enzymes (for

example, proteases used in the manufacture of laundry detergents)

(Refs. 29 through 31). These reports relate primarily to workers in

production plants (Ref. 30) and are not relevant to an evaluation of

[[Page 19891]]

the safety of ingestion of such enzymes in food.

The 1977 report of the Select Committee on GRAS substances

concerning the plant enzyme papain (Ref. 29) supports the view that the

ingestion of an active protease at levels found in food products is not

likely to affect the human gastrointestinal tract, where many proteases

already exist at levels adequate to digest food:

In common with other proteolytic enzymes, papain digests the

mucosa and musculature of tissues in contact with the active enzyme

for an appreciable period. Because there is no food use of papain

that could result in the enzyme preparation occurring in sufficient

amount in foods to produce these effects, this property does not

pose a dietary hazard.

FDA concludes that generally available and accepted data and

information corroborate the safety of the enzyme components of the

enzyme preparations that are the subject of this document by

establishing that these enzyme components are identical or

substantially equivalent to enzymes that are known to have been safely

consumed in the diet for many years. FDA also concludes that generally

available and accepted data and information corroborate that the enzyme

components of the enzyme preparations that are the subject of this

document are nontoxigenic and nonallergenic when ingested.

2. Enzyme Sources, Manufacturing Methods, and Processing Aids

Enzyme preparations used in food processing are usually not

chemically pure; in addition to the enzyme component(s), they may

contain other components derived from the production organism and the

fermentation medium, residual amounts of processing aids, and

substances added as stabilizers, preservatives, or diluents. The agency

has concluded that the enzyme components of the carbohydrase and

protease enzyme preparations derived from B. subtilis or B.

amyloliquefaciens do not raise safety concerns; therefore, the

remaining safety issue is whether other components of the enzyme

preparations are toxic or raise other safety concerns.

a. Antibiotics. Some microorganisms are capable of producing

antibiotics, which are a special class of metabolites that can inhibit

the growth of, or kill, other microorganisms. Some microorganisms have

genetic traits that make them resistant to one or more antibiotics such

as penicillin, tetracycline, and kanamycin. These traits or markers are

often located on plasmids (extrachromosomal pieces of deoxyribonucleic

acid (DNA) that are easily transferred to other microorganisms in the

environment (e.g., in the gastrointestinal tract). The presence of

antibiotics in the food supply would be expected to favor the growth of

microorganisms resistant to the antibiotic, and thus could accelerate

the spread of antibiotic resistance among microorganisms, including

human pathogens, rendering them resistant to therapy with antibiotic

drugs. Therefore, experts have recommended that microbial-derived

enzyme preparations that are intended for food use not contain

clinically important antibiotics (Refs. 1 and 32).

Accordingly, FDA has evaluated the potential for carbohydrase or

protease enzyme preparations derived from B. subtilis or B.

amyloliquefaciens to contain antibiotics as contaminants derived from

the bacterial source. Although Bacillus species are capable of

producing a number of linear or cyclic polypeptide antibiotics

following the exponential phase of growth as part of the process of

spore formation (Ref. 33), the production of antibiotics can be

repressed by selection of strains that produce low or undetectable

levels of antibiotics as well as by strict control of the growth

conditions. In addition, the enzyme preparations can be tested for the

presence of antibiotic activity by routine methods (Ref. 34) to ensure

that they do not contain antibiotics. Because of safety concerns about

the presence of antibiotics in substances added to food, a condition of

agency affirmation of GRAS status for the enzyme preparations that are

the subject of this document is that the enzyme preparations not

contain antibiotics.

b. Toxicity and pathogenicity. A published scientific review

article (Ref. 23) states that Bacillus species, with the exception of

the B. cereus group (which does not include B. subtilis or B.

amyloliquefaciens) do not produce toxins. Another published scientific

review article on the safety of B. subtilis and B. amyloliquefaciens

(Ref. 35) notes that B. subtilis is consumed in large quantities in the

Japanese food natto. Further, according to a monograph on microbial

enzymes that was prepared under the auspices of the agency-initiated

review of GRAS substances conducted during the 1970's, there had been

no reported problems of pathogenicity or toxicity with enzyme

preparations derived from B. subtilis for use in food as of the time of

that review (Ref. 12).

