Safety of Microbial Enzymes Used in Food: FDA Perspective

1
Safety of Microbial Enzymes Used in Food FDA
Perspective

• Robert I. Merker and Zofia S. Olempska-Beer
• U.S. Food and Drug Administration
• Center for Food Safety and Applied Nutrition
• Office of Food Additive Safety

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Overview

• REGULATORY OPTIONS
• Food additive (petition)
• subject to premarket review
• GRAS determination optional notice
• not subject to premarket review
• SAFETY ASSESSMENT
• Information provided in GRAS notices
• Case studies
• Lipase from a conventional microorganism
• Chymosin from bioengineered microorganisms
• a-amylase from a bioengineered microorganism

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Regulatory Basis

• 1958 Food Additive Amendments to Federal Food
  Drug and Cosmetic Act
• In the United States, a food additive is defined
  as
• any substance the intended use of which results
  or may reasonably be expected to result, directly
  or indirectly, in its becoming a component or
  otherwise affecting the characteristics of any
  food if such substance is not generally
  recognized to be safe under the conditions of
  its intended use
• FFDCA 201 (s)

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U.S. Regulatory Processes for Food Ingredients

• Food Additive Petition
• Regulation in 21 CFR 172 or 173
• GRAS Affirmation Petition
• Regulation in 21 CFR 184
• GRAS Notice
• Primary route for submitting information on
  microbial enzymes to FDA
• Information available on the Internet

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GRAS Criteria Comparing a GRAS Substance to a
Food Additive
Food Additive
GRAS Substance
Review Approval by FDA
Evidence of Safety
General Recognition
Evidence of Safety
Widely known
Consensus
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The GRAS Proposal62 FR 18938 April 17, 1997

• Proposed notification program for GRAS substances
  
• Notifier prepares summary of basis for its GRAS
  determination
• Not an iterative process
• Three categories of response letters
• No questions
• No basis
• Withdrawn by notifier

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The Summary of GRAS Notices

• At http//www.cfsan.fda.gov/rdb/opa-gras.html
• Lists all Notices and information about each
  notice
• Currently, 24 out of 101 notices are about
  enzymes
• Includes a link to the text of FDAs response
  letter

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GRAS Notices Enzymes on the Internet
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GRAS Affirmation Regulations on Microbial Enzymes
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Some Microbial Enzymes are Approved Food Additives
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Enzyme Submissions in 2002

• GRAS Notices - most commonly used means for
  submitting information about microbial enzymes to
  FDA
• Include published and non-published information
• Consistency in the categories of information
  provided in GRAS notices

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Information Provided About Microbial Enzyme
Preparations in GRAS Notices

• Safety of the enzyme
• Safety of the production microorganism
• Safety of the manufacturing process
• Safety of the enzyme preparation

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The Enzyme

• Identity
• (name, IUB classification, CAS Reg. No.)
• Enzymatic activity
• Physico-chemical characteristics
• (e.g., MW, thermal stability, optimum pH)
• Genetic modifications at the DNA and amino acid
  levels (if applicable)

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The Production Microorganism

• Name and taxonomic classification
• Source
• Important characteristics
• Nonpathogenic
• Nontoxigenic
• History of safe use
• Modifications (classical mutagenesis genetic
  engineering)

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For a Bioengineered Production Microorganism

• Characterization of the host strain
• Description of the expression plasmid (genetic
  material, its identity, organization, and origin)
• Description of the gene encoding the enzyme
  (source(s), modifications)
• Introduction of hereditary materials (DNA) into
  the host and characterization of the production
  strain

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The Manufacturing Process

• Fermentation (type of process, raw materials,
  controls)
• Removal of the microorganism
• Recovery (process, raw materials)
• Purification and concentration
• Formulation and standardization (e.g., water,
  sodium chloride, sucrose)
• Use of food grade materials

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The Enzyme Preparation

• Composition (including total organic solids
  TOS)
• Specifications (Food Chemicals Codex, 4th
  edition, 1996 JECFA, 2001)
• Absence of transformable DNA (bioengineered
  microorganisms)

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The Enzyme Preparation, cont.

• Use in food
• Level in food (generally very low)
• Estimation of daily intake
• Toxicological studies (case-by case basis may
  include e.g., gene mutation, chromosomal
  aberrations, toxicity in rats)
• Consideration of constituents derived from
  source, and manufacturing process

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Case studies

• Lipase enzyme preparation from Penicillium
  camembertii
• Chymosin enzyme preparation from bioengineered
  Escherichia coli K-12
• ?-amylase enzyme preparation from bioengineered
  Bacillus licheniformis

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Case Studies Lipase

• GRN 000068 Lipase enzyme preparation derived
  from Penicillium camembertii
• Intended Use Production of fatty acids from
  fats and oils
• Similar to other lipases used in food processing
• Source Organism (published information)
• Nontoxigenic
• Nonpathogenic
• Long used in the production of Camembert cheese

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Lipase The Manufacturing Process

• Major steps
• Fermentation using food grade ingredients
• Enzyme secreted into medium
• Filtration and heat remove fungal cells
• EtOH and acid precipitation
• Centrifuged, dried, crushed, blended with diluent
  to desired activity

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Lipase The Enzyme Preparation

