Food

Agency Response Letter GRAS Notice No. GRN 000649

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CFSAN/Office of Food Additive Safety

November 28, 2016

Richard F. Mann
Keller and Heckman LLP
1001 G St. NW, Suite 500 West
Washington, DC 20001

Re: GRAS Notice No. GRN 000649

Dear Mr. Mann:

The Food and Drug Administration (FDA, we) completed our evaluation of GRN 000649. We received the notice, dated April 25, 2016, that you submitted on behalf of GenoFocus, Inc. (GenoFocus), 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) on April 26, 2016, and filed it on May 25, 2016. We received amendments containing clarifying information on September 30, 2016, and October 31, 2016.

FDA published the GRAS final rule on August 17, 2016 (81 FR 54960), with an effective date of October 17, 2016. As GRN 000657 was pending on the effective date of the GRAS final rule, we requested some additional information consistent with the format and requirements of the final rule. We received an amendment responding to this request on November 1, 2016.

The subject of the notice is beta-galactosidase II enzyme preparation produced by Bacillus subtilis carrying a beta-galactosidase II gene from B. circulans (beta-galactosidase II enzyme preparation). The notice informs FDA of the view of GenoFocus that beta-galactosidase II enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the production of galacto-oligosaccharides (GOS) at levels up to 0.3% of the lactose starting material.

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 components derived from the manufacturing process, e.g., constituents of the fermentation media or the residues of processing aids. GenoFocus’ notice provides information about each of these components in the beta-galactosidase II enzyme preparation.

According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, beta-galactosidase II is identified by the Enzyme Commission Number 3.2.1.23. The accepted name for the enzyme is beta-galactosidase; and the systematic name is beta-D-galactoside galactohydrolase. This enzyme is also known as lactase; β-lactosidase; maxilact; hydrolact; β-D-lactosidase; S 2107; lactozym; trilactase; β-D-galactanase; oryzatym; sumiklat. The CAS Registry Number for beta-galactosidase is 9031-11-2. Beta-galactosidase II enzyme is a hydrolase that catalyzes the transfer of non-reducing beta-D- galactose residues in beta-D-galactosides (such as lactose) to water. Under specific processing conditions (e.g., high lactose concentration) beta-galactosidase II enzyme can utilize lactose as an alternative acceptor to form GOS. GenoFocus has calculated the molecular mass of beta- galactosidase to be 155977.22 Da based on its primary amino acid sequence.

GenoFocus describes the construction of the B. subtilis production strain that contains the beta- galactosidase II gene (bgaII) from a B. circulans donor strain (ATCC31382); the donor strain was isolated from soil. The beta-galactosidase expression module consisted of a promoter, mRNA stabilizing element, and the coding sequence of codon-optimized bgaII gene; the components of the expression module along with a homologous pair of B. subtilis thrC genes were cloned into an E. coli B. subtilis shuttle plasmid that was transformed into the B. subtilis recipient strain (ATCC 6051). In addition to this insertion, several genes encoding extracellular proteases and sporulation were deleted. GenoFocus states that the introduced genetic material is commonly used in molecular biology and well documented in scientific literature. GenoFocus confirmed the insertion and deletion sites, the absence of antibiotic resistance genes, and the integrity of the expression module by PCR analysis. GenoFocus confirmed that the production strain with the introduced and deleted genetic sequences is stable for up to 50 generations, via PCR analysis. GenoFocus states that both the recipient and donor organisms are nonpathogenic and nontoxigenic, and widely used in the safe production of food-grade enzymes.

GenoFocus states that the beta-galactosidase II enzyme preparation is produced by a controlled fermentation of a selected pure culture of the production strain. The manufacture of beta- galactosidase II enzyme preparation includes fermentation, processing, and formulation of the final product. Appropriate measures are set in place to control identity, purity, and enzyme- generating ability of the production strain during and after fermentation. The produced enzyme is separated from the production organism by centrifugation, followed by microfiltration. The ensuing filtrate is subject to ultrafiltration. The enzyme concentrate is then freeze-dried prior to formulation with food-grade dextrin. According to GenoFocus, the raw materials used in the fermentation, recovery, and formulation processes are food grade. The entire process is performed in accordance with good manufacturing practice. GenoFocus also states that the manufacturing process will remove any unused fermentation components from the final product, and that the beta-galactosidase II enzyme preparation will not contain any production organism or any major food allergens from the manufacturing process.

