Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1994-03-25
2001-08-14
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S155000, C424S774000, C424S725000, C426S650000, C426S655000
Reexamination Certificate
active
06274358
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to flavors and fragrances, and in particular to methods for providing flavorgenic alcohols and aldehydes using plant tissue.
Leaf alcohol (i.e., cis-3-hexen-1-ol) and leaf aldehyde (i.e., trans-2-hexenal) are present in a wide variety of fresh leaves, vegetables and fruits. These compounds are responsible for a so-called “green odor”, “green aroma”, “fresh note” or “green note”. Related compounds present in many plants include trans-2-hexenol, cis-2-hexenol, trans-3-hexenol, 1-hexanol, 1-hexanal and cis-3-hexenal. Green note compounds and uses thereof are set forth by Morris,
Perfumer
&
Flavorist
, Vol. 6, No. 1 (1981) and Clark,
Perfumer
&
Flavorist
, Vol. 15 (1990). Green note compounds exhibit organoleptic characteristics which can be characterized as fresh or grassy in nature. Such compounds can be used to sharpen and enhance flavored products, such as those products having fruit flavors.
Green note compounds have been isolated from plants or chemically synthesized, as reported by Bedoukian,
Amer. Perf
. Vol. 78, p. 31 (1963) and U.S. Pat. No. 3,839,457 to Jouffret. Green note compounds are present in plant essential oils (e.g., sage, citrus and mint), and have been obtained by steam distillation of plant material followed by fractional distillation or “topping” techniques. Green note compounds also have been biosynthetically produced, as reported by Muller et al,
Adv. Flav. Res. Tech. Symposium
(1993); Hatanaka el al,
Phytochem
., Vol. 17, p. 869 (1978); Hatanaka,
Bull. Inst. Chem. Res., Kyoto Univ
., Vol. 61, p. 180 (1983); Lumen et al.,
J.Food Sci
., Vol. 43, p. 698 (1978); Hatanaka et al.,
Phytochem
., Vol. 17, p. 869 (1978); Sekiya et al.,
Phytochem
., Vol. 23, p. 2439 (1984); Hatanaka et al.,
ACS Symposium Biogeneration of Aromas
(1985); French Patent Application No. 2,652,587; U.S. Pat. No. 4,769,243 to Kanisawa et al and U.S. Pat. No. 4,806,379 to Goers et al; and WO 93/24,644. The biosynthetic reaction involving the unsaturated C-6 hydroperoxydismutation of linolenic acid is carried out using a series of enzymatic steps. In particular, lipoxygenase forms a hydroperoxide moiety at a double bond of linolenic acid. The hydroperoxide lyase cleaves the hydroperoxide to produce a C-6 unsaturated aldehyde, in particular, cis-3-hexen-1-al. Then, aldehyde isomerase, when present in the plant material and under certain conditions, catalyzes the formation of a trans-2-hexenal from the cis-3-hexen-1-al. Cis-3-hexen-1-ol and other green note alcohols are formed by the action of alcohol dehydrogenase, which reduces the aldehydes to alcohols.
It would be highly desirable to provide an efficient and effective method for biosynthetically producing natural green note compounds, such as cis-3-hexen-1-ol. In particular, it would be highly desirable to provide green note compounds, such as shortchain saturated and unsaturated alcohols and aldehydes, which result from the enzymatic oxidation of a fatty acid substrate.
SUMMARY OF THE INVENTION
The present invention relates to a method for providing green note compounds. The process, in one aspect, involves the steps of:
a) providing unsaturated fatty acid, such as linolenic acid;
b) providing plant biomass, such as fresh watermelon foliage, having active levels of lipoxygenase and hydroperoxide lyase enzymes;
c) providing alcohol dehydrogenase, such as in the form of active yeast;
d) providing a mixture by contacting the plant biomass and unsaturated fatty acid in the presence of the alcohol dehydrogenase and an aqueous liquid under conditions sufficient to:
i) provide release of lipoxygenase and hydroperoxide lyase from the plant biomass, and
ii) provide reaction of the fatty acid with the lipoxygenase, hydroperoxide lyase and alcohol dehydrogenase to provide a green note compound;
e) collecting aqueous phase containing green note compound; and
f) separating green note compound from the aqueous phase.