More recently, de Boer and Diderichsen (Ref. 35) searched the

scientific literature for references that might implicate B. subtilis

or B. amyloliquefaciens as a cause of human disease. These authors

characterized B. subtilis as an opportunistic microorganism with no

pathogenic potential to humans. Although they reported that cultures

from some patients with opportunistic infections have revealed the

presence of B. subtilis along with other microorganisms, they

attributed the presence of B. subtilis in these cultures to the virtual

ubiquity of this microorganism in the environment (e.g., B. subtilis

commonly occurs in the soil and can be isolated in the home environment

from sites such as the kitchen and bathroom). De Boer and Diderichsen

also noted that only patients treated with immunosuppressive drugs

appeared to be susceptible to such infections. Moreover, viable cells,

which are not present in finished enzyme preparations, would be a

prerequisite for any opportunistic infection in an immunocompromised

patient. De Boer and Diderichsen also reported that their search for

references on B. amyloliquefaciens infections revealed no such cases.

As discussed in section IV.A.2 of this document, any references to B.

amyloliquefaciens prior to the late 1980's would be expected to occur

under the name B. subtilis.

A few reports have implicated B. subtilis as a potential source of

food poisoning when present as a contaminant in food (Refs. 36 and 37).

However, a particular strain of virtually any microorganism may, under

certain circumstances, mutate to become an opportunistic pathogen.

Therefore, FDA considered these reports in the context of: (1) The

information summarized in the monograph on microbial enzymes (Ref. 12);

(2) the scientific review article describing Bacillus species other

than those in the B. cereus group as nontoxigenic (Ref. 23); (3) the

documented consumption of B. subtilis bacteria in the Japanese food

natto (Ref. 35); and (4) the characterization by de Boer and

Diderichsen of B. subtilis as an opportunistic microorganism with no

pathogenic potential to humans (Ref. 36). Based on this information,

FDA concludes that nontoxigenic and nonpathogenic strains of B.

subtilis are widely available and have been safely used in a variety of

food applications. Because an enzyme preparation derived from a

toxigenic or pathogenic source would not be GRAS, a condition of agency

affirmation of GRAS status for the enzyme preparations that are the

subject of this document is that the bacterial strains used as a source

of these enzyme preparations be nontoxigenic and nonpathogenic.

c. Manufacturing methods and processing aids. Enzyme preparations

[[Page 19892]]

that are manufactured in accordance with CGMP using the methods

described in section III.B of this document meet the general

requirements and additional requirements in the monograph on enzyme

preparations in the Food Chemicals Codex, 4th ed. (Ref. 3). Such enzyme

preparations are produced using substances that are acceptable for use

in foods and under culture conditions that ensure a controlled

fermentation, thus preventing the introduction of extraneous

microorganisms that could be the source of toxic materials and other

toxic substances (Ref. 3).

FDA concludes that generally available and accepted data and

information corroborate the safety of carbohydrase and protease enzyme

preparations derived from nontoxigenic and nonpathogenic strains of B.

subtilis or B. amyloliquefaciens and manufactured in accordance with

CGMP by establishing that any added substances or impurities derived

from the enzyme source or introduced during the manufacturing of such

enzyme preparations would not be expected to present health concerns.

V. Comments

FDA received seven comments in response to the filing notice and

none in response to the amendment notices. Of these, FDA received two

comments from food manufacturers, two from trade associations, one from

a manufacturer of enzymes for use in animal feed, one from a

pharmaceutical manufacturer, and one from a consumer group. Six

comments supported the petition for GRAS affirmation, stating that the

enzyme preparations included in the petition have a long history of use

in foods such as cheese, bread, and corn syrup.

