USOO5695973A United States Patent (19) 11 Patent Number: 5,695,973 Subbiah 45 Date of Patent: Dec. 9, 1997 54 ISOLATED ALCOHOLDEHYDROGENASE T.W. Pfeiffer et al., "Inheritance of a Lipoxygenase-1 Alloz PRODUCING MIOLD yme in Soybean.” Crop.Sci. 33,691-693 (1993). A. Hatanaka et al., “Distribution of and Enzyme System 75 Inventor: Wen Subbiah, Edenton, N.C. Producing cis-3-Hexenal and n-Hexenal From Linoleic and 73 Assignee: R.J. Reynolds Tobacco Company, Linoleic Acids in Some Plants.” Phytochemistry 17, Winston-Salem, N.C. 869-872 (1978). A. Hatanaka et al., "Fatty Acid Hydroperoxide Lyase in 21 Appl. No.: 674,920 Plant Tissues,” in American Chemical Society, Biogenera tion of Aromas, 167-75 (1986). 22 Filed: Jul. 3, 1996 A. Hatanaka, "Biosynthesis of Leaf Alcohol.” Bull. Inst. 1511 Int. Cl. .............. C12P 7/02; C12N 1/00 Chem. Res., 61(2) 180-192 (1983). 52 U.S. Cl. .......................... 435/155; 435/134; 435/911; J. Sekiya et al., “Distribution of Lipoxygenase and Hydro 435/254.1; 426/52 peroxide Lyase in the Leaves of Various Plant Species.” 58) Field of Search ................................. 435/155, 134, Phytochemistry 22(9), 1867-1869 (1983). 435/911, 254.1; 426/52 E. Gotz-Schmidt et al., "CVolatiles in Homogenates from Green Leaves: Localization of Hydroperoxide Lyase Activ 56 References Cited ity.” Lebensm-Wiss Technol. 19(2), 152-155 (1986). U.S. PATENT DOCUMENTS A.J. McLeod et al., "Formation of (E)-Hex-2-enal and 3,839,457 10/1974 Jouffret ................................... 260601 (Z)-Hex-3-en-1-ol by Fresh Leaves of Brassica olera 3.962,354 6/1976 Dahil, Jr. ........ ... 260/638 cea,” J. Agric. Food. Chem. 27(3), 469–75 (1979). 4,769.243 9/1988 Kanisawa et al. ........................ 426/33 R. Saijo et al., “Increase of Cis-3-Hexen-1-ol Content in 4,806,379 2/1989 Goers et al. ............ ... 42.67650 Tea Leaves Following Mechanical Injury.” Phytochemistry 5,017,386 5/1991 Hildebrand et al. ...................... 426/18 5,464,761 11/1995 Muller et al. ........................... 435/47 14, 181-182 (1975). E.J. Stone et al. "Formation of Aldehydes and Alcohols in FOREIGN PATENT DOCUMENTS Tomato Fruit From U-'C-Labeled Linolenic and Linoleic 89 1290 4f1991 France. Acids.” J. Food. Sci. 40, 1138-1141 (1978). 93(24644 12/1993 WIPO. A. Hatanaka et al., "Alcohol Dehydrogenase from Thea OTHER PUBLICATIONS sinensis Seeds.” Agr: Biol. Chem, 38(10), 1835-1844 (1974). Pelczar et al., Fungi: the Molds, In: Microbiology, 4th A. Hatanaka et al., “Formation of Cis-3-Hexenal, edition, McGraw-Hill Book Company, pp. 285-301, 1977. Trans-2-Hexenal and Cis-3-Hexenal In Macerated Thea Charton et al., Specifities of immobilized Geotrichum can sinensis Leaves,” Phytochemistry 12, 2341-2346 (1973). didum CMICC 335426 lipases A and B in hydrolysis and ester sysnthesis reactions in organic solvents, Enzyme A. Hatanaka et al., “Purification and Properties of Alcohol Microb. Technol., vol. 15, pp. 489-493, 1993. Dehydrogenase from Tea Seeds,” Agr. Biol. Chem, 36(11), H.W. Gardner, "Flavors and Bitter Tastes from Oxidation of 2033-2035 (1972). Lipids by Enzymes,” in Flavor Chemistry of Fats and Oils, A. Hatanaka et al., “Phytochemical somerization of Leaf 189–206 (eds. D.B. Min and T.H. Smouse, 1985). Aldehyde,” Agr: Biol. Chem, 36(7), 1263-1264 (1972). J. Sekiya et al., "Seasonal Changes in Activities of Enzymes Responsible for the Formation of Caldehydes and R.T. Major et al., “Formation of 2-Hexenal From Linolenic C-alcohols in Tea Leaves, and the Effects of Environmental Acid By Macerated Ginkgo Leaves," Phytochemistry 11, Temperatures on the Enzyme Activities." Plant Cell. 611-617 (1972). Physiol. 25(2)269-280 (1984). Sigma Catalog, p. 74 (1992). Primary Examiner-Leon B. Lankford, Jr. The New Royal Horticultural Society Dictionary of Garden Assistant Examiner-Christopher R. Tate ing vol. 1, 144-145 (ed. A. Huxley, 1992). Attorney, Agent, or Firm-Myers Bigel Sibley & Sajovec, J.M. Olias et al., “Aroma of Virgin Olive Oil: Biogenesis of LLP the Green Odor Notes,” J. Agric. Food Chem, 41, 57 ABSTRACT 2368-2373 (1993). D.R. Phillips et al., “Partial Purification and Properties of a The present invention relates to a microorganism capable of Cis-3:Trans-2-Enal somerase From Cucumber Fruit,” producing alcohol dehydrogenase (ADH). The microorgan Phytochemistry 18, 401–404 (1979). ism of the invention is a non-yeast mold obtained from the J. Sekiya et al., "Cis-3-Hexenal and n-Hexanal Formation kale plant. In a preferred embodiment of the invention, the From Linolenic And Linoleic Acids in Alfalfa Cells Cultured mold is Geotrichium candidian M 369326. The microor In Vitro,” Plant Science Letters 19, 165-169 (1977). ganism of the invention is particularly useful in the green B.O. de Lumen et al., “Formation of Volatile Flavor Com note processing industry as a substitute for yeast. For pounds In Green Beans From Linoleic Acid and Linolenic example, the microorganism is useful as a substitute for Acids,” J. Food Sci. 43(3), 698-702 (1978). yeast in a process of converting a leaf aldehyde to a leaf J. Sekiya et al., “Fatty Acid Hydroperoxide Lyase in alcohol. Tobacco Celis Cultured In Vitro,” Phytochemistry, 23(11), 2439-2443 (1984). 11 Claims, No Drawings 5,695,973 1. 2 SOLATED ALCOHOLDEHYDROGENASE alcohol dehydrogenase (ADH), instead of live yeast. PRODUCING MOLD Accordingly, the present invention provides a non-yeast mold microorganism that is capable of producing alcohol FIELD OF THE INVENTION dehydrogenase (ADH). In one preferred embodiment, the 5 moldis obtained from the kale plant, and in a more preferred The present invention relates to an isolated embodiment, is the strain Geotrichium candidum IMI microorganism, and more specifically to an isolated mold 369326. useful in producing green note compounds. The present invention also provides a process for the BACKGROUND OF THE INVENTION conversion of a leaf aldehyde to a leaf alcohol, comprising 10 contacting the leaf aldehyde with a microorganism capable Leaf alcohol (i.e., cis-3-hexen-1-ol) and leaf aldehyde of producing alcohol dehydrogenase, whereby the microor (i.e., trans-2-hexenal) are present in a wide variety of fresh ganism is a non-yeast mold derived from the kale plant, and leaves, vegetables and fruits. These compounds are respon under conditions sufficient to support the conversion reac sible for a so-called "green aroma," "green odor," "fresh tion. In one preferred embodiment of the present invention, note,” or "green note.” Related compounds present in many 15 the microorganism is derived from the kale plant, and in a plants include trans-2-hexenol, cis-2-hexenol, trans-3- more preferred embodiment, is the strain Geotrichium can hexenol, 1-hexenol, 1-hexenal, and cis-3-hexenal. Green did in IMI 369326. note compounds and uses thereof are set forth by Morris, The present invention also relates to a method for pro Perfumer & Flavorist, Vol. 6, No. 1 (1981) and Clark, viding green note compounds. The process, in one aspect, Perfumer and Flavorist, Vol. 15 (1990). Green note com 20 involves the steps of providing at least one unsaturated fatty pounds exhibit organoleptic characteristics which can be acid, plant biomass having active levels of lipoxygenase and characterized as fresh or grassy in nature. Such compounds hydroperoxide lyase enzymes, and active alcohol dehydro can be used to sharpen and enhance flavored products, such genase in the form of a non-yeast mold capable of producing as those products having fruit flavors. alcoholdehydrogenase. These components are provided as a Green note compounds have been isolated from plants or 25 mixture by simultaneously contacting the fatty acid, plant chemically synthesized, as reported by Bedoukian, Amer: biomass and alcohol dehydrogenase in the presence of an Perf. 78, 31 (1963) and U.S. Pat. No. 3,839,457 to Jouffret. aqueous liquid under conditions sufficient to release lipoxy Green note compounds are present in plant essential oils genase and hydroperoxide lyase from the plantbiomass, and (e.g., sage, citrus, and mint) and have been obtained by allow reaction of the fatty acid with the lipoxygenase, steam distillation of plant material followed by fractional 30 hydroperoxide lyase and alcohol dehydrogenase to provide distillation or "topping” techniques. Green note compounds green note compounds. After the mixture is reacted, the have also been biosynthetically produced, as reported by aqueous phase containing green note compound is collected Muller et al., Ady, Flay. Res. Tech. Symposium (1993); and the green note compound separated from the aqueous Hatanaka et al., Phytochem. 17,869 (1978); Lumen et al., J. phase. Food Sci., 43, 698 (1978); and U.S. Pat. Nos. 4.769.243 to 35 The plant biomass (i.e. plant material), unsaturated fatty Kaniswa et al. and 4,806,379 to Goers et al. acid and alcohol, dehydrogenase are combined in the pres One green note-producing biosynthetic reaction involves ence of an aqueous liquid. The ingredients are most prefer the unsaturated C-6 hydroperoxydismutation of linolenic ably contacted with one another in an essentially simulta acid and is carried out using a series of enzymatic steps. In neous fashion; or the plant material is combined with a particular, lipoxygenase forms a hydroperoxide moiety at a mixture of unsaturated fatty acid, a non-yeast mold that double bond of a fatty acid (e.g. linolenic acid). The enzyme produces ADH, and water. Thus, the unsaturated fatty acid hydroperoxide lyase cleaves the hydroperoxide to produce a is converted to a green note compound. That is, the lipoxy C-6 unsaturated aldehyde, in particular, cis-3-hexen-1-al. genase and fatty acid interact to form a fatty acid Aldehyde isomerase, when present in the plant material and hydroperoxide, which is cleaved by the action of the hydro under certain conditions, catalyzes the formation of trans 45 peroxide lyasetoforman aldehyde, which is converted to an 2-hexenal from the cis-3-hexen-1-al.