Agency Response Letter
GRAS Notice No. GRN 000120

Robert Biwersi
Lesaffre Yeast Corporation
433 East Michigan Street
Milwaukee, WI 53202

Re: GRAS Notice No. GRN 000120

Dear Mr. Biwersi:

The Food and Drug Administration (FDA) is responding to the notice, dated January 2, 2003, 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 January 7, 2003, filed it on January 10, 2003, and designated it as GRAS Notice No. GRN 000120.

The subject of the notice is Saccharomyces cerevisiae strain ML01 (S. cerevisiae strain ML01) carrying a gene encoding malolactic enzyme from Oenococcus oeni and a gene encoding malate permease from Schizosaccharomyces pombe. The notice informs FDA of the view of Lesaffre Yeast Corporation (Lesaffre) that S. cerevisiae strain ML01 is GRAS, through scientific procedures, for use in winemaking as a yeast starter culture for grape must fermentation. Lesaffre recommends using between 0.1 to 0.2 grams of active dry yeast per liter of wine.

Lesaffre describes generally available information about traditional manufacturing processes for the production of wine from grapes. These processes include the harvesting, de-stemming and crushing of grapes (resulting in must), the separation of the juice from the skins and seeds, one or more distinct types of microbial fermentation, clarification, stabilization, and bottling. Winemakers may vary the sequence of operational steps or modify procedures, depending upon the desired characteristics and nature of the wine.

Lesaffre describes generally available information about two distinct fermentation processes (i.e., alcoholic fermentation and malolactic fermentation) that occur either through the action of microorganisms that already are present on the grapes or through the action of microorganisms that are specifically added by the winemaker. Alcoholic fermentation (i.e., a process whereby the sugars glucose and fructose are converted to ethanol) is mediated by metabolic pathways associated with yeast (usually S. cerevisiae or closely related species). Malolactic fermentation (i.e., a process whereby the dicarboxylic acid malic acid is decarboxylated to the monocarboxylic acid lactic acid) is mediated by lactic acid bacteria through the combined action of a protein (called malate permease) that transports malic acid from the wine into the bacteria and an enzyme (called malolactic enzyme) that converts the malic acid to lactic acid. Because malolactic fermentation reduces the number of carboxylic acid groups on organic acids present in the wine, it reduces the acidity of the must. Although alcoholic fermentation is an inherent process associated with all winemaking, malolactic fermentation is a secondary process that may or may not be induced by the winemaker, depending on the desired characteristics and nature of the wine.

Lesaffre describes generally available information about clarification of wine, which can occur either at the end of the alcoholic fermentation or after the wine has been kept on the lees (the sediment formed by spent yeast cells and grape particulate matter). Wine clarification encompasses the removal of solid particles in the wine via gravity or centrifugation and subsequent elimination of the sediment or pellet. When clarification occurs at the end of fermentation, the clarification process removes most yeast cells. When the wine is kept on the lees before clarification, the yeast cells undergo autolysis, which releases cellular material that ultimately is degraded through the action of enzymes such as proteases.

Lesaffre describes generally available information about stabilization processes, which differ depending on whether the wine is a white wine or a red wine. For white wine, stabilization involves removing proteins via filtration with bentonite. For red wine, stabilization involves adding gelatin or egg white albumin to precipitate colloidal structures that include tannin-protein complexes. Prior to bottling, most wines undergo filtration (e.g., with diatomaceous earth, cellulose filters, or membrane filters) that eliminates any remaining yeast cells.

Lesaffre describes published articles about bioengineered strains of S. cerevisiae, including strains of S. cerevisiae that have been modified to conduct malolactic fermentation. Lesaffre notes that the use of bioengineered strains that can conduct both alcoholic and malolactic fermentation eliminates the need for separate additions of two distinct microorganisms (i.e., yeast and lactic acid bacteria).

