Agency Response Letter GRAS Notice No. GRN 000587
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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
May 17, 2016
77 Perry Chapel Church Rd
P. O. Box 576
Franklinton NC 27525
Re: GRAS Notice No. GRN 000587
Dear Ms. Oesterling:
This letter corrects our letter dated September 08, 2015, sent in response to GRAS Notice No. GRN 000587. The purpose of this revised letter is to correct information regarding the margin of safety calculations in Footnote 4, reported in our previous letter.
The Food and Drug Administration (FDA) is responding to the notice, dated June 30, 2015, that you submitted 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 July 8, 2015, filed it on July 22, 2015, and designated it as GRAS Notice No. GRN 000587.
The subject of the notice is acetolactate decarboxylase produced by Bacillus licheniformis carrying an acetolactate decarboxylase gene from Bacillus brevis (acetolactate decarboxylase enzyme preparation). The notice informs FDA of the view of Novozymes North America Inc. (Novozymes) that acetolactate decarboxylase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the processing of beer up to levels of 0.133 milligrams total organic solids per liter (mg TOS/L) of fermented wort.
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 components derived from the production organism and from the manufacturing process, e.g., constituents of the fermentation media or the residues of processing aids. Novozymes’ notice provides information about the components in the acetolactate decarboxylase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, acetolactate decarboxylase is identified by the Enzyme Commission Number 18.104.22.168. The accepted name for the enzyme is acetolactate decarboxylase, and the systematic name is (2S)- 2-hydroxy-2-methyl-3-oxobutanoate carboxy-lyase [(3R)-3-hydroxybutan-2-one-forming]. Other common names include a-acetolactate decarboxylase; (S)-2-hydroxy-2-methyl-3-oxobutanoate carboxy- lyase; (S)-2-hydroxy-2-methyl-3-oxobutanoate carboxy-lyase [(R)-2-acetoin-forming]. The CAS Registry Number for acetolactate decarboxylase is 9025-02-9. Acetolactate decarboxylase catalyzes the hydrolysis of (2S))-2-hydroxy-2-methyl-3-oxobutanoate to (3R)-3-hydroxybutan-2-one, with the production of CO2; i. e., it catalyzes the decarboxylation of alpha-acetolactate to acetoin.
Novozymes states that the B. licheniformis production strain is obtained from the recipient strain B. licheniformis AEB1763. AEB1763 was modified at several chromosomal loci to delete genes encoding anumber of proteases, a gene essential for sporulation, and genes encoding certain proteins to be expressed that can affect product purity. Novozymes describes B. licheniformis as a non-pathogenic, non- toxigenic, well-characterized production organism, with a history of safe industrial use (GRASP 3G0026, GRASP 0G0363, GRAS Notice No. GRAS Notice No. 22, GRAS Notice No. GRN 24, GRAS Notice No. GRN 72, GRAS Notice No. GRN 79, and GRAS Notice No. GRN 472)
Novozymes describes the construction of the production strain using the plasmid pJA3860 for the transformation of B. licheniformis AEB1763. Plasmid pJA3860 contains an expression cassette consisting of a fragment of a hybrid bacillus promoter, the acetolactate decarboxylase coding sequence, and a transcriptional terminator sequence. The presence of a non-coding DNA sequence enables the targeted integration of the expression cassette into the genome of the recipient strain. Novozymes confirms the presence of the introduced DNA via Southern blot analysis, PCR, and DNA sequencing. Novozymes also confirmed the transformation, stability, and poor mobility of the introduced DNA, using Southern hybridization.
Novozymes states that acetolactate decarboxylase enzyme is produced by the submerged fed-batch pure culture fermentation of the production strain. The fermentation is carried out under controlled conditions and the culture is periodically tested until the desired enzyme production is achieved. During fermentation, acetolactate decarboxylase enzyme is secreted to the fermentation broth. After fermentation, the enzyme is recovered from the fermentation broth by adjusting the pH, prior to filtration or centrifugation; this is followed by ultrafiltration and/or evaporation to concentrate the enzyme. The concentrated liquid enzyme is filtered, and then stabilized by the addition of glycerol. This is followed by the addition of potassium sorbate as a preservative, and water, as the standardizing agent. Novozymes states that the entire process is performed in accordance with current good manufacturing practices using raw materials of food grade quality. Novozymes also states that the final enzyme preparation contains no major food allergens from the fermentation medium.
Novozymes has established food grade specifications and notes that the acetolactate decarboxylase enzyme preparation conforms to specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2014), 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). Novozymes provides analytical data from three batches of acetolactate decarboxylase enzyme preparation to demonstrate consistency with the set specifications. Novozymes confirms the absence of functional antibiotic resistance genes, DNA encoding transferable antibiotic resistance genes, and the presence of any production organism in the final enzyme preparation.
