STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH Not applicable.

BACKGROUND OF THE INVENTION Hops used in brewing mainly add bitterness and impart a hop flavor (e. g. , taste and smell) to the final product. Hops derived from the fruit (cones) of hop plants are composed of soft resins, hard resins, hop oils, waxes, lipids, and carbohydrates. The soft resins consist of a-acids and (3-acids. The soft resins and a hop oil fraction are extractable by organic solvents, or by liquid and supercritical CO2. In conventional brewing, the a-acids are converted into iso-a-acids, which are responsible for the bitterness taste. Although there is a lack of concensus as to how best to describe the character of-hop flavor in beer, it is universally recognized that the hop flavor (or the kettle hop flavor) is an essential aspect of the total organoleptic impact of beer.

U. S. Serial No. 08/838,217, which is incorporated by reference herein, disclosed that water soluble glycosides comprising an aromatic compound bonded to a mono-, di-, or trisaccharide may be extracted from hop or hop solids.

The hop glycosides thus obtained are precursors to essences and flavorants and can be used to flavor beer.

Hydrolysis of the glycosidic bond of the glycosides results in the formation of aglycons and free sugars. It is the volatile aromatic compounds that help confer flavor to beverages such as beer. Conversion of glycosides to their constituent aglycons and sugars can be achieved chemically by acid hydrolysis, enzymatically through the action of specific glycosidases, or

fermentatively using whole cell biocatalysts such as yeast. The aromatic aglycons may be added to unhopped beer to impart a flavor after fermentation. The use of these kettle hop essences and flavorants provides economy, efficiency, consistency, flexibility, convenience, and quality to the brewing process.

The isolation of hop glycoside flavor precursors from hop cones as a source of flavorants used as additives to flavor beer was an improvement over the prior art. However, there remains a need for further improvements in the brewing process.

Traditionally, hops used in brewing are derived from the fruit or hop cones of hop plants, and the hop plant leaves and stems are discarded. However, the use of hop cones in brewing has certain disadvantages. The cones of hop plants contain certain hop resins, which can indirectly confer an excessively bitter taste to beer.

Derivatives of hop resins are susceptible to degradation upon exposure to light, which results in the formation of compounds that impart an undesirable flavor to beer or similar beverages comprising the compounds. Therefore, beer containing hop resins is susceptible to staling or developing undesirable flavors over time, particularly if the beer is exposed to light.

There is an ongoing interest within the brewing industry to develop new methods that afford improved efficiency, cost savings, and processing advantages over prior art methods, or which yield a product having desirable characteristics, such as improved taste, reduced sensitivity to light, or an extended shelf life.

What is needed in the art is a method of isolating hop glycosides from hop plant parts that are substantially free of resins.

BRIEF SUMMARY OF THE INVENTION We have discovered that kettle hop flavor essence and flavorant precursors can be isolated from hop plant parts other than hop cones, including hop plant leaves and stems, at acceptable yields and essentially free of hop resins.

One aspect of the invention provides a kettle hop essence and flavorant precursor prepared from hop leaves or stems, and a method of making the precursor preparation, comprising the steps of extracting hop plant leaves or stems with an aqueous alcohol to produce an extract containing polar, water soluble compounds; concentrating the polar, water soluble compounds in the extract; and purifying the kettle hop essence and flavorant precursor in the concentrated extract.

Another aspect of the invention is a kettle hop essence and flavorant preparation, and method of preparing the essence and flavorant preparation from the essence and flavorant precursors of the present invention, the method comprising the step of treating the precursors under conditions suitable to cause hydrolysis of the glycosidic bond between the aglycon and sugar moiety of at least a portion of the glycosides in the precursor.

Optionally, the kettle hop essence and flavorant preparation may be modified by adding to the preparation at least one hop fraction selected from hop oil, iso-a- acids, dihydroiso-a-acids, tetrahydroiso-a-acids, and hexahydroiso-a-acids. The kettle hop essence and flavorant preparation may be modified by adding to the preparation a kettle hop essence and flavorant derived from hop cones.

