Agency Response Letter GRAS Notice No. GRN 000267
CFSAN/Office of Food Additive Safety
July 15, 2009
John B. Dubeck
Keller & Heckman
1001 G Street N. W.
Suite 500 West
Washington, DC 20001
Re: GRAS Notice No. GRN 000267
Dear Mr. Dubeck:
The Food and Drug Administration (FDA) is responding to the notice, dated November 10, 2008, that you submitted on behalf of Amano Enzyme Inc. (Amano) 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 November 14, 2008, filed it on November 14, 2008, and designated it as GRAS Notice No. GRN 000267. On May 7, 2009, you sent additional communication limiting the intended use to those protein materials and foods described in the following paragraph.
The subject of the notice is a protein glutaminase enzyme preparation derived from Chryseobacterium proteolyticum (protein glutaminase enzyme preparation). The notice informs FDA of the view of Amano that the protein glutaminase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme to deamidate vegetable proteins (cereal, soy, and pea), milk proteins, and yeast proteins; and, to deamidate proteins in baked goods, dairy foods such as yogurt, cheese, and ice-cream, egg-based foods such as mayonnaise, and cakes.
Commercial enzyme preparations that are used in food typically contain an enzyme component, 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. In its notice, Amano provides information about all components of the protein glutaminase enzyme preparation.
21 CFR 101.4 states that all ingredients must be declared by their common or usual name. In addition, 21 CFR 102.5 outlines general principles to use when establishing common or usual names for nonstandardized foods. Our use of protein glutaminase enzyme preparation in this letter should not be considered an endorsement or recommendation of that term as an appropriate common or usual name for the purpose of declaring the substance in the ingredient statement of foods that contain that ingredient. Issues associated with labeling and the appropriate common or usual name of a food are the responsibility of the Office of Nutrition, Labeling, and Dietary Supplements in the Center for Food Safety and Applied Nutrition.
The protein glutaminase catalyzes the deamidation of glutaminyl residues in the substrate polypeptide, resulting in the conversion of glutaminyl residues to glutamyl residues and release of ammonia. According to the classification of enzymes established by the International Union of Biochemistry and Molecular Biology, the enzyme is identified by the Enzyme Commission number 18.104.22.168. Its accepted name is protein-glutamine glutaminase and its systematic name is protein-L-glutamine amidohydrolase. Amano describes the identity and properties of the protein glutaminase as a monomeric polypeptide constituted of 185 amino acids, with a molecular mass of 10860 atomic mass units and isoelectric point of 10.0. The enzyme is optimally active at pH 5–7 and a temperature of 50–60° Celsius.
Amano describes the isolation and identification of the production strain. A new bacterium designated as strain 9670 was isolated from soil in a rice field in Tsukuba, Japan. It was subsequently identified taxonomically as a new species belonging to the genus Chryseobacterium and family Flavobacteriaceae based on published studies that included phenotypic characterization, 16S rRNA sequencing, and DNA-DNA hybridization. Strain 9670 was designated as the type strain of the new Chryseobacterium species, which was named C. proteolyticum. Amano states that the C. proteolyticum strain 9670 has been deposited in the Patent Microorganism Depository, National Institute of Bioscience and Human Technology (Tsukuba, Japan) as strain FERM P-17664. Amano describes this strain as a Gram-negative, non-spore forming, and non-motile rod-shaped bacterium.
Amano states that C. proteolyticum strain 9670 is nonpathogenic and nontoxigenic based on published studies. Amano evaluated C. proteolyticum for potential pathogenicity via intravenous and oral inoculation studies in mice. In addition, Amano measured endotoxin levels of cell lysates; Amano concluded that C. proteolyticum is safe because the endotoxin levels are comparable to those found in drinking water and teas.