Novozymes proposes to use acetolactate decarboxylase enzyme preparation as an enzyme in brewing alcoholic and non-alcoholic beverages during fermentation, in order to reduce the formation of diacetyl, and to decrease the length of maturation time. Novozymes proposes to use acetolactate decarboxylase at up to 0.133 mg TOS/L of fermentation wort. Novozymes notes that acetolactate decarboxylase will not be present in the final food as it will be removed and/or denatured during the processing. Novozymes also states that acetolactate decarboxylase, if present in the final food, will be digested like all other proteins in the gastrointestinal system. However, in order to estimate dietary exposure to acetolactate decarboxylase enzyme preparation, Novozymes assumes that the enzyme preparation will be used at the maximum intended use level, and that all of the enzyme preparation will remain in the final food. Novozymes estimates an intake of acetolactate decarboxylase enzyme preparation from all intended uses to be 0.0008 mg TOS per kg bodyweight per day (mg TOS/kg bw/d) based on the mean consumption of alcoholic beverages by adults 21 years and older, that was obtained from 2003-2006 NHANES, National Health and Nutrition Examination Survey.
Novozymes summarizes corroborative toxicological studies using the acetolactate decarboxylase liquid enzyme concentrate to support the safety of the enzyme for the proposed uses. Tests conducted using bacterial cells showed that acetolactate decarboxylase did not induce gene mutations, both in the presence and absence of S-9 mix; i. e., it is not mutagenic. Novozymes also demonstrates that the enzyme is not clastogenic based on in vitro micronucleus testing in human lymphocytes. A 13-week sub-chronic oral toxicity study (90-day study) conducted in rats showed that consumption of acetolactate decarboxylase enzyme liquid concentrate did not cause any treatment-related adverse effects up to the highest dose tested, i.e., 1018 mg TOS/kg bw/d. Based on the highest dose tested in the 90-day study, and the estimated daily intake from the proposed use levels of acetolactate decarboxylase enzyme preparation, Novozymes calculates a margin of safety to be approximately 1260000.
Novozymes discusses potential food allergenicity of acetolactate decarboxylase enzyme. Novozymes conducted an 80-amino acid sequence homology search for acetolactate decarboxylase enzyme against known allergens stored in the Food Allergy Research and Resource Program, FARRP, database, and the World Health Organization and International Union of Immunological Societies, WHO/IUIS, Allergen Nomenclature Sub-committee allergen protein database. Novozymes identified no significant sequence homology between acetolactate decarboxylase and any of the allergens in these databases. Additionally, no matches of contiguous stretches of eight amino acids were observed. Novozymes further cites the conclusions of several organizations and working groups about the low risk of allergenicity posed by enzymes due to their low use levels and extensive processing of the enzyme-containing foods during manufacturing. Based on the information available, Novozymes concludes that it is unlikely that oral consumption of acetolactate decarboxylase will result in any allergenic responses.
Based on the data and information summarized above, Novozymes concludes that acetolactate decarboxylase enzyme preparation is GRAS for its intended use.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FD&C Act)
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 Novozymes’ notice that acetolactate decarboxylase 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 acetolactate decarboxylase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain acetolactate decarboxylase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Standards of Identity
In the notice, Novozymes Inc. states its intention to use acetolactate decarboxylase enzyme preparation in 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.
Based on the information provided by Novozymes, as well as other information available to FDA, the agency has no questions at this time regarding Novozymes’s conclusion that acetolactate decarboxylase produced by B. licheniformis carrying an acetolactate decarboxylase gene from B. brevis is GRAS under the intended of use. The agency has not, however, made its own determination regarding the GRAS status of the subject use of acetolactate decarboxylase produced by B. licheniformis carrying an acetolactate decarboxylase gene from B. brevis. As always, it is the continuing responsibility of Novozymes North America Inc. 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 00 0587, 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
 B. licheniformis AEB1763 is a natural isolate of B. licheniformis strain DSM 9552. DSM 9552 has been used to produce B. licheniformis strains that have been shown to be safe for industrial production of enzymes marketed and sold by Novozymes.
 The hybrid promoter contains promoter elements from B. licheniformis, B. amyloliquefaciens and B. thuringiensis.
 FDA calculated a mean and 90th percentile consumption of alcoholic beverages in the United States to be 6.6 g/kg bw/d and 15.8 g/kg bw/d, respectively, using NHANES 2003-12 combined 2-day consumption data. FDA calculated the estimated daily intake of acetolactate decarboxylase to be 0.002 mg/kg-bw/d, based on the 90th percentile consumption of alcoholic beverages.
 Based on the estimated daily intake calculated by FDA, the margin of safety using the NOAEL from the 90-day study would be approximately 510000.