• Specifications
• Complies with general and additional requirements
  of Food Chemicals Codex, 4th edition (published)
• Does not contain detectable antibiotics or
  mycotoxins (unpublished)
• Estimated intake 1 mg/person/day (unpublished)

23
Lipase Studies

• Pathogenicity study (published) organism (P.
  camembertii) injected into mice no effects, not
  recovered
• Toxicological studies (published) using
  concentrated lipase enzyme preparation
• 90 day gavage study in rats up to 2000 mg/kg/d
  no effect
• Bacterial mutagenicity no effects

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Lipase FDA letter

• Based on the information provided by Amano
  Enzyme, Inc., as well as other information
  available to FDA, the agency has no questions at
  this time regarding Amanos conclusion that the
  lipase enzyme preparation derived from P.
  camembertii is GRAS under the intended conditions
  of use. The agency has not, however, made its own
  determination regarding the GRAS status of the
  subject use of this lipase preparation. As
  always, it is the continuing responsibility of
  Amano to ensure that food ingredients that the
  firm markets are safe, and are otherwise in
  compliance with all applicable legal and
  regulatory requirements.

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Case Studies Chymosin from Escherichia coli K-12

• First FDA regulation issued on an enzyme
  preparation produced from a bioengineered
  organism (21 CFR 184.1685)
• Bovine enzyme expressed in E. coli K-12
• Subsequent regulations
• Bovine chymosin from Kluyveromyces marxianus
• Bovine chymosin from Aspergillus niger

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Chymosin from E. coli K-12

• The host strain E. coli K-12 JA198
• Nonpathogenic
• Nontoxigenic
• The production strain E. coli K-12 GE81
• Contains the expression plasmid pPFZ-87A based on
  the E. coli vector pBR322
• The plasmid carries a gene encoding bovine
  prochymosin

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Chymosin from E. coli K-12

• The manufacturing process
• Pure culture fermentation of E. coli K-12 GE81
• Prochymosin accumulates within cells as
  inclusion bodies
• Prochymosin is isolated, purified, and converted
  to chymosin
• Chymosin is purified and shown to be identical to
  bovine chymosin present in rennet (published
  information)

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Case Studies Bioengineered ?-Amylase

• GRAS Notice 000022 ?-Amylase enzyme preparation
  derived from a bioengineered strain of Bacillus
  licheniformis
• Bioengineered for enhanced stability at low pH,
  low calcium concentration and high temperature
• Intended use starch hydrolysis in the production
  of syrups (e.g., high-fructose corn syrup) and
  alcohol

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Bioengineered ?-Amylase The Enzyme

• Gene encoding ?-amylase derives from B.
  licheniformis.
• Modifications for enhanced stability and a lower
  calcium requirement (as compared with other
  ?-amylases)
• 35 amino acids at amino-terminal region from B.
  amyloliquefaciens ?- amylase
• Five additional altered amino acids
• DNA sequence highly homologous to those encoding
  other ?-amylases
• Same enzymatic function as other ?-amylases that
  have a history of safe use in food (published)

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Bioengineered ?-Amylase The Production Strain

• B. licheniformis
• Nonpathogenic (published)
• Nontoxigenic (published)
• History of use as a source of food enzymes
  (published)
• Host strain B. licheniformis strain SJ1707
  (sporulation deficient and protease negative)
• Production strain LiH 1159
• contains plasmid pLiH1108 stably integrated into
  the host chromosome.

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Bioengineered ?-Amylase The Bioengineered Strain

• Plasmid pLiH1108 contains
• The bioengineered ?-amylase gene
• Selectable marker the kanamycin resistance gene
  encoding aminoglycoside 3-phosphotransferase II
• DNA sequences necessary for plasmid integration
• DNA sequences from the certified cloning vectors
  pE194 and pUB110

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Plasmid Map pLiH1108
pE194 origin
pUB110(kan)
B. licheniformis 3 region
pLiH1108
B. licheniformis 5 region
?-amylase coding sequence
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Bioengineered ?-Amylase The Manufacturing Process

• Pure culture fermentation of the bioengineered
  production strain LiH 1159
• ?-Amylase is recovered from the fermentation
  broth, concentrated, and formulated with sodium
  chloride, sucrose, and water
• Materials used in fermentation and recovery are
  food grade

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Bioengineered ?-Amylase The Enzyme Preparation

• Derived from the safe production strain.
• Contains the safe active component, bioengineered
  ?-amylase.
• Complies with FCC and JECFA specifications
  (published).
• Does not contain the production strain
  (unpublished).

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Bioengineered ?-Amylase The Enzyme Preparation,
cont.

• Does not contain plasmid DNA (unpublished)
• Does not contain APH(3)II (unpublished)
• Is not carried over to food (syrups and alcohol)
• Estimated intake negligible (unpublished)
• Toxicological studies gene mutation, chromosomal
  aberrations, subchronic toxicity in rats, all
  negative (unpublished)

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Summary

• In the U.S.A., microbial enzymes intended for use
  in food are most commonly submitted to FDA as the
  subjects of GRAS notices, which do not require
  premarket review.
• GRAS Notices commonly contain published and
  unpublished information about
• The Safety of the Enzyme
• The Safety of the Production Microorganism
• The Safety of the Manufacturing Process
• The Safety of the Enzyme Preparation