The plant biomass (i.e., plant material), unsaturated fatty acid and alcohol dehydrogenase are combined in the presence of an aqueous liquid. The ingredients most preferably are contacted with one another in an essentially simultaneous fashion; or the plant material is combined with a mixture of unsaturated fatty acid, a source of alcohol dehydrogenase and water. In a highly preferred aspect, the ingredients are combined in a continuous manner in controlled amounts. The ingredients most preferably are subjected to high shear agitation. As such, the plant material is macerated in order to release the enzymes and provide for mixing of ingredients involved in the biochemical reaction. Thus, the unsaturated fatty acid is converted to a green note compound. That is, the lipoxygenase and fatty acid interact to form a fatty acid hydroperoxide, which is cleaved by the action of the hydroperoxide lyase to form an aldehyde, which is converted to an alcohol by the action of the alcohol dehydrogenase. As such, the biosynthesis of the green note compound occurs in such a manner so as to result in a high yield of that compound relative to the amount of plant material employed. Green note compounds produced according to the process steps of this aspect of the present invention include cis-3-hexen-1-ol as the primary green note compound component.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Green note compounds capable of being provided using the process of the present invention can vary. Exemplary compounds include cis-3-hexen-1-ol, trans-2-hexen-1-ol, n-hexanal, trans-3-hexen-1-ol, cis-3-hexenal, trans-2-hexenal, trans-3-hexenal, 1-hexanol, cis-3-penten-3-ol and cis-2-penten-1-ol. Preferred green note compounds include cis-3-hexen-1-ol, trans-2-hexenol and trans-2-hexenal as primary green note components. When the fatty acid is linolenic acid, and all of the ingredients are combined in an essentially simultaneous fashion, the green note compounds consist predominantly of cis-3-hexen-1-ol; and depending upon the purity of that acid (i.e., the presence of certain amounts of other fatty acids), relatively minor amounts of other green note compounds (e.g., trans-2-hexenal and 1-hexanol) can be produced. When the alcohol dehydrogenase is combined with the other ingredients after those other ingredients have been combined, the green note compounds which result have a tendency to consist of complex mixtures of green note compounds, which mixtures can vary depending upon factors such as selection of fatty acid, amount of yeast employed, and timing of yeast addition to the other ingredients of the biochemical reaction. Typical green note compounds are volatile, and are highly flavorful and aromatic. Desired green note compounds are those which exhibit fresh, green and leafy aroma and flavor properties and characteristics.
Plant materials useful in carrying out the process of the present invention can vary. Plants having relatively highly accessible amounts of enzymes (e.g., lipoxygenase and hydroperoxide lyase) are preferred. The plant material also can contain aldehyde isomerase enzyme, which has the effect of transforming an aldehyde in a cis form to a trans form. Typically, the amount of aldehyde isomerase in most plant materials useful in accordance with the present invention is sufficient to provide transformation of about 2 to about 50 percent of cis-aldehyde to trans-aldehyde. Plant materials such as alfalfa have relatively high levels of aldehyde isomerase, while plant materials such as watermelon foliage have relatively low levels of aldehyde isomerase. The plant material can be provided by a single plant variety or by a mixture of two or more plant types, each of which has a desirable level of lipoxygenase, hydroperoxide lyase or aldehyde isomerase. For example, soybeans which have not been heat treated, defatted or solvent treated can have relatively high levels of lipoxygenase; and warm weather-grown watermelon vine and cold weather-grown spinach have relatively high levels of hydroperoxide lyase. If desired, various portions of different types of plants can be mixed to
Garger Stephen John
Holtz Richard Barry
McCulloch Michael Jay
Phillips Harriet Flannery
Teague Richard King
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