The invention includes a method of making kettle hop flavored beverages using the kettle hop essence and flavorant precursors, and a fermentable growth medium comprising the steps of adding a kettle hop essence and

flavorant precursors of the present invention to the medium prior to fermentation.

The present invention includes beverages flavored with a kettle hop essence and flavorant preparation derived from essence and flavorant precursors isolated from hop plant parts other than cones, and methods of making the beverages comprising adding the preparation to the beverage in an amount sufficient to achieve the desired intensity and character of flavor. For beverages prepared from a fermentable growth medium, the flavorant preparation is preferably added after the medium has been fermented.

Optionally, the beverage of the present invention may be prepared using a kettle hop essence and flavorant that has been modified to contain at least one hop fraction selected from hop oil, a-acids, iso-a-acids, dihydroiso-a-acids, tetrahydroiso-a-acids, and hexahydroiso-a-acids, or a flavorant derived from hop cones.

It is an object of the invention to provide a method that will allow hop plant parts other than hop cones to be used for preparing kettle hop essences and flavorants.

It is a further object of the invention to provide a method of preparing fermented beverages flavored with flavorant precursors extracted from hop plant leaves or stems or kettle hop essences and flavorants prepared by hydrolysing and/or fermenting at least a portion of the glycosides present in the essence and flavorant precursors of the present invention.

It is an advantage of the present invention that the yield of purified kettle hop essences and flavorants per hop plant is increased.

It is a further advantage that the hop stems and leaves from which the essence and flavorant precursors of the present invention are prepared contain essentially none of the resins that can contribute to excessively

bitter taste, off flavor, and staling of fermented beverages.

It is a further advantage that kettle hop essence and flavorant precursors to be added into a fermentable beverage media prior to fermentation for preparing fermented beverages.

It is advantageous that by using hop plant leaves and stems the need for extraction of hops with organic solvents or liquid/supercritical CO2 used to remove hop oil and resins is eliminated, which simplifies the purification process.

Another advantage of the invention is that hops essences and flavorants prepared from precursors isolated from hop plant stems and leaves may confer a different desirable flavor to fermented beverages comprising the hops essences and flavorants than are provided by hops essences and flavorants derived from hops cones.

It is a further advantage that the method of the present invention increases the yield of hop flavorant precursor by using leaves and stems that are normally discarded.

Other objects, features, and advantages of the present invention will become apparent from review of the specification, drawings, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS Not applicable.

DETAILED DESCRIPTION OF THE INVENTION Traditionally, flavorings for beers have been prepared using hop solids, including whole hop cones or hop pellets'that are prepared from hop cones extracted with a non-polar inorganic or organic solvent such as carbon dioxide (liquid or supercritical) or hexane, or hop extracts prepared from hop cones extracted with a non-polar inorganic or organic solvent such as carbon

dioxide (liquid or supercritical) or hexane. The present invention allows the isolation of kettle hop essences and flavorants from hop plant parts other than cones.

In plants, various glycosides are stored for a particular function and purpose. Glycosides or glycoconjugates constitute a class of complex carbohydrates composed of various organic compounds that have glycosidic bonds to monosaccharides (mostly glucose), disaccharides, trisaccharides and or longer chain polysaccharides. The organic non carbohydrate moieties of the molecules (called aglycons) can be freed by biological or chemical reactions that break the glycosidic bond. Certain aglycons have aromatic potential. Many flavor characteristics of fruits are due to the presence of aromatic aglycons that are formed by hydrolysis of the glycosidic bond in glycosides comprising aromatic moieties.

By"essence"it is meant a compound that can be perceived through the sense of smell, and by"flavorant" it is meant a compound that can be perceived by its aroma and taste.

The examples demonstrate the purification of kettle hops essence or flavorant precursors in acceptable yields from the leaf of hop plants, specifically, from frozen or fresh Cascade hop leaves. It should be appreciated that the method of the present invention may also be employed to purify kettle hops essence or flavorant precursors from dried leaves, or from"spent"leaves (e. g. , leaves that had been previously extracted with a nonpolar inorganic or organic solvent). It is reasonably expected that the method of the invention may be practiced using any hop plant part other than hop cones, including stems.