One comment stated that B. subtilis has a history of use in animal

feed and requested GRAS affirmation for this use. However, the petition

is for the use of certain enzyme preparations in human food, and not in

animal feed. Therefore, the agency finds that this comment is not

relevant to the petition.

One comment asserted that enzyme preparations should not be

considered GRAS. The comment further asserted that the use of enzyme

preparations should be declared on the label of foods and that

consumers should be warned about hazards inherent in their use. The

comment stated that enzyme preparations are rarely purified to any

significant degree and contain a variety of cellular constituents and

metabolic debris. The comment further argued that, although enzyme

preparations are used at low levels and are inactivated after the

treatment of food, they may elicit allergic reactions and other

biological activities which could be detrimental to human health. In

support of this statement, the comment cited a published scientific

article (Ref. 38) that reported that enzyme preparations from B.

subtilis caused temporary weight loss and aggravated infection in mice

when injected into the abdominal cavity and caused hemolysis and

hemagglutination of sheep erythrocytes in in vitro studies.

FDA has evaluated the comment and the article it cited. For the

following two reasons, FDA concludes that the study cited by the

comment is not relevant to food uses of the bacterial enzyme

preparations that are the subject of this document.

First, the paper did not identify the composition of the B.

subtilis enzyme preparations tested. The preparations were intended for

use in laundry detergents; such nonfood grade enzyme preparations need

not conform to specifications for enzyme preparations used in food

processing. For example, nonfood grade enzyme preparations may include

processing aids that are not acceptable for food use. Because of such

differences, the results from the testing of laundry cleaning enzyme

preparations have little value in the safety assessment of food-

processing enzyme preparations.

Second, in the cited study, adverse effects were observed in mice

after the intraperitoneal administration of B. subtilis autolysates.

However, exposure to enzyme preparations in food occurs by ingestion

and not by injection. The difference in the route of exposure is

particularly significant for assessing the significance of

immunological effects. With intraperitoneal administration, the

components of the immune system are directly exposed to a high level of

the test compound. This contrasts with exposure to enzyme preparations

in food, whereby low levels of the enzyme preparations are ingested and

undergo hydrolysis by digestive enzymes before any interaction with the

immune system. Pariza and Foster (Ref. 1) note that there are no

confirmed reports of allergic reactions in consumers caused by enzymes

used in food processing.

Moreover, a report of the Joint Food and Agriculture Organization/

World Health Organization Expert Committee on Food Additives (JECFA)

corroborates the safety of food uses of enzyme preparations from B.

subtilis (Ref. 39). This report concluded that results from a 90-day

feeding study in rats showed no adverse effects. The test diet was meat

protein-based and supplemented with a protease enzyme preparation from

B. subtilis at a 1-percent level (equivalent to approximately 1 gram of

enzyme preparation per kilogram of body weight per day). This level is

more than 300 times greater than the highest level that would be

expected in the human diet (200 mg/person/day, or 3.3 mg/kg body weight

per day for a 60 kg person), as estimated in section IV.B of this

document.

