Agency Response letter GRAS Notice No. GRN 000485
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CFSAN/Office of Food Additive Safety
April 15, 2014
1620 Pinetree Drive
Pittsburg, PA 15241
Re: GRAS Notice No. GRN 00485
Dear Mr. Moore:
The Food and Drug Administration (FDA) is responding to the notice, dated July 29, 2013, that you submitted on behalf of Clasado Inc. (Clasado) in accordance with the agency’s proposed regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS); the GRAS proposal). FDA received the notice on September 4, 2013, filed it on September 24, 2013, and designated it as GRAS Notice No. GRN 000485.
The subject of the notice is beta-galactosidase enzyme preparation. The notice informs FDA of the view of Clasado that beta-galactosidase enzyme preparation from recombinant Escherichia coli BL21 (DE3) is GRAS, through scientific procedures, for use as an enzyme in the production of galacto-oligosaccharides (GOS) from lactose at use levels of 0.1 to 0.22 gram (g) per kilogram (kg) of lactose.
Commercial enzyme preparations that are used in food processing typically contain an enzyme component that catalyzes the chemical reaction as well as substances used as stabilizers, preservatives, or diluents. Enzyme preparations may also contain constituents derived from the production organism and constituents derived from the manufacturing process, e.g., components of the fermentation media or the residues of processing aids. Clasado’s notice provides information about each of these components of beta-galactosidase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, beta-galactosidaseis identified by the Enzyme Commission number 22.214.171.124. The accepted name for the enzyme is beta-galactosidase, and the systematic name is β-D-galactoside galactohydrolase. The CAS Registry Number for beta-galactosidase is 9031-11-2. Beta-galactosidase catalyzes the hydrolysis of terminal non-reducing beta-D-galactose residues in simple disaccharides (e.g., lactose), and longer chain galacto-oligosaccharides. At high concentrations of the lactose substrate, beta-galactosidase also catalyzes the transfer of one or more D-galactosyl units onto the D-galactose substituent of lactose, resulting in the production of GOS.
Clasado states that E. coli BL21 (DE3) is the host organism used to develop the production strain. E. coli BL21 (DE3) was obtained from a commercial culture collection (Life Technologies Cat #: C6000-03). The complete gene sequence of E. coli BL21 (DE3) has been published, including comprehensive bioinformatics analyses. The microbe is gram-negative, non-spore forming, facultative anaerobe, nonpathogenic and nontoxigenic, with a long history of safe industrial use.
Clasado describes the construction of the production strain that contains the gene encoding beta-galactosidase. Beta-galactosidase gene from Bifidobacterium bifidum NCIMB1 41171 (Genbank accession number: DQ443548.2) was synthesized for expression in E. coli. The native beta-galactosidase gene was obtained from a fecal sample obtained from a healthy volunteer and the strain is maintained within NCIMB, a commercial culture collection.It was codon optimized for expression in E. coli, and cloned into a T7 promoter expression vector, pET21a, a commercial expression vector (Novagen). The resulting plasmid was transformed into the production organism, E. coli BL21 (DE3). Using the plasmid, the microorganism was grown to an appropriate level. Ampicillin was used during fermentation to stabilize growth of the organism. Since the expression of the beta-galactosidase enzyme is under control of the Lac repressor gene, after optimal growth was achieved, the expression of the beta-galactosidase enzyme was induced by the addition of isopropylthio-beta-D-galactopyranoside.
Clasado states that the beta-galactosidaseenzyme is produced by batch fermentation of a pure culture of the production strain. The production strain is grown in an aqueous solution and appropriate measures are taken to control for identity, purity, and enzyme-generating ability before use. After fermentation is stopped, the enzyme is released by cell disruption. The homogenized biomass is treated with buffered solutions and the biomass is removed by a series of sedimentation and filtration processes to remove all debris greater than the molecular weight of beta-galactosidase. The filtrate is concentrated using ultrafiltration and it is dia-filtered to remove all components less-than 10 kDa. Finally, the retentate is filtered through a 0.22 micrometer bacto-filtration unit and freeze dried with sucrose (added as a stabilizer) to a light-beige powder. According to Clasado, the raw materials used in the fermentation, recovery, and formulation processes are food grade. Clasado states that the final enzyme preparation contains no major food allergens.
The product is tested to ensure compliance with specifications prior to release. Clasado confirmed the absence of intact full-length copies of antibiotic resistance gene, antibiotics, and the production organism in the final enzyme preparation. Clasado established food grade specifications for the beta-galactosidase enzyme preparation. Clasado notes that the beta-galactosidaseenzyme preparation conforms to the specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 8th edition, 2012), and to the current General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives (JECFA, 2006). Clasado provided analytical data from three batches of beta-galactosidase enzyme preparation to demonstrate consistency with the specifications.
Clasado proposes to use beta-galactosidase enzyme preparation in the manufacture of GOS from lactose at levels of 0.1 to 0.22 g/kg lactose. After the enzymatic reaction, Clasado uses a 10 kDa ultrafiltration membrane to remove the residual enzyme, therefore, Clasado does not expect any dietary exposure to the beta-galactosidase enzyme preparation from the intended food uses of GOS. Clasado provides SDS PAGE analytical data demonstrating the exclusion of residual enzyme in the finished GOS product.2
Although Clasado states that the beta-galactosidase enzyme preparation is not expected to be in the final food based on the processing steps involved in the manufacturing of GOS, they discuss published literature describing the safety of microbial and non-microbial enzymes used in food production, in general. Clasado notes that active beta-galactosidases of microbial (e.g., Bifodobacterium and Lactobacillus species) and human origin are naturally present within the gastrointestinal tract of all individuals. Clasado also discusses lack of potential toxicity of whole cell isolates and enzyme preparations containing beta-galactosidases from 90-day repeat dose studies. In addition, Clasado cites two regulations (21 CFR 184.1387 and 21 CFR 184.1388) describing the use of beta-galactosidase enzyme preparations for use as direct food ingredients. Clasado discusses potential food allergenicity of beta-galactosidase. Clasado conducted an 80 amino acid sliding window analysis of beta-galactosidase against the allergen database using FASTA3. No amino acid identity matches greater than 35% over 80 amino acids were found. Based on this information, Clasado concludes that it unlikely that oral consumption of beta-galactosidase will result in any allergic responses.
Based on the data and information summarized above, Clasado concludes that beta-galactosidaseenzyme preparation is GRAS for its intended use.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FD&C Act)
The Food and Drug Administration Amendments Act of 2007 which was signed into law on September 27, 2007, amends the FD&C Act to, among other things, add section 301(ll). Section 301(ll) of the FD&C Act prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FD&C Act, a biological product licensed under section 351 of the Public Health Service Act, or a drug or a biological product for which substantial clinical investigations have been instituted and their existence made public, unless one of the exemptions in section 301(ll)(1)-(4) applies. In its review of Clasado’s notice that beta-galactosidase enzyme preparation is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing beta-galactosidase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain beta-galactosidase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Clasado, as well as other information available to FDA, the agency has no questions at this time regarding Clasado’s conclusion that beta-galactosidase enzyme preparation 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 beta-galactosidase enzyme preparation. As always, it is the continuing responsibility of Clasado to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.
In accordance with proposed 21 CFR 170.36(f), a copy of the text of this letter responding to GRN 000485, as well as a copy of the information in this notice that conforms to the information in the GRAS exemption claim (proposed 21 CFR 170.36(c)(1)), is available for public review and copying at www.fda.gov/grasnoticeinventory.
Dennis M. Keefe, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition