Contains Nonbinding Recommendations  
Guidance for Industry
Enzyme Preparations: 
Recommendations for Submission of 
Chemical and Technological Data for Food 
Additive Petitions and GRAS Notices  
Additional copies are available from: 
Office of Food Additive Safety 
Division of Biotechnology and GRAS Notice Review, HFS-255 
Center for Food Safety and Applied Nutrition 
Food and Drug Administration 
5001 Campus Drive
College Park, MD 20740 
(Tel) 301-436-1200 
http://www.fda.gov/FoodGuidances 
You may submit written comments regarding this guidance at any time.  Submit written 
comments on the guidance to the Division of Dockets Management (HFA-305), Food and Drug 
Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852.  All comments should be 
identified with the title of the guidance document. 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Food Safety and Applied Nutrition 
January 1993; Revised July 2010  Contains Nonbinding Recommendations 
 
Table of Contents 
 
I.  Introduction 
II.  Background 
III.  Discussion 
A.  Petitions for Enzyme Preparations 
B.  GRAS Notices for Enzyme Preparations 
IV.   Recommendations for Petition or GRAS Notice Preparation 
A. Identity 
B. Manufacturing Process 
  C.  Specifications for Identity and Purity 
  D.  Intended Technical Effects and Use 
  E.  Intake Estimate 
V.  Additional Information 
A. Enzyme Preparations Used in Meat and Poultry Processing 
B. Enzyme Preparations Containing Allergenic Ingredients 
 
 
 
 
 
 
 
 
  2 Contains Nonbinding Recommendations 
 
1
Guidance for Industry  
Enzyme Preparations: Recommendations for Submission of 
Chemical and Technological Data for Food Additive Petitions and 
GRAS Notices 
 
This guidance represents the Food and Drug Administrations current thinking on this topic.  It 
does not create or confer any rights for or on any person and does not operate to bind FDA or the 
public.  You can use an alternative approach if the approach satisfies the requirements of the 
applicable statutes and regulations.  If you want to discuss an alternative approach, contact the 
FDA staff responsible for implementing this guidance.  If you cannot identify the appropriate 
FDA staff, call the telephone number listed on the title page of this guidance. 
 
I. Introduction 
 
This  document  describes  chemical  and  technological  data  that  the  FDAs  Office  of  Food 
Additive Safety (OFAS) in the Center for Food Safety and Applied Nutrition considers when 
evaluating food additive petitions and GRAS notices for enzyme preparations.  
 
This guidance does not discuss data and information relevant to microbiological, toxicological 
and environmental considerations. It also does not consider the safety of substances used to 
immobilize enzymes. Such substances should be approved through either a food additive petition 
or  a  food  contact  notification.  This  guidance  will  be  updated  as  needed  to  reflect  new 
developments in the manufacture and use of enzyme preparations.  
 
This document revises and supersedes the guidance entitled Enzyme Preparations: Chemistry 
Recommendations for Food Additive and GRAS Affirmation Petitions dated January 1993. 
  
FDAs  guidance  documents,  including  this  guidance,  do  not  establish  legally  enforceable 
responsibilities. Instead, guidances describe the Agencys current thinking on a topic and should 
be viewed only as recommendations, unless specific regulatory or statutory requirements are 
cited. The use of the word should in Agency guidances means that something is suggested or 
recommended, but not necessarily required. 
 
II. Background
 
  
Enzyme preparations used in food processing contain an active enzyme that is responsible for the 
intended technical effect in food. In some instances, enzyme preparations may contain a blend of 
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two or more active enzymes. Enzymes present in enzyme preparations may be derived from a 
variety  of  biological  sources,  such  as  plants,  animal  tissues,  or  microorganisms.  Enzymes 
obtained  from  these  sources  are  formulated  with  intentionally  added  ingredients,  such  as 
diluents,  preservatives,  stabilizers  or  other  substances  suitable  for  use  in  food.  Enzyme 
preparations  may  also  contain  constituents  derived  from  the  enzyme  source  and  the 
manufacturing process, for example, the residues of a fermentation medium used for growing the 
microorganism that produces the enzyme. Enzyme preparations are used in food processing at 
very low levels and often are removed from the final food or inactivated. For example, enzymes 
used  in  processing  starch  into  glucose  or  high  fructose  corn  syrup  are  removed  during 
purification  of  the  syrup,  while  enzymes  used  in  baked  goods  are  inactivated  by  high 
temperatures during baking.     
 