With respect to the comment's assertion that enzyme preparations

should be declared on the label of foods in which they are used, the

agency notes that under certain circumstances, applicable regulations

already require use of an enzyme preparation in a food to be declared

on the label, depending upon the nature of the enzyme preparation's use

and technical effect in the food. Section 403(i)(2) of the Federal

Food, Drug, and Cosmetic Act (21 U.S.C. 343(i)(2)) requires that all

ingredients of multi-ingredient foods be listed on the label of the

food. By regulation, FDA has exempted certain ingredients that are used

only as processing aids from this requirement. Section

101.100(a)(3)(ii)(a) and (a)(3)(ii)(c) (21 CFR 101.100(a)(3)(ii)(a) and

(a)(3)(ii)(c)) provides an exemption from the ingredient listing

requirement for processing aids that are added to a food for their

technical or functional effect during processing, but are either

removed from the food before packaging or are present in the finished

food at insignificant levels and do not have any technical or

functional effect in the finished food. Although many enzyme

preparations are used as processing aids in food (e.g., amylase

preparations used in the manufacture of glucose syrup and protease

preparations used in the manufacture of protein hydrolysates), other

enzyme preparations that are added during processing (e.g., protease

preparations used in tenderizing meat) are not processing aids as

defined in Sec. 101.100(a)(3)(ii) because they remain active in the

finished food product. For example, enzymes used in the manufacture of

swiss and cheddar cheese remain active in the finished cheese,

enhancing body, flavor, and aroma (49 FR 29242, July 19, 1984). Because

such effects in the finished food remove the enzymes from the

ingredient listing exemption for processing aids in

Sec. 101.100(a)(3)(ii)(c), the use of such enzymes must be declared on

the label. Therefore, whether a label declaration is needed for the use

of an enzyme preparation in a food will depend upon its function and

effect in the food.

[[Page 19893]]

VI. Conclusions

The petitioner has provided generally available evidence

demonstrating that carbohydrase and protease enzyme preparations from

B. subtilis were in common use in food prior to 1958. FDA has

determined, under Sec. 170.30(a) and (c)(1), that this information

provides an adequate basis upon which to conclude that the safety of

these enzyme preparations for use in food is generally recognized among

the community of experts qualified by scientific training and

experience to evaluate the safety of food ingredients.

The petitioner has also provided generally available evidence

demonstrating that the bacterium now known as B. amyloliquefaciens was

formerly included within the B. subtilis classification. Based on its

analysis of the data submitted, the agency concludes that the evidence

of common use in food pertains to carbohydrase and protease enzyme

preparations from the bacterium now known as B. amyloliquefaciens as

well as to carbohydrase and protease enzyme preparations from B.

subtilis.

This evidence of common use in food prior to 1958 is corroborated

by information that the enzymes themselves and the sources from which

they are derived are nontoxic and nontoxicogenic, and that

manufacturing will not introduce impurities that would adversely affect

the safety of the finished enzyme preparations. Moreover, the

carbohydrase and protease enzyme preparations from B. subtilis and B.

amyloliquefaciens are substantially equivalent to enzymes naturally

present in foods that have been safely consumed in the human diet for

many years.

Having evaluated the information in the petition, along with other

available information related to the use of these enzyme preparations,

the agency concludes that carbohydrase enzyme preparation and protease

enzyme preparation derived from either B. subtilis or B.

amyloliquefaciens are GRAS under conditions of use consistent with

CGMP. The agency is basing its conclusion on evidence of a substantial

history of safe consumption of the enzyme preparations in food by a

significant number of consumers prior to 1958, corroborated by the

other evidence summarized in section IV.B of this document.

FDA is affirming that the use of these bacterially-derived

carbohydrase and protease enzyme preparations in food is GRAS with no

limits other than CGMP (21 CFR 184.1(b)(1)). To clarify the identity of

each enzyme preparation, the agency is including in Secs. 184.1148(a)

and 184.1150(a) the EC numbers of the enzymes that supply the

characterizing enzyme activities of each preparation. In order to make

clear that the affirmation of the GRAS status of these enzyme

preparations is based on the evaluation of specific uses, the agency is

including in Secs. 184.1148(c) and 184.1150(c) the technical effect and

the specific substances on which each enzyme preparation acts, although

the data show no basis for a potential risk from any foreseeable use of

these enzyme preparations.

