AU2006200153A1

AU2006200153A1 - Vegetable tenderizer

Info

Publication number: AU2006200153A1
Application number: AU2006200153A
Authority: AU
Inventors: Uchenna N. Chukwu
Original assignee: CHI S RES Corp
Current assignee: CHI'S RESEARCH Corp
Priority date: 2003-12-29
Filing date: 2006-01-13
Publication date: 2007-01-11
Anticipated expiration: 2023-12-29
Also published as: AU2006200153B2

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Vegetable tenderizer The following statement is a full description of this invention, including the best method of performing it known to us: 004760952v2.doc lA VEGETABLE TENDERIZER BACKGROUND OF THE INVENTION The present invention generally relates to using enzymes to degrade and tenderize raw vegetables prior to application of conventional processing techniques. More specifically, the present invention relates to forming a enzyme-degraded vegetable product with improved processing and nutritional characteristics and to a method of making the vegetable product.

During the last several years, consumer interest in eating foods that are nutritionally balanced with an adequate source of protein, fat, carbohydrates, fiber, vitamins and minerals has increased. Growing concern over chronic diseases, such as cancer, diabetes and heart disease have motivated consumers to seek foods for consumption that are effective in treating chronic diseases while promoting a healthier lifestyle.

Consumption of vegetables having phytochemicals may pose several problems. The presence of anti-nutritional components such as indigestible sugars, enzyme inhibitors, nutrient-binding substances or toxic compounds typically render a vegetable containing the beneficial phytochemicals unfit for consumption. Low concentrations of the desired phytochemical in the vegetable is another problem for consumers, especially if the concentration of the phytochemical is considered too low to deliver a health benefit.

Heat and/or pressure processing of vegetables to eliminate antinutritional components in the vegetable prior to consumption is the traditional approach used by food manufacturers. However, heat and/or pressure processing may eliminate most, if not all phytochemical levels during the manufacturing process. In addition, the manufacturing process may require physical and/or chemical pre-treatment strategies, such as cooking, boiling, application of strong acids, and/or hydration of the raw vegetable prior to processing, in order to adequately process the vegetable. Unfortunately, physical and/or chemical pretreatment strategies of the vegetable prior to processing may include complicated steps that increase the overall costs associated with vegetable production.

004760952v2.doc 2 BRIEF SUMMARY OF THE INVENTION The present invention includes a method of processing vegetables prior to human consumption by applying enzyme(s) to a raw vegetable for a time that is sufficient to form an enzyme-degraded vegetable under normal atmospheric pressures followed by deactivation of the enzyme(s).

In a first aspect the present invention provides a method of enzymatically degrading a raw vegetable composition for subsequent human consumption, the method comprising: providing a raw vegetable composition having a moisture content of less than about 30 weight percent; applying an aqueous enzyme composition comprising water and a cellulase to the raw vegetable composition under normal atmospheric pressures for more than about 2 hours, wherein the aqueous enzyme composition is at an initial pH of between about 2.0 and 7.0; and deactivating the aqueous enzyme composition.

Optionally, the method further includes applying a second aqueous enzyme composition to the raw vegetable composition.

Preferably, the second aqueous enzyme composition is selected from the group consisting of alpha-galactosidase, mannanase, beta-giuconase, betagluconase, arabinase, xylanase, beta-galactosidase, invertase, betafructofuranosidase, alpha-amylase, beta-amylase, pectinase, pectin depolymerase, pectin methyl esterase, pectin lyase, glucoamylase, oligo-1,6 glucosidase, lactase, beta-rf-glucosidase, and any combination thereof.

In a further aspect the present invention provides a method of enzymatically processing a vegetable composition for subsequent human consumption, the method comprising: providing a raw vegetable composition having a moisture content of less than about 30 weight percent; applying a first enzyme composition comprising water and a cellulase to the raw vegetable composition under normal atmospheric pressures for about 2 hours to about 24 hours, wherein the first enzyme composition is at an initial pH of between about 2.0 and applying a second enzyme composition comprising water and a carbohydrase to the enzyme-degraded raw vegetable composition; and 004760952v2.doc 3 deactivating the first enzyme composition and the second enzyme composition.

In another aspect the present invention provides a method of processing a vegetable composition for subsequent human consumption, the method comprising: providing a raw vegetable composition having a moisture content of less than about 40 weight percent; applying an enzyme composition having a pH of between about 2.0 and to the raw vegetable composition under normal atmospheric pressures for more than about 2 hours, wherein the enzyme composition includes water, a first enzyme component, and a second enzyme component, wherein the first enzyme component includes a cellulase that degrades the raw vegetable composition, and wherein the second enzyme component includes a protease that degrades a protein or a peptide; and deactivating the enzyme composition.

Preferably, the raw vegetable composition is a legume, a soybean, grain, an edible seed, a green or unfermented cocoa bean, or any combination thereof.