Enzyme preparations can be regulated as secondary direct food additives under Title 21 of the 
Code of Federal Regulations, Part 173 (21 CFR 173). The regulatory status of food additives, 
including secondary food additives, is established through the petition process. According to 
Section 409(b)(1) of the Federal Food, Drug, and Cosmetic Act (the Act) [21 U.S.C. 348(b)(1)], 
anyone may file a petition proposing the issuance of a regulation. Section 409(b)(2) of the Act 
[21 U.S.C. 348(b)(2)] prescribes the statutory requirements for food additive petitions. The 
requirements for food additive petitions are discussed in greater detail in 21 CFR 171.1.  
 
Section 201(s) of the Act [21 U.S.C. 321(s)] exempts the use(s) of a substance that is generally 
recognized as safe (GRAS) from the definition of a food additive. A substance can be determined 
to be GRAS under the intended conditions of use if there is evidence of its safety (the technical 
element of the GRAS standard) and a basis to conclude that this evidence is generally known 
and accepted by qualified experts. Title 21 of the CFR contains regulations for food ingredients 
that are either listed as GRAS or affirmed as GRAS through GRAS affirmation petitions. In the 
past, FDA reviewed GRAS affirmation petitions for enzyme preparations. A successful review of 
a GRAS affirmation petition resulted in a regulation in 21 CFR, Part 184. The GRAS affirmation 
process has been replaced by a voluntary notification program under the agencys proposed 
regulation  (Proposed  21  CFR  170.36  (62  FR  18938;  April  17  1997;  Substances  Generally 
Recognized as Safe (GRAS)). 
 
  
III. Discussion 
 
As noted above, an enzyme preparation may be either regulated as a secondary direct food 
additive through a premarket approval process or determined to be GRAS. In either case, the 
safety determination is limited to the intended conditions of use of an enzyme preparation. 
 
A. Petitions for Enzyme Preparations 
 
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Section 409(b)(2) of the Act describes the statutory requirements for food additive petitions, 
which encompass five general areas of information: 
 
1. The identity of the additive. 
2. The proposed use of the additive. 
3. The intended technical effect of the additive. 
4. A method of analysis for the additive in food.  
5. Full reports of all safety investigations with respect to the additive. 
 
21 CFR 171.1(c) describes in greater detail the data to be provided in food additive petitions, 
including administrative information. Food additive petitions for enzyme preparations should 
contain data and information pertinent to the above five basic areas. However, the specific 
information  within  each  of  these  areas  should  be  compatible  with  the  nature  of  enzyme 
preparations  which,  in  addition  to  the  enzyme(s)  of  interest,  contain  intentionally  added 
ingredients and may also contain unidentified constituents derived from the enzyme source and 
manufacturing process. The experimental data related to chemistry issues should be described in 
a clear and concise manner and supported with raw data and appropriate analytical methods.   
 
As described in 21 CFR 171.1(h), certain data and information contained in food additive 
petitions are available for public disclosure, while other data and information are not. Questions 
in this regard should be directed to OFAS. 
 
B. GRAS Notices for Enzyme Preparations 
 
General recognition of safety may be based either on scientific procedures (21 CFR 170.30(b)) 
or, for substances used in food prior to January 1 1958, on experience based on common use in 
food  (21  CFR  170.30(c)).  A  GRAS  determination  through  scientific  procedures  requires 
common knowledge about the substance and its safety under the intended conditions of use 
throughout the scientific community knowledgeable about the safety of substances directly or 
indirectly added to food.  This determination can be made by qualified experts outside of 
government.  
 