For simplicity, FDA is affirming the GRAS status of both

carbohydrase enzyme preparations in a single combined regulation that

describes the source of the enzyme as B. subtilis or B.

amyloliquefaciens, rather than affirming the GRAS status of

carbohydrase derived from B. subtilis separately from that of

carbohydrase derived from B. amyloliquefaciens. Likewise, FDA is

affirming the GRAS status of both protease enzyme preparations in a

single combined regulation that describes the source of the enzyme as

B. subtilis or B. amyloliquefaciens.

To ensure that the enzyme preparations are of suitable purity for

use in food, FDA is including in the regulations the general

requirements and additional requirements for enzyme preparations in the

monograph ``Enzyme Preparations'' in the Food Chemicals Codex, 4th ed.

(1996) as general specifications for these enzyme preparations.

Furthermore, to ensure that the use of these enzyme preparations does

not promote the development of antibiotic resistance, the agency is

specifying that the enzyme preparations must be free of antibiotic

activity as determined by a suitable method (e.g., the method described

in Ref. 34).

VII. Environmental Considerations

The agency has determined under 21 CFR 25.32(f) 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.

VIII. Analysis for Executive Order 12866

FDA has examined the impacts of this final rule under Executive

Order 12866. Executive Order 12866 directs Federal agencies to assess

the 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). According

to Executive Order 12866, a regulatory action is significant if it

meets any one of a number of specified conditions, including having an

annual effect on the economy of $100 million, adversely affecting in a

material way a sector of the economy, competition, or jobs, or raising

novel legal or policy issues. FDA finds that this final rule is not a

significant regulatory action as defined by Executive Order 12866. In

addition, the agency has determined that this final rule is not a major

rule for the purpose of Congressional review.

The primary benefit of this action is to remove uncertainty about

the regulatory status of the petitioned substances. No compliance costs

are associated with this final rule because no new activity is required

and no current or future activity is prohibited by this rule.

IX. Regulatory Flexibility Analysis

FDA has examined the impacts of this final rule under the

Regulatory Flexibility Act. The Regulatory Flexibility Act (5 U.S.C.

601-612) requires agencies to consider alternatives that would minimize

the economic impact of their regulations on small entities. No

compliance costs are associated with this final rule because no new

activity is required and no current or future activity is prohibited.

Accordingly, under the Regulatory Flexibility Act (5 U.S.C. 605(b)),

the agency certifies that this final rule will not have a significant

economic impact on a substantial number of small entities.

X. Paperwork Reduction Act of 1995

This final rule contains no collections of information. Therefore,

clearance by the Office of Management and Budget under the Paperwork

Reduction Act of 1995 is not required.

XI. 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)).

XII. References

The following references have been placed on display in the Dockets

Management Branch (address above) and may be seen by interested persons

[[Page 19894]]

between 9 a.m. and 4 p.m., Monday through Friday.

1. Pariza, M. W., and E. M. Foster, ``Determining the Safety of

Enzymes Used in Food Processing,'' Journal of Food Protection, vol.

46, pp. 453-468, 1983.

2. Scott, D., ``Enzymes, Industrial,'' Encyclopedia of Chemical

Technology, edited by H. F. Mark et al., John Wiley and Sons, New

York, 3d ed., vol. 9, pp. 173-224, 1978.

3. Monograph on ``Enzyme Preparations,'' Food Chemicals Codex,

National Academy Press, Washington, DC, 4th ed., pp. 131 and 133-

134, 1996.

4. Underkofler, L. A., and W. J. Ferracone, ``Commercial

Enzymes--Potent Catalyzers that Promote Quality,'' Food Engineering,

vol. 29, pp. 123, 125-126, 130, and 133, 1957.

5. Underkofler, L. A., R. R. Barton, and S. S. Rennet,

``Microbiological Process Report--Production of Microbial Enzymes

and Their Applications,'' Applied Microbiology, vol. 6, pp. 212-221,

1958.

6. Beckhorn, E. J., M. D. Labee, and L. A. Underkofler,

``Production and Use of Microbial Enzymes for Food Processing,''

Journal of Agricultural and Food Chemistry, vol. 13, pp. 30-34,

1965.