Because it is reasonably expected that any hop plant species or variety may be used in the practice of the invention, the invention is not limited to the Cascade hop plant.

As described in the examples, the essence and flavorant obtained from Cascade hop leaves has a very fruity aroma, and a strong grape aroma and flavor. It should be appreciated that using the method of the present invention, it should be possible to obtain hop essences and flavorants from different varieties of plants, and that these essences and flavorants may have different flavor profiles than those of essences and flavorants isolated from cascade hop plants : In the examples, the kettle hops essence or flavorant precursors were extracted from hop leaves using an excess volume of an aqueous solution comprising 80% methanol. One of ordinary skill in the art would appreciate that other low molecular weight alcohols, including ethanol, as well as a range of concentrations of alcohol in the extraction solution, would be equally suitable for the practice of the invention.

Preferably, the ratio of hop components (stems, leaves, or parts other than cones) to aqueous alcohol is 1: 1 to 1: 100 w/v. It is also preferred that the aqueous alcohol contains 0. 1% to 100% v/v water and that the alcohol is methanol or ethanol. More preferably, the alcohol is in the range of from about 20% to about 80 (v/v). Preferably, the concentrating step is accomplished by evaporation.

The purifying step is preferably accomplished by the steps of (a) feeding the extract to a liquid chromatography column having a packing material selected from the group consisting of polymeric resins, activated carbon, molecular sieves, silica gels, derivatized silica gels, celluloses, and derivatized celluloses whereby kettle hop essence and flavorant precursors are retained on the packing material; (b) eluting the kettle hop essence and flavorant precursors from the packing material to obtain an eluent containing the precursors; and (c) further concentrating the precursors in the eluent by evaporation.

The purifying step can also be accomplished by the steps of (a) feeding the extract to a membrane process selected from the group consisting of filtration, reverse osmosis, ultrafiltration, size-exclusion, dialysis, electro-dialysis, and osmosis whereby the kettle hop essence and flavorant precursors are retained in a retentate and (b) further concentrating the compounds in the retentate by evaporation.

Preferably, the above purification steps allow the removal of at least a portion of the free carbohydrates and inorganic salts from the extract.

Essence and flavorant precursors of the present invention may be used directly or indirectly to flavor beverages. In order to yield a flavor, the glycosides present in the essence and flavorant precursors must be hydrolyzed to yield aromatic compounds, which are primarily responsible for essence and flavor. The essence and flavorant precursors may be added to fermentable beverages (e. g. , beer, wine, or cider) prior to fermentation, with flavor being produced upon modification by yeast enzymes.

Preferably, the precursor is converted to an essence and flavorant before being added to the beverage. In the examples below, this conversion was accomplished by adding the precursors to a yeast fermentation in a model wort (with or without sugar). The flavorant produced by fermentation in this model wort could in turn be used to flavor either fermentable or nonfermentable beverages.

Alternatively, the precursor may be treated to achieve release of aglycons by acid or enzymatic hydrolysis of the glycosidic bonds, and the flavorant thus produced may be used to flavor beverages, either with or without further fermentation. This allows the flavorant to be used in nonfermented beverages, such as water and soda. The option of adding flavorants postfermentatively to fermented beverages such as wine or beer affords greater flexibility in the production of these beverages.

One wishing to flavor beer by adding the kettle hop essences and flavorants to unhopped beer may first convert at least a portion of the glycosides present in the kettle hop essence and flavorant precursors to aglycons by breaking the attachment of the sugar moiety to the aglycon. Such a converting step can be accomplished by treating the precursor with an enzyme selected for its ability to hydrolyze the glycosidic bond attaching the sugar moiety to the aglycon. As discussed extensively in U. S. Serial No. 08/838, 217, conversion can also be accomplished by fermentation in the presence of yeast and model wort; by fermentation in the presence of yeast and wort; by acid hydrolysis; or by any other suitable method for hydrolyzing glycosidic bonds.

As the examples below demonstrate, the essence and flavorant of the present invention may be produced by including the flavorant precursors in a fermentation in a model wort in the presence or absence of sugar. It was noted that a stronger flavor was obtained in the model wort buffer that was not supplemented with sugar.