FDA has proposed a voluntary notification program whereby a person may notify FDA of its 
determination that a substance is GRAS (Proposed 21 CFR 170.36 (62 FR 18938; April 17, 
1997; Substances Generally Recognized as Safe (GRAS)) (available at: Federal Register Notice 
 the GRAS Proposal or the GPO website; search for 62 FR 18938) If the data and information 
provided in the GRAS notice do not raise safety concerns about the use of the substance at the 
time of the review, FDA issues a letter to the notifier stating that the agency has no questions 
regarding the notifiers conclusion that the substance is GRAS under the intended conditions of 
use.  
 
In  recent  years,  numerous  enzyme  preparations  intended  for  use  in  food  processing  were 
evaluated on the basis of scientific procedures through the GRAS notification program. GRAS 
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notices  for  enzyme  preparations  contain  a  GRAS  exemption  claim,  technical  information 
showing that there is a reasonable certainty in the minds of competent scientists that the enzyme 
preparation is safe under the intended conditions of use, and evidence that this information is 
generally known and accepted by the scientific community. The technical information may 
include but is not limited to the identity, method of manufacture, specifications, use levels, 
dietary exposure, and toxicological studies.   
 
The technical information in a GRAS notice is essentially the same as in a food additive petition. 
However, FDA encourages the notifiers to submit only a summary of data and information in 
GRAS notices and retain the detailed records and raw data for FDA review upon request. FDA 
discourages including proprietary information in GRAS notices, as the notices are available for 
public disclosure in compliance with the Freedom of Information Act. If a GRAS notice contains 
proprietary information, the notifier should clearly designate the information as confidential. 
FDA will review the designated information, determine whether that information is exempt from 
public disclosure under 21 CFR part 20, and release or protect the information in accordance 
with that determination.  
 
A GRAS notice should also contain a discussion of any other generally available and accepted 
data and information that the notifier relied upon to support the GRAS determination of an 
enzyme preparation under the intended conditions of use, as well as any information that may 
appear to be inconsistent with the GRAS determination. Not all GRAS notices will need to be 
supported by the same quantity and quality of data. Questions regarding the tailoring of the data 
package for specific cases may be discussed with agency personnel prior to submitting a GRAS 
notice. The notifiers may also review FDAs information on the GRAS notification program.  
 
IV. Recommendations for Petition and GRAS Notice Preparation
  
 
A. Identity 
 
In  the  "Identity"  section  of  a  petition  or  GRAS  notice,  the  enzyme  preparation  should  be 
characterized as completely as possible. The characterization should include the identity and 
characterization of the enzyme, enzyme source, and the final enzyme preparation. The items to 
be considered under "Identity" are given below.  
1.  Identity of the enzyme 
  Accepted  name,  systematic  name,  and  the  Enzyme  Commission  (EC)  number 
according to the recommendations of the International Union of Biochemistry and 
Molecular Biology.2 
  Other name(s).  
  Chemical Abstract Service Registry Number (CAS No.), if available. 
  6 Contains Nonbinding Recommendations 
 