7. Comments of Ad Hoc Enzyme Technical Committee regarding FDA's

draft final regulations, entitled ``Enzymes Proposed for Affirmation

as GRAS,'' with a letter dated December 21, 1984, from Roger D.

Middlekauff, Ad Hoc Enzyme Technical Committee, to Kenneth A. Falci,

FDA.

8. Response of the Enzyme Technical Association to the letter

dated December 13, 1985, of Lawrence Lin regarding GRASP 3G0016,

received February 18, 1986.

9. Smythe, C. V., ``Microbiological Production of Enzymes and

Their Practical Applications,'' Economic Botany, vol. 5, pp. 126-

144, 1951.

10. Reed, G., ``Enzymes, Industrial,'' Encyclopedia of Chemical

Technology, edited by R. E. Kirk and D. F. Othmer, Interscience

Encyclopedia, Inc., New York, 1st supplemental vol., pp. 294-312,

1957.

11. Fogarty, W. M., editor, Microbial Enzymes and Biotechnology,

Applied Science Publishers, New York, NY, pp. 1-11, 22-25, 34-35,

111-113, 115-118, 162-173, 259-260, 282-286, 1983.

12. Rogers, R.W., ``Monograph on Microbial Enzymes,''

Informatics, Inc., vol. 1, pp. 1-16, 1977.

13. Reed, G., ``Industrial Enzymes--Now Speed Natural

Processes,'' Food Engineering, vol. 24, pp. 105-109, 1952.

14. Response of the Enzyme Technical Association to the letter

dated June 26, 1986, of Lawrence J. Lin regarding GRASP 3G0016,

received with a letter dated October 3, 1986, from Roger D.

Middlekauff of the Enzyme Technical Association, to Lawrence J. Lin,

FDA.

15. Letter dated August 17, 1995, from Gary L. Yingling, Enzyme

Technical Association, to Alan M. Rulis, FDA.

16. Letter dated April 16, 1996, from Alice J. Caddow, Enzyme

Technical Association, to Linda Kahl, FDA.

17. Priest, F. G., M. Goodfellow, L. A. Shute, and R. C. W.

Berkeley, ``Bacillus amyloliquefaciens sp. nov., nom. rev.,''

International Journal of Systematic Bacteriology, vol. 37, pp. 69-

71, 1987.

18. Priest, F. G., M. Goodfellow, and C. Todd, ``A numerical

classification of the genus Bacillus,'' Journal of General

Microbiology, vol. 134, pp. 1847-1882, 1988.

19. Baptist, J. N., M. Mandel, and R. J. Gherna, ``Comparative

zone electrophoresis of enzymes in the genus Bacillus,''

International Journal of Systematic Bacteriology, vol. 28, pp. 229-

244, 1978.

20. Gordon, R. E., W. C. Haynes, and C. Hor-Nay Pang, ``The

genus Bacillus,'' U.S. Department of Agriculture, 1973.

21. Welker, N. E., and L. L. Campbell, ``Unrelatedness of

Bacillus amyloliquefaciens and Bacillus subtilis,'' Journal of

Bacteriology, vol. 94, pp. 1124-1130, 1967.

22. Welker, N. E., and L. L. Campbell, ``Comparison of the

alpha-amylase of Bacillus amyloliquefaciens and Bacillus subtilis,''

Journal of Bacteriology, vol. 94, pp. 1131-1135, 1967.

23. Aunstrup, K., ``Production, Isolation, and Economics of

Extracellular Enzymes,'' Applied Biochemistry and Bioengineering,

vol. 2, pp. 27-69, 1979.

24. Memorandum dated June 6, 1985, from John Modderman, Food

Additive Chemistry Evaluation Branch, to L. Lin, GRAS Review Branch.

``Enzymes proposed for GRAS affirmation based on history of use.''