GenoFocus states that the beta-galactosidase II enzyme preparation conforms to the specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 10th edition, 2016), 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). GenoFocus provides data from five non-consecutive batches of beta-galactosidase II enzyme preparation to demonstrate that the manufacturing process conforms to set specifications.

GenoFocus intends to use beta-galactosidase II enzyme preparation at levels up to 0.3% of the lactose starting material. GOS may be used in a variety of food applications, including products consumed by children <1 year of age. [1] GenoFocus does not expect any dietary exposure to the beta-galactosidase II enzyme preparation from the intended food uses of GOS based on the steps employed in the manufacture of GOS, which include use of a 20 kDa ultrafiltration membrane to remove any residual enzyme. In addition, GenoFocus provides analytical data from a quantitative colorimetric method demonstrating that the residual enzyme in the finished GOS product is less than 1 milligram per kilogram, the limit of detection of the method. If any active beta- galactosidase enzyme preparation remains in the final food, GenoFocus states that any hydrolysis products occurring in food due to the presence of residual beta-galactosidase II would already be expected to be part of the human diet.

GenoFocus relies on published information for the safety of microbial enzyme preparations used in food processing and summarizes toxicity assessment of the beta-galactosidase II enzyme based on bioinformatics analyses. GenoFocus reports the absence of sequence homology between glucoamylase and known toxins on the publically available database, Toxin and Toxin Target Database, T3DB. GenoFocus also found no homology between the predicted peptide sequences of beta-glucosidase II and any known toxic peptides in the publically available database; Toxin Pred. GenoFocus used the publically available bioinformatics tool PeptideCutter to obtain the predicted peptide fragments formed during digestion by major proteases present in the human gastrointestinal tract. Additionally, search results showed no homology of the amino acid sequence of beta-galactosidase II enzyme with any known toxic peptides. This data supports that the intact enzyme and its metabolized products are not expected to be toxigenic, in case of the possibility of only partial digestion of protein by newborn babies as a result of their immature digestive systems.

GenoFocus discusses potential food allergenicity of beta-galactosidase II enzyme preparation. GenoFocus did not find any positive match over an 80-amino acid sequence homology search comparing the sequence of beta-galactosidase II enzyme against sequences of known allergens stored in the Food Allergy Research and Resource Program database. GenoFocus also performed a sequence identity search over a contiguous stretch of 8 amino acids, to amino acid sequences of known allergens, and did not observe any sequence matches. Based on the totality of the information available, GenoFocus concludes that it is unlikely that oral consumption of beta- galactosidase II enzyme will result in allergenic responses.

Based on the data and information summarized above, GenoFocus concludes that the beta- galactosidase II 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 our evaluation of GenoFocus’ notice concluding that beta glucosidase II enzyme preparation is GRAS under its intended conditions of use, we did not consider whether section 301(ll) or any of its exemptions apply to foods containing beta glucosidase II enzyme preparation. Accordingly, our response should not be construed to be a statement that foods containing beta glucosidase II enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).

Conclusions

Based on the information that GenoFocus provided, as well as other information available to FDA, we have no questions at this time regarding GenoFocus’ conclusion that beta-galactosidase II enzyme preparation produced in B. subtilis carrying a beta-galactosidase II gene from B. circulans is GRAS under its intended conditions of use. This letter is not an affirmation that beta-galactosidase II enzyme preparation produced in B. subtilis carrying a beta-galactosidase II gene from B. circulans is GRAS under 21 CFR 170.35. Unless noted above, our review did not address other provisions of the FD&C Act. Food ingredient manufacturers and food producers are responsible for ensuring that marketed products are safe and compliant with all applicable legal and regulatory requirements.

In accordance with 21 CFR 170.275(b)(2), the text of this letter responding to GRN 000649 is accessible to the public at www.fda.gov/grasnoticeinventory.

Sincerely,

Dennis M. Keefe, Ph.D. Director
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition

[1] GenoFocus incorporates uses of GOS from GRN 000236.

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