  Enzymatic activity, substrate specificity, and the reaction catalyzed. 
  Other properties, such as specific activity, molecular weight, amino acid sequence, 
and  stability  and  activity  as  a  function  of  temperature  and  pH,  if  available  or 
necessary to support the enzyme identity and its intended uses. 
  Structural  modifications  introduced  by  chemical  or  genetic  methods  that  affect 
enzyme performance under the intended conditions of use. 
  The characteristics of the enzyme that may be useful in assessing if the enzyme is 
likely to be a food allergen. The information relevant to the assessment of potential 
allergenicity should be consistent with current views of the scientific community and 
may  include  the  considerations  regarding  the  enzyme  source,  comparison  of  the 
amino acid sequence of the enzyme to the amino acid sequences of known allergens, 
susceptibility of the enzyme to pepsin digestion, and other relevant information, if 
available.  
2.  Characterization of the enzyme source 
Enzymes currently used in food processing are derived from animal tissues, plant material, and 
microorganisms. These sources should comply with the principles outlined in the monograph 
Enzyme Preparations (section Other Requirements) of the 6th (2008) or current edition of the 
Food Chemicals Codex (FCC).3 Animal tissues used to produce enzymes should comply with 
the applicable U.S. meat inspection requirements and should be handled in accordance with good 
hygienic practices. Plant material used to produce enzymes and culture media used to grow 
microorganisms should consist of components that leave no residues harmful to health in the 
finished food under normal conditions of use. The fermentation process should be conducted 
under controlled conditions to prevent contamination with microorganisms that could be the 
source of toxic and other undesirable substances.  
Microorganisms used in the production of enzymes should be taxonomically identified and 
shown  to  be  nonpathogenic  and  nontoxigenic.  However,  certain  strains  of  microorganisms 
normally considered to be nontoxigenic may be capable of producing toxins when cultured under 
conditions that are conducive to toxin synthesis. When such microorganisms are used as sources 
of  enzymes,  the  fermentation  conditions  should  be  adjusted  to  prevent  toxin  synthesis  and 
appropriate tests should be conducted to assure that the final enzyme preparations do not contain 
toxins at unsafe levels. Alternatively, such microorganisms may be genetically modified to 
inactivate biochemical pathways involved in toxin synthesis. This can be accomplished either by 
classical mutagenesis or genetic engineering.  All the information relevant to the identity and 
safety of the microorganisms used as sources of enzymes should be described, including current 
and previous uses in food or in the production of food ingredients, if applicable.  
Additional  information  should  be  provided  for  enzymes  derived  from  genetically  modified 
microorganisms  (GMMs).4  Such  microorganisms  should  be  thoroughly  characterized  with 
respect to any introduced DNA. The source(s) of the introduced DNA including the gene(s) 
encoding the enzyme(s) of interest, any other genes (e.g., genes encoding selectable markers), 
and regulatory DNA sequences necessary for gene expression should be identified. The enzyme-
encoding genes can be derived from known organisms, unidentified organisms sampled from the 
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environment, or generated from a pool of genes from various sources via molecular evolution 
also known as gene shuffling. The enzyme-encoding genes can also be synthesized or modified 
by traditional or site-specific mutagenesis to adapt the enzyme properties to the specific food 
application conditions or to enhance the enzyme production.  
 
The host microorganisms can also be modified by inactivating or deleting certain endogenous 
genes, for example, to prevent the synthesis of potentially harmful secondary metabolites (e.g., 
mycotoxins)  or  to  minimize  the  production  of  other  enzymes  that  may  interfere  with  the 
production of the enzyme of interest or its function in food processing. All approaches and steps 
involved in the production of enzymes from GMMs should be described. 
3.  Composition of the enzyme preparation 
Commercial enzyme preparations that are used in food processing typically contain an enzyme, 
which catalyzes the chemical reaction that is responsible for its technical effect, as well as 
substances used as stabilizers, preservatives or diluents. Enzyme preparations may also contain 
constituents derived from the production organism and manufacturing process. To characterize 
the enzyme preparation the following information should be provided: 
  Identities and levels of diluents, stabilizers, preservatives, and any other substances 
used in the enzyme formulation. 
  Information on secondary enzymes derived from the production organism(s) that may 
be present in the enzyme preparation. 
  Information  on  the  residues  of  other  metabolites  derived  from  the  production 
organism(s) and substances used in enzyme isolation and purification. 
  The  content  of  total  organic  solids  (TOS)  for  both  the  commercial  enzyme 
preparation and the enzyme batch used in toxicology studies. TOS is the sum of all 
organic  compounds  present  in  the  enzyme  preparation  derived  from  the  enzyme 
source and manufacturing process.5  TOS is calculated as follows: 
TOS (%) = 100 - A - W - D  
Where:  
A = % ash  
W = % water  
D = % diluents and other formulation ingredients 
 
B. Manufacturing Process 
 
Regardless of the source material, enzyme preparations should be produced in accordance with 
the current good manufacturing practice (cGMP). For enzyme preparations obtained from animal 
  8 Contains Nonbinding Recommendations 
 
or plant materials including tissue culture, the source materials should be characterized and the 
procedures for enzyme isolation and purification should be described.   
 