Amano states that the production strain is not genetically modified and that it was developed from the wild type strain 9670 by UV mutagenesis followed by N-methyl-N'-nitro-nitrosoguanidine mutagenesis. Amano describes the manufacture of protein glutaminase using controlled fermentation of the C. proteolyticum production strain. Each production batch is initiated from a primary culture. The protein glutaminase is produced at the full-scale culture step and is subsequently recovered via several filtration, desalting and concentration steps. The ensuing liquid concentrate is purified by ion exchange, and subsequently filtered and powdered using a spray dryer. The powdered enzyme is mixed with dextrin as an excipient to make the homogeneous enzyme preparation. The typical total organic solids (TOS) content of the protein glutaminase enzyme preparation is 3.4 percent. The protein glutaminase enzyme preparation is also formulated to a lower activity product by further dilution with dextrin. Amano states that both protein glutaminase enzyme preparations conform to the general and additional requirements for enzyme preparations as described in the Food Chemicals Codex (5th edition).
The protein glutaminase is used to improve functional properties of protein via deamidation. The intended use level of the enzyme preparation is 0.49 percent by weight of the protein in foods and 0.09 percent by weight of the protein in finished foods. Amano estimates a maximum intake of the enzyme preparation of 334 milligram per person per day (mg/p/d; equivalent to 11.3 mg TOS/p/d) based on a 2000 calorie diet. Amano estimates this intake under the conservative assumption that all protein consumed in the diet (100 grams/person/day (g/p/d)) is treated with protein glutaminase and that 100 percent of the enzyme TOS would remain in foods.1
Amano summarizes unpublished toxicological studies conducted with the protein glutaminase enzyme preparation, which include a 90-day oral toxicity study in rats, a bacterial reverse mutation assay (Ames test), and an in vitro mammalian chromosomal aberration test. Amano states that the enzyme preparation was not toxic in the 90-day toxicity study at the highest dose tested of 93 mg TOS/p/d and not mutagenic in the in vitro mutagenicity studies.
Amano evaluated the potential allergenicity of protein glutaminase via digestion studies in simulated gastric and intestinal fluids, and by comparison with known allergens for sequence homology. Amano concludes that the enzyme is rapidly digested in simulated gastric fluid and that its digestibility in simulated intestinal fluid is close to that of food proteins ribulose 1, 5-diphosphate carboxylase (Rubisco) from spinach and ovoalbumin from egg white. In addition, Amano compares the amino acid sequence of protein glutaminase to the sequences of known allergens contained in five allergen databases using search criteria including a match for 6-, 7- and 8-consecutive amino acid sequences and a greater than 35 percent homology within a sliding window of 80 amino acids. Amano concludes that the enzyme does not match the amino acid sequences of any known food allergens when tested according to the FAO/WHO (2001) recommendations.
Standards of Identity
In the notice, Amano states its intention to use protein glutaminase enzyme preparation in several food categories, including foods for which standards of identity exist, located in Title 21 of the Code of Federal Regulations. We note that an ingredient that is lawfully added to food products may be used in a standardized food only if it is permitted by the applicable standard of identity.
Section 301 (ll) of the Federal Food, Drug, and Cosmetic Act (FFDCA)
Section 301(ll) of the FFDCA prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FFDCA, 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 Amano’s notice that the protein glutaminase enzyme preparation is GRAS for use in various foods, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the protein glutaminase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the protein glutaminase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Amano, as well as other information available to FDA, the agency has no questions at this time regarding Amano’s conclusion that the protein glutaminase 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 the protein glutaminase enzyme 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. In accordance with proposed 21 CFR 170.36(f), a copy of the text of this letter responding to GRN 000267, 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 via the FDA home page at http://www.fda.gov. To view or obtain an electronic copy of the text of the letter, follow the hyperlinks from the “Food” topic to the “Food Ingredients and Packaging” section to the “Generally Recognized as Safe (GRAS)” page where the GRAS Inventory is listed.
Laura M. Tarantino, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
1The FDA calculations of the mean and 90th percentile intake of proteins based on a 2000 calorie diet are 75.5 g/p/d and 119.3 g/p/d, respectively. Using the 90th percentile value, FDA calculated the estimated maximum daily intake of protein glutaminase to be 392 mg/p/d (equivalent to 13.2 mg TOS/p/d) at the intended use levels of the enzyme preparation.