It is recognized in the art that kettle hop flavor is probably not due to a single compound, but rather, depends on a mixture of aromatic and non-aromatic substances.

Each compound has its own flavor threshold level in beer.

Because multiple flavoring compounds present at low concentrations are responsible for the kettle hop flavor, chemical analysis is very difficult. Therefore, a combination of concentration and fractionation, sensorial and chemical analyses, direct and indirect characterization of the flavoring precursors and flavoring compounds are necessary.

To complement chemical analysis, a routine sensorial analysis was used to identify the kettle hop flavor in an established fermentation system having a simple sugar, selected hop components, yeast, inorganic salts and water.

Using a simple sugar in the model wort rather than a real wort simplifies the system and allows more meaningful

comparisons to be made between kettle hop flavor preparations.

To isolate hop essence and flavorant precursors, aqueous alcohol extracts of hop plant parts were concentrated and partially purified by a preparative liquid column chromatography. A column packed with a polyvinyl resin or Amberlite XAD-2 (styrene- divinylbenzene) was used to concentrate/purify the extract. These resins are capable of retaining polar compounds from aqueous solutions while excluding non-polar compounds such as free aglycons or glycoside degradation products.

In general, the concentration and purification of the aqueous extract of hop plant parts can be accomplished by using materials such as: (1) polymeric adsorbent materials such as Amberlite XAD series resins from Rohm & Haas (XAD-2, XAD-4, XAD-16, etc.) ; (2) derivatized silica gel with hydrocarbons (C-2, C-4, C-18, etc. ) ; (3) activated carbon; and (4) cellulose, as well as membrane processes such as reverse osmosis, ultrafiltration, size exclusion, etc. Using a combination of an aqueous alcohol extraction and one of the above methods, the concentration of the polar, water soluble kettle hop essence and flavorant precursors present in hop plant parts may be increased considerably. It should be understood that the above purification methods are capable of removing at least a portion of the free carbohydrates and inorganic salts from the aqueous alcohol extract of hop plant parts.

The polar compounds retained on the column can be eluted by alcohol and the eluent can be concentrated by evaporation to yield a concentrate designated as XAD-2 fraction. A carbohydrate composition analysis of a completely hydrolyzed XAD-2 fraction can be performed employing an ion-exchange HPLC with a sensitive pulse amperometric detector-an electrochemical (EC) detector A completely hydrolyzed XAD-2 fraction can be comprised of glucose and minor components such as galactose, fucose,

glucosamine, and galactosamine, and aglycones, which can indicate that the XAD-2 fraction consists primarily of glycosides.

Due to the non-volatile character (multiple OH groups) of glycosides, a reversed phase C-18 LC/Electrospray MS technique can be used for the direct analysis of glycosides in the XAD-2 fraction. Glycoside compounds can be identified by this method.

Also due the non-volatile character (multiple OH groups) of glycosides, trifluoroacetyl (TFA) derivatization GC/MS technique can be used for the analysis of whole intact glycosides in the XAD-2 fraction.

Glycoside compounds can be identified by this method.

In addition to the Amberlite XAD-2 (polyvinyl resin), activated carbon, reversed phase silica gels (C-18, C-2, NH2), Sephadex, and Cellulose may also be used to concentrate and fractionate the aqueous alcohol extract of hop plant parts.

The conditions employed for fermentation of the XAD-2 fraction were slightly different from those used in conventional beer fermentation. A model wort buffer solution containing inorganic salts or inorganic salts and glucose (0. 11 g/ml) were used rather than real wort, because the flavor complexity of real wort makes characterization of extracts of hop plant parts more difficult. Fermentations were conducted under aerobic conditions at 15°C. The fermentation temperature was not found to be especially critical but can be conducted in a range from about 10°C to about 30°C.

The model wort fermentation was found to yield a concentrate with direct, pure, and unambiguous kettle hop flavor having an intense fruity aroma, a pleasant, strong grape sweet aroma, and a strong grape or grape soda flavor. The flavor profile of a model wort fermentation comprising an XAD-2 concentrate prepared from hop plant leaves differed from that of a control fermented without the XAD-2 fraction, which produced a product perceived as

winey, sulfidic, and yeasty, with some estery notes. The grape flavor produced by the wort buffer fermentation was reported to be more intense than that produced in the wort buffer + glucose fermentation.