For enzyme preparations obtained from microorganisms, the fermentation process should be 
described, including all steps and controls necessary to maintain the proper growth conditions 
and purity and genetic stability of the culture. The isolation of the enzyme from the cellular 
material or from the fermentation broth (depending on whether the enzyme is intracellular or 
secreted)  should  be  described,  including  all  chemical  and  physical  treatments  and  quality 
controls.  All  materials  used  in  the  fermentation  and  subsequent  downstream  processing 
(including antifoaming and flocculating agents, if used) should be identified and shown to be 
suitable for use in food processing.  
   
C. Specifications for Identity and Purity 
 
Enzyme  preparations  should  conform  to  the  purity  specifications  for  enzyme  preparations 
provided in the monograph Enzyme Preparations of the 6th (2008) or current edition of the 
Food Chemicals Codex (FCC). Petitioners and notifiers should rely on the analysis of several 
batches of the enzyme preparation to establish conformance with the specifications. The standard 
analytical methods (e.g., those described in FCC) used in the specifications should be referenced, 
while  the  non-standard  methods,  such  as  those  elaborated  by  the  manufacturer,  should  be 
validated and described and the unit of activity should be defined.     
 
Enzyme preparations obtained from microbial sources should not contain antibiotics, toxins (e.g., 
protein toxins or mycotoxins) or transformable DNA coding for protein toxins or proteins that 
inactivate therapeutic antibiotics. If the absence of these substances cannot be established on the 
basis of the characteristics of the production strain, appropriate tests should be performed to 
show that the enzyme preparation does not contain these substances at biologically significant 
levels. 
 
D. Intended Technical Effects and Use 
 
All foods, or groups of foods, in which the enzyme preparation is used or intended to be used 
should be specified and the technical effects of the enzyme should be described. The use level, or 
range of levels, should be provided on the basis of TOS for each food or food group and 
expressed in metric units (e.g., in milligrams of TOS per kilogram of food). 
 
The fate of the enzyme preparation in food should be described. The enzyme preparation may be 
carried over to food or be removed from food prior to consumption, or the enzyme may be 
  9 Contains Nonbinding Recommendations 
 
inactivated during food preparation (e.g., baking) or remain active in the final food. If the 
enzyme preparation is removed from food, the residual level of the enzyme preparation TOS in 
food "as consumed" should be estimated, if possible.  
 
The reaction products formed in food as a result of enzyme activity should be identified and their 
safety should be addressed. 
 
E. Intake Estimate 
 
A food additive petition or GRAS notice should contain an estimate of the dietary exposure to 
the enzyme preparation, i.e., an estimated daily intake (EDI). The EDI should be based on food 
intake and the level of enzyme TOS in food.  If the actual TOS level in the final food is not 
available, the EDI may be calculated on the basis of the use level expressed as TOS. The sources 
of food consumption data should be referenced and briefly described.  
 
V. Additional Information 
 
A. Enzyme Preparations Used in Meat, Poultry, and Egg Products 
 
FDA and FSIS developed a Memorandum of Understanding (MOU),6 which provides for a 
simultaneous review of substances intended for use in or on meat and poultry products that are 
subjects of food and color additive petitions and GRAS notices. If an enzyme preparation is 
intended for use in or on meat and poultry products, FDA will consult with the Food Safety and 
Inspection Service (FSIS) of the U.S. Department of Agriculture consistent with this MOU.  
 
