U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
Office of Food Additive Safety
February 7, 2002

Mr. David R. Joy
Keller and Heckman, LLP
1001 G St., N.W.
Suite 500 West
Washington, DC 20001

Re: GRAS Notice No. GRN 000081

Dear Mr. Joy:

The Food and Drug Administration (FDA) is responding to the notice, dated August 31, 2001, that you submitted on behalf of Amano Enzymes, 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 September 5, 2001, and designated it as GRAS Notice No. GRN 000081.

The subject of the notice is lipase enzyme preparation from Candida rugosa. The notice informs FDA of the view of Amano that this lipase enzyme preparation is GRAS, through scientific procedures, for use on fats and oils to produce fatty acids or glycerides at a level of up to one percent of the weight of the fat or oil, or for use in the esterification of fatty acids to produce glycerides at a level of up to one percent of the weight of the fatty acids. Amano estimates the dietary exposure to this enzyme preparation from its intended use would be a maximum of 4.5 milligrams per person per day.

Commercial enzyme preparations that are used in food processing 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 that derive from the source organism and constituents that derive from the manufacturing process, e.g., components of the fermentation media or the residues of processing aids. Amano’s notice provides information about each of these components of lipase enzyme preparation from C. rugosa.

Amano describes published and unpublished information about the technical effects of lipases, which catalyze hydrolysis, esterification, and transesterification of esters formed from glycerol or substituted glycerol and long-chain fatty acids. Lipases often can catalyze these reactions on a broad range of substrates, including phospholipids and other compounds, often selectively for specific positions and chain lengths of the acyl groups. Thermal factors, pH stability and dependence, and other physical factors may also differentiate these enzymes. They are employed in a number of industrial applications, and often are used in the cheese industry.

Amano describes generally available information about lipases from C. rugosa, indicating that C. rugosa produces at least 5 lipase isoenzymes. Lipase enzyme preparations from C. rugosa have been used in Japan and the United States to prepare flavoring ingredients from milk for about twenty years. A published review discusses the uses of lipases from C. rugosa in production of fats and oils, fermented foods, ice cream, pharmaceuticals and cosmetics.

Amano describes the manufacturing process for lipase enzyme preparation, which is produced by a controlled fermentation of the C. rugosa strain with conditions specified for time, temperature, aeration, and rate of rotation of the culture. Amano uses food-grade ingredients in the fermentation medium. The production strain secretes the enzyme into the medium. Amano separates the enzyme from the yeast cells by filtration (using a press filter), followed by concentration by ultrafiltration (MW 6000), and additional filtration (press filter and ceramic filter). The remaining protein is then precipitated from the supernatant with the addition of ethanol. The precipitated protein is centrifuged, dried (under vacuum), crushed, sieved, and mixed, then blended with a diluent to the desired enzyme activity for the finished product. To dilute the enzyme preparation to its final activity, Amano uses dextrin, which FDA has affirmed as GRAS (21 CFR 184.1277). The final enzyme preparation is sieved through 42 mesh screen.

Amano provides specifications for lipase enzyme preparation from C. rugosa, which comply with the general and additional requirements for enzyme preparations provided in the Food Chemicals Codex (4th edition, 1996; FCC IV). Amano notes that FCC IV includes a specific listing for lipase enzyme preparation from C. rugosa and that its lipase enzyme preparation is consistent with that listing.

Amano describes published information pertaining to the safety of the production strain, the yeast C. rugosa. Amano describes a published study in which mice were inoculated with fifteen million viable spores of C. rugosa. Because C. rugosa cells were not recovered from the mice and apparent adverse effects were not observed, Amano concludes that the mouse immunological system rapidly inactivated C. rugosa spores. Amano also discusses published reports regarding opportunistic infections in immunologically compromised patients and in the contamination of a catheter. Amano concludes that such opportunistic infections seldom occur and are not inconsistent with the safety of the microorganism as a source of food ingredients.

Amano includes a published article that describes studies performed with lipase enzyme preparation from C. rugosa. These studies include a 14-day oral toxicity study conducted in rats and mice, a thirteen-week oral gavage study in rats, a bacterial reverse mutation study and an in vitro chromosome aberration test. Amano found no adverse effects at any level tested in these studies.

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 lipase enzyme preparation from C. rugosa 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 lipase enzyme preparation from C. rugosa. 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, as well as a copy of the information in the notice that conforms to the information in proposed 21 CFR 170.36(c)(1), is available for public review and copying on the homepage of the Office of Food Additive Safety (on the Internet at http://www.cfsan.fda.gov/~lrd/foodadd.html).

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