Lesaffre describes the development of its own bioengineered strain of S. cerevisiae. The host strain, S. cerevisiae strain S92, was isolated from the Champagne region in France and is closely related or identical to commercial strains commonly used in winemaking. The microbial source of malate permease (i.e., Schizosaccharomyces pombe), is a yeast⁽¹⁾ that was first isolated from African beer and has frequently been found in sugar-containing products in tropical and sub-tropical regions and in grape must and cider in moderate climates. The microbial source of malolactic enzyme (i.e., Oenococcus oeni) is a lactic acid bacterium that has been isolated from wines and related habitats such as wineries and vineyards. It is the preferred, and most commonly used, lactic acid bacterium for malolactic fermentation of wines. The malate permease is a 49 kDa protein with a hydrophobicity profile typical of membrane transport proteins. It contains a peptide sequence (composed of proline, glutamic acid, serine and threonine) that characterizes proteins with a rapid turnover. The malolactic enzyme is a dimer with a total molecular weight of approximately 130 kDa.

Lesaffre describes the construction of an integration cassette that contains genes encoding malate permease from S. pombe and the malolactic enzyme from O. oeni, regulatory sequences associated with the expression of these genes, and sequences used for integration into an appropriate chromosomal site in S. cerevisiae strain S92. Lesaffre also describes the transformation strategy that it used to reduce the numbers of potentially transformed yeasts that needed to be screened for the successful integration of the integration cassette. This strategy involved co-transformation of S. cerevisiae strain S92 with a plasmid (pUT322) that carries a selectable marker conferring resistance to the antibiotic phleomycin and was based on the hypothesis that cells transformed with plasmid pUT322 are more likely to also have been transformed with the integration cassette. Using this strategy, Lesaffre first screened transformed yeast for resistance to phleomycin and then screened the selected phleomycin-resistant yeast for the ability to produce lactic acid. Lesaffre obtained a phleomycin-sensitive isolate and confirmed that it is free of plasmid pUT332 sequences. Lesaffre designated this strain as ML01.

Based on DNA analysis, Lesaffre concluded that the chromosomal patterns of S. cerevisiae strains S92 and ML01 are the same except for the presence of the integration cassette. Lesaffre found that the integration cassette remained stably incorporated after 100 generations. Lesaffre also found that S. cerevisiae strain ML01 functions as intended in that it efficiently degrades malic acid. Based on studies that evaluated yeast physiology under different culture conditions, Lesaffre concluded that S. cerevisiae strain ML01 has the same growth kinetics, fermentation rate, and ethanol yield as S. cerevisiae strain S92 under winemaking conditions and that uptake and utilization of malic acid did not confer a growth advantage to S. cerevisiae strain ML01.

Lesaffre describes the method for routine production of S. cerevisiae strain ML01 and notes that this method is based on well-established procedures for the production of active dry yeast. The yeast is grown primarily under aerobic conditions to promote yeast propagation rather than alcohol production. The yeast is harvested via centrifugation and is subsequently dewatered with a rotary vacuum filter, processed through an extruder, and dried, resulting in active dry yeast. The yeast is packaged in vacuum foil pouches prior to shipping.

Lesaffre discusses potential dietary intake of S. cerevisiae strain ML01 and of the proteins that Lesaffre has introduced into that strain. Lesaffre considers that exposure to the yeast itself or to the newly introduced proteins would be negligible because the processing procedures used in winemaking remove intact yeast cells, debris associated with autolyzed yeast cells, and proteins released during autolysis of yeast cells.

Based on the information provided by Lesaffre, as well as other information available to FDA, the agency has no questions at this time regarding Lesaffre's conclusion that Saccharomyces cerevisiae strain ML01 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 S. cerevisiae strain ML01. As always, it is the continuing responsibility of Lesaffre 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).

Sincerely,    /s/ Laura M. Tarantino, Ph.D. Acting Director Office of Food Additive Safety Center for Food Safety      and Applied Nutrition

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⁽¹⁾Although malolactic fermentation is usually mediated by lactic acid bacteria, Lesaffre chose a yeast (rather than a lactic acid bacterium) as a source of the permease, because the permease must function in the membrane of the yeast S. cerevisiae.