25. De Becze, G. I., ``Food Enzymes,'' Critical Reviews in Food

Technology, vol. 1, pp. 479-518, 1970.

26. Phaff, H. J., M. W. Miller, and E. M. Mrak, ``The Life of

Yeasts, Their Nature, Activity, Ecology, and Relation to Mankind,''

Harvard University Press, Cambridge, MA, pp. 1-6, 133-149, 1966.

27. Wilcox, G. ``Manufacture of Yogurt'' from ``Eggs Cheese and

Yogurt Processing,'' Noyes Data Corp., p. 269, 1971.

28. ``Biotechnology and Food Safety,'' Food and Agriculture

Organization, Rome, 1996.

29. ``Evaluation of the Health Aspects of Papain as a Food

Ingredient,'' Select Committee on GRAS Substances, Washington, DC,

available through U.S. Department of Commerce, National Technical

Information Service, Order No. PB-274-174, 1977.

30. Fulwiler, R. D., ``Detergent Enzymes--An Industrial Hygiene

Challenge,'' American Industrial Hygiene Association Journal, vol.

32, pp. 73-81, 1971.

31. ``Enzyme-containing Laundering Compounds and Consumer

Health,'' National Research Council/National Academy of Sciences,

National Technical Information Service, Washington, DC, Order No.

PB-204-118, 1971.

32. Reanney, D., ``Extrachromosomal Elements as Possible Agents

of Adaptation and Development,'' Bacteriological Reviews, vol. 40,

pp. 552-590, 1976.

33. Fogarty, W. M., P. J. Griffine, and A. M. Joyce, ``Enzymes

of Bacillus species--Part 1,'' Process Biochemistry, vol. 9, pp. 11-

13, 15, 17-18, 24, 1974.

34. ``Determination of Antibiotic Activity,'' Compendium of Food

Additive Specifications, vol. 2, Joint FAO/WHO Expert Committee on

Food Additives (JECFA), Food and Agriculture Organization of the

United Nations, Rome, 1992.

35. De Boer, A. S., and B. Diderichsen, ``On the Safety of

Bacillus subtilis and Bacillus amyloliquefaciens: A Review,''

Applied Microbiology and Biotechnology, vol. 36, pp. 1-4, 1991.

36. Sneath, P. H. A., ``Endospore-forming Gram-Positive Rods and

Cocci,'' in Bergey's Manual of Systematic Bacteriology, edited by P.

H. A. Sneath et al., Williams & Wilkins, vol. 2, pp. 1104-1139,

Baltimore, 1986.

37. Gilbert, R. J, P. C. B. Turnbull, J. M. Parry, and J. M.

Kramer, ``Bacillus cereus and other Bacillus Species: Their Part in

Food Poisoning and other Clinical Infections,'' in The Endospore-

forming Bacteria, edited by Berkeley and Goodfellow, Academic Press,

London, pp. 297-314, 1981.

38. Dubos, R., ``Toxic Factors in Enzymes Used in Laundry

Products,'' Science, vol. 173, pp. 259-260, 1971.

39. ``Toxicological Evaluation of Some Enzymes, Modified

Starches, and Certain Other Substances,'' 15th report of the Joint

Food and Agriculture Organization (FAO)/World Health Organization

(WHO) Expert Committee on Food Additives, WHO Technical Report

Series, FAO Nutrition Meetings Report Series, pp. 3-10, 1972.

List of Subjects in 21 CFR Part 184

Food additives, 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, 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: 21 U.S.C. 321, 342, 348, 371.

2. Section 184.1148 is added to subpart B to read as follows:

Sec. 184.1148 Bacterially derived carbohydrase enzyme preparation.

(a) Bacterially derived carbohydrase enzyme preparation is obtained

from the culture filtrate resulting from a pure culture fermentation of

a nonpathogenic and nontoxigenic strain of Bacillus subtilis or B.

amyloliquefaciens. The preparation is characterized by the presence of

the enzymes -amylase (EC 3.2.1.1) and -glucanase (EC

3.2.1.6), which catalyze the hydrolysis of O-glycosyl bonds in

carbohydrates.