The flavor profile of the fermentation comprising the XAD-2 concentrate prepared from hop plant leaves differed from a fermentation comprising a similar concentrated extract derived from hop cones.

Additional prototype essences or flavorants can be created by dosing the above essences with hop oils. This different hop flavor prototype of post-fermentation essence and flavorant is suitable for making products with heavy hop flavor, late hopping or dry hopping character.

In addition, essences or flavorants may be modified to include combinations of essences and flavorants obtained from hop cones and hop plant parts other than hop cones.

The isolation of essences and flavorants from hop plant parts other than cones has resulted in the discovery of a unique flavorant that can be used alone or in combination with other hop oils, essences, or flavorants to flavor beers. The addition of these purified essences and flavorants postfermentatively provides a convenient method for obtaining a variety of beers having unique flavor profiles from a single fermentation.

The following nonlimiting examples are intended to be purely illustrative.

EXAMPLES Determination of water content of hop leaves Frozen fresh Cascade hop leaves were employed. The hop leaf sample contained some stems, but consisted primarily of leaf material.

To determine the dry weight of the leaf material in the frozen fresh hop leaves (FHL), a sample of frozen fresh hop leaves (123.6 g) was dried over time in the lab.

The weight of the sample was monitored periodically, and the drying process was allowed to continue until there was almost no further reduction in weight. After about 3

weeks, the sample weighed 41.2 g, or approximately one third of its original wet weight.

Extraction of frozen hop leaves Frozen fresh hop leaves were extracted as follows: A sample of frozen fresh hop leaves (8. 6 kg wet weight, equivalent to 2.9 kg dry hop leaves) was ground using a 4 L capacity blender. For each batch, the blender was filled to 80% capacity and about 1 L of a 80% methanol/20% Millipore water solution was included to obtain a puree. A total of 15 L of the 80% methanol aqueous solution was used to grind and extract the leaves.

The purees were pooled and mixed with stirring for 2 h for extraction. Following extraction, the mixture was vacuum filtered using a Buchner funnel and cheesecloth. Solids in the funnel were rinsed with 1 L of 80% methanol aqueous solution and hand pressed until no solution came out. A total of five large Buchner funnels full of the mixture was filtered and pressed, using a total of 20 L 80% methanol solution in the extraction and washing steps.

The filtered liquid extract was vacuum filtered using a Buchner funnel and Whatman #541 paper.

A total of 17.025 L of the leaf extract solution was obtained. A 5.9 L portion of this extract (the equivalent of an extract from one kg dry hop leaves) was further processed through the XAD procedure to isolate and concentrate glycosides. Briefly, the extract was stripped of methanol using a Rotovap, and concentrated extract was passed through a column packed with Amberlite XAD-2, and the retained extract was eluted from the column with methanol. The eluted fraction was stripped of methanol to produce the glycoside fraction. The glycoside fraction was contained in a 600 ml volume. A 100 ml portion of the fresh hop leaves glycoside fraction was freeze dried to yield 1.08 g solid material.

HPLC glycosyl-glucose analysis was performed on the extract to estimate glycoside concentration and derivatized GC/MS was performed on the freeze-dried (FD)

material to determine glycoside constituents. Both methods used established procedures.

Two sets of fermentation experiments (with or without sugar) were performed using the freeze dried XAD concentrate of fresh hop leaves to evaluate flavor development.

Fermentation in model wort buffer without sugar 1. Fresh hop leaf (XAD freeze dried material (0.4 mg/ml) ; model wort buffer and yeast (100 million cells/ml).

2. Control-model wort buffer + yeast (100 million cells/ml) 3. Control-buffer only Fermentation reactions 1-3 were conducted at 15° C with aeration by shaking for 3 days.

Fermentation in model wort buffer with alucose 4. Fresh hop leaf XAD freeze dried material (0.4 mg/ml); glucose (0.. 11 g/ml); model wort buffer; and yeast (12 million cells/ml).