According to the MOU, FDA evaluates information related to the safety of the substance under 
the intended conditions of use, while FSIS evaluates data that support the suitability of the use of 
the substance. Therefore, a food additive petition or a GRAS notice for an enzyme preparation to 
be used in or on meat and poultry products should contain information pertinent to both, the 
safety and suitability of the use of the enzyme preparation. Regarding the suitability of the use of 
an enzyme preparation, data should be provided showing that the enzyme preparation will be 
used at the lowest level necessary to achieve the intended technical effect under the proposed 
conditions of use. These data should be provided for each meat and poultry category in which the 
use of an enzyme preparation is intended. FDA will provide this information to FSIS and 
coordinate a joint review consistent with the MOU. 
 
  10 Contains Nonbinding Recommendations 
 
FSIS also evaluates the suitability of substances in the formulation of egg products as defined in 
9 CFR 590.5. Therefore, a food additive petition or a GRAS notice submitted to FDA for an 
enzyme  preparation  intended  for  use  in  egg  products  regulated  by  FSIS  should  contain 
information on the suitability of the use of the enzyme preparation in egg products.  
 
B. Enzyme Preparations Containing Allergenic Ingredients 
 
A food additive petition or a GRAS notice for an enzyme preparation should contain information 
on the presence of proteins derived from major food allergens that fall under the provisions of 
the Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA). FALPCA 
requires that the labels of all FDA-regulated foods that are or contain a major food allergen must 
disclose the food source name of the allergen, unless the ingredient has been exempted from 
labeling through either a petition or notification process. According to FALCPA, a major food 
allergen is defined as one of the following foods and ingredients that contain protein from them: 
milk, egg, peanuts, tree nuts, soybeans, wheat, fish, and Crustacean shellfish. The food source 
names of the major food allergens are milk, egg, peanuts, the specific type of tree nut (e.g., 
almonds), soybeans, wheat, the specific species of fish (e.g., flounder) and the specific species of 
Crustacean shellfish (e.g., crab). 
 
If an enzyme preparation contains proteins derived from a major food allergen and is used in 
foods that normally do not contain this allergen, such an enzyme preparation may fall under the 
provisions of FALCPA unless it is granted an exemption from allergen labeling as a result of a 
petition or a notification. For example, such a situation may occur if an enzyme preparation has 
been formulated with wheat flour and is intended for use in foods that normally do not contain 
wheat protein. More information on food allergen labeling requirements may be found in the 
FDA Guidance documents for Food Labeling and Nutrition.7   
 
 
 
 
 
                                                 
1 This guidance has been prepared by the Division of Biotechnology and GRAS Notice Review 
in the Center for Food Safety and Applied Nutrition at the U.S. Food and Drug Administration. 
 
2 http://www.chem.qmul.ac.uk/iubmb/enzyme/search.html 
 
3Food Chemicals Codex, 6th Edition (2008). The United States Pharmacopeial Convention. 
Available in print or online at: http://online.foodchemicalscodex.org 
 
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4 The term genetically modified microorganism (GMM) means for the purpose of this 
document a microorganism that has been modified by modern recombinant DNA techniques. 
However, the scientific meaning of this term is much broader and encompasses any intentional 
genetic modification accomplished by either traditional or rDNA methods. 
 
5 NRC/NAS, 1981. The 1978 Enzyme Survey Summarized Data.  National Research 
Council/National Academy of Sciences.  Washington, D.C., 1981. U.S. Department of 
Commerce.  National Technical Information Service. 
 
6 Memorandum of Understanding Between The Food Safety and Inspection Service, United 
States Department of Agriculture, and The Food and Drug Administration, United States 
Department of Health and Human Services. 
http://origin-www.fsis.usda.gov/Regulations_&_Policies/Labeling_FDA_MOU/index.asp 
 
http://www.fda.gov/AboutFDA/PartnershipsCollaborations/MemorandaofUnderstandingMOUs/
DomesticMOUs/ucm112604.htm 
 
7http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/Fo
odLabelingNutrition/ucm059116.htm 
 
 
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