(b) The ingredient meets the general requirements and additional

requirements in the monograph on enzyme preparations in the Food

Chemicals Codex, 4th ed. (1996), pp. 128-135, which is incorporated by

reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies

are

[[Page 19895]]

available from the National Academy Press, 2101 Constitution Ave. NW.,

Washington, DC 20418, or may be examined at the Center for Food Safety

and Applied Nutrition's Library, 200 C St. SW., rm. 3321, Washington,

DC, or at the Office of the Federal Register, 800 North Capitol Street,

NW., Suite 700, Washington, DC. In addition, antibiotic activity is

absent in the enzyme preparation when determined by an appropriate

validated method such as the method ``Determination of antibiotic

activity'' in the Compendium of Food Additive Specifications, vol. 2,

Joint FAO/WHO Expert Committee on Food Additives (JECFA), Food and

Agriculture Organization of the United Nations, Rome, 1992. Copies are

available from Bernan Associates, 4611-F Assembly Dr., Lanham, MD

20706, or from The United Nations Bookshop, General Assembly Bldg., rm.

32, New York, NY 10017, or by inquiries sent to ``http://www.fao.org''.

Copies may be examined at the Center for Food Safety and Applied

Nutrition's Library, 200 C St. SW., rm. 3321, 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 practice.

The affirmation of this ingredient as GRAS as a direct 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 to hydrolyze polysaccharides (e.g.,

starch).

(2) The ingredient is used in food at levels not to exceed current

good manufacturing practice.

3. Section 184.1150 is added to subpart B to read as follows:

Sec. 184.1150 Bacterially-derived protease enzyme preparation.

(a) Bacterially derived protease enzyme preparation is obtained

from the culture filtrate resulting from a pure culture fermentation of

a nonpathogenic and nontoxigenic strain of Bacillus subtilis or B.

amyloliquefaciens. The preparation is characterized by the presence of

the enzymes subtilisin (EC 3.4.21.62) and neutral proteinase (EC

3.4.24.28), which catalyze the hydrolysis of peptide bonds in proteins.

(b) The ingredient meets the general requirements and additional

requirements in the monograph on enzyme preparations in the Food

Chemicals Codex, 4th ed. (1996), pp. 128-135, which is 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 Center for Food

Safety and Applied Nutrition's Library, 200 C St. SW., rm. 3321,

Washington, DC, or at the Office of the Federal Register, 800 North

Capitol Street, NW., Suite 700 Washington, DC. In addition, antibiotic

activity is absent in the enzyme preparation when determined by an

appropriate validated method such as the method ``Determination of

antibiotic activity'' in the Compendium of Food Additive

Specifications, vol. 2, Joint FAO/WHO Expert Committee on Food

Additives (JECFA), Food and Agriculture Organization of the United

Nations, Rome, 1992. Copies are available from Bernan Associates, 4611-

F Assembly Dr., Lanham, MD 20706, or from The United Nations Bookshop,

General Assembly Bldg., rm. 32, New York, NY 10017, or by inquiries

sent to ``http://www.fao.org''. Copies may be examined at the Center

for Food Safety and Applied Nutrition's Library, 200 C St. SW., rm.

3321, 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 practice.

The affirmation of this ingredient as GRAS as a direct 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 to hydrolyze proteins or polypeptides.

(2) The ingredient is used in food at levels not to exceed current

good manufacturing practice.

Dated: March 26, 1999.

L. Robert Lake,

Director, Office of Policy, Planning and Strategic Initiatives, Center

for Food Safety and Applied Nutrition.

[FR Doc. 99-10011 Filed 4-22-99; 8:45 am]

BILLING CODE 4160-01-F