5. Control-glucose (0.11 g/ml) ; model wort buffer ; and yeast (12 million cells/ml) Fermentation reactions 4-5 were conducted at 15° C for 7 days with aeration.

One liter of model wort buffer contains the following components in 1 L of Millipore water: KH2PO 1.03 g CaS04-2H20 0.31 g MgC12 6 H20 1.41 g NaCl 0.11 g

(NH4) 2SO4 1.79 g For volatile analysis the above fermentation samples were extracted by a C18 cartridge extraction method and analyzed by GC/MSD.

RESULTS The results of HPLC glycosyl-glucose analysis are as shown in Table 1. Results obtained using Galena hop solids (solid residues remaining after extraction of hop cones or pellets with liquid/supercritical C02 or a nonpolar organic solvent) are shown for purposes of comparison. Based on HPLC glycosyl-glucose analysis, fresh hop leaves contained a far smaller quantity of glycosides than hop solids.

Table 1. Samples Free gluc Total gluc Glyc-gluc g/Kg* g/Kg* g/Kg* Cascade FHL 0.4 0. 5 0. 1 Galena hop 0 2.7 2.7 solids * g/Kg of equivalent dry hop leaves or CDH Table 2 shows ion intensities (as determined by the mass fragment peak area count of TFA derivatized GC/MS analysis) of selected glycosides in Cascade FHL and Mt.

Hood hop solids.

Table 2. Compounds Cascade FHL Mt. Hood hop solids 3-methyl butanol 449940 139964 glucose 4TFA Benzyl alcohol glucose 15342336 964657 4TFA 2-phenyl ethanol 6534319 288187 glucose 4TFA 1-octanol glucose 4TFA 0 73348 Vanillin glucose 4TFA 245462 127699 Linalol glucose 4 TFA 59055 44698 Alpha-terpineol 21451 277018 glucose 4TFA TOTAL 22652533 1915571

It is interesting to note that there are significant differences in the compositions of glycosides between FHL and hop solids. For example, benzyl alcohol glycoside is present in a very high concentration in FHL, whereas octanol glycoside was undetectable, and based on TFA derivatized GC/MS analysis, the concentration of total glycosides was greater in fresh leaves than in the spent cones.

Aroma evaluation results of the fermented samples The results of aroma evaluations, conducted by five people, are summarized in Tables 3 and 4.

Table 3. Wort buffer Aroma Evaluation 1. FHL XAD FD + buffer + Intense fruity aroma, strong yeast grape or grape soda, beer aroma, very fruity, the best of three 2.. Control-buffer + Fruity, yeasty, off-aroma yeast 3. Control-buffer only Green dried hay, grassy, hop leaf extract aroma

Table 4. Wort buffer + glucose Aroma Evaluation 4. FHL XAD FD + sugar + Very fruity, pleasant, strong buffer + yeast grape sweet aroma, grape aroma may not be strong as #1 sample, tasted same as #5 control but with grape flavor, no bitter taste 5. Control-sugar + Fermented model wort aroma, buffer + yeast winey and yeasty, tasted same as #4 without any grape flavor Fermentations 1 and 4 produced a very pleasant, strong, sweet grape aroma. The intensity of this aroma seemed to be stronger in the sample without sugar (#1), where yeast was forced to metabolize FHL XAD FD. There was no bitter taste imparted by FHL XAD FD in the sample #4 (sample #1 was not tasted) as is typically found in samples prepared with the hop glycosides derived from hop solids.

Extracts prepared from FHL have significantly different glycoside compositions than those prepared from hop solids. An important distinction between fermentation products using FHL or hop solids are that inclusion of FHL in fermentation results in a product having a very intense, pleasant grape aroma; in contrast, no such grape aroma is produced by fermentations including hop solids.

At present, the compound responsible for the intense grape aroma has not been unequivocally established.

Another important finding is that FHL does not impart a bitter taste that is characteristic of the glycosides from hop solids.

The present invention is not limited to the exemplified embodiments, but is intended to encompass all such modifications and variations as come within the scope of the following claims.