In yet another aspect the present invention provides a method of processing a vegetable composition for subsequent human consumption, the method comprising: providing a raw vegetable composition having a moisture content of less than about 30 weight percent; applying an enzyme composition having an initial pH of between about and 7.0 to the raw vegetable composition for about 2 hours to about 24 hours, wherein the enzyme composition includes water, a first enzyme component, and a second enzyme component, wherein the first enzyme component includes a cellulase that is effective to degrade the raw vegetable composition, wherein the second enzyme component is effective to degrade methylxanthine; and deactivating the enzyme composition.

In yet another aspect the present invention provides a method of tenderizing a vegetable composition for subsequent human consumption, the method comprising: applying an enzyme composition that includes water and a cellulase to a raw vegetable composition under normal atmospheric pressures for more than 004760952v2.doc 4 about 2 hours, wherein the enzyme composition has an initial pH of between about 2.0 and 7.0, and wherein the enzyme composition is effective to tenderize the raw vegetable composition; and deactivating the enzyme composition.

In a further aspect the present invention provides a method of modifying a raw vegetable composition comprising: applying an enzyme composition that includes water, at least one protease, and at least one carbohydrase to a raw vegetable composition under normal atmospheric pressure for more than about 2 hours, wherein the enzyme composition has a pH of between about 2.0 and 7.0, and wherein the enzyme composition is effective to hydrolyze the protein content of the raw vegetable composition; and deactivating the enzyme composition.

Optionally, the enzyme composition is applied for about 2 hours to about 24 hours. Alternatively, the enzyme composition is applied for at least about 24 hours. In a further alternative, the enzyme composition is applied for no more than 7 days.

In yet another aspect the present invention provides a method of modifying a raw vegetable composition comprising: applying an enzyme composition that includes water, at least one lipase, and at least one carbohydrase to a raw vegetable composition under normal atmospheric pressures for more than about 2 hours, wherein the enzyme composition has a pH of between about 2.0 and 7.0, and wherein the enzyme composition is effective to hydrolyze the lipid content of the raw vegetable composition; and deactivating the enzyme composition.

In a further aspect the present invention provides an enzyme-degraded vegetable composition comprising a raw vegetable composition degraded by an enzyme composition, comprising water, at least one carbohydrase and at least one protease, for more than about 2 hours at an initial pH of about 2 to about 7.

004760952v2.doc In yet another aspect the present invention provides an enzyme-degraded vegetable composition comprising a raw vegetable composition degraded by an enzyme composition, comprising water, at least one carbohydrase and at least one lipase, for more than about 2 hours at an initial pH of about 2 to about 7.

In yet a further aspect the present invention provides an enzymedegraded vegetable composition comprising a raw vegetable composition degraded by an enzyme composition, comprising water and cellulose, for more than about 2 hours at an initial pH of about 2 to about 7.

In another aspect the present invention provides a method of modifying a raw vegetable composition comprising: applying an enzyme composition that includes water, at least one lipase, at least one protease, and at least one carbohydrase to a raw vegetable composition under normal atmospheric pressures for more than about 2 hours, wherein the enzyme composition has a pH of between about 2.0 and wherein the enzyme composition is effective to hydrolyze the lipid content of the raw vegetable composition; and deactivating the enzyme composition.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

DETAILED DESCRIPTION The present invention includes a method of processing vegetables. In the method, an aqueous enzyme composition is applied to a raw vegetable composition under normal atmospheric pressure for a time that is effective to form an enzyme-degraded vegetable composition. After degrading, the enzymedegraded vegetable composition can be processed by one or more additional processing steps that transforms the enzyme-degraded into a vegetable product destined for human consumption. The present invention preferably uses enzymes to degrade, hydrolyze, modify and/or tenderize the raw vegetable composition.

004760952v2.doc 6 Traditional vegetable processing techniques often require the use of high temperatures and/or high pressure during the manufacturing process due in part to the presence of a tough outer layer on vegetables that functions as a barrier.

Such high temperatures and/or pressures increase the cost and complexity of processing vegetables. In addition, such high temperatures and/or high pressures may ultimately reduce the nutritional quality of processed vegetables by lowering phytochemical levels in a manner that reduces consumer acceptability and consumption.

The present invention includes enzymatic degradation of raw vegetables under normal atmospheric pressures prior to human consumption or the use of more traditional processing techniques that may require high pressures and/or temperatures to complete production. Additionally, the present invention represents a novel approach that helps to reduce the need for high temperatures and/or pressures during vegetable processing. In addition, since enzymatic processing of vegetables in accordance with the present invention typically occurs under normal atmospheric pressure, specialized equipment is typically not required and subsequent reduction in the cost and complexity of manufacturing vegetables may be realized. Furthermore, enzymatic degradation of vegetables prior to using more traditional processing techniques may also permit a reduction in time, energy and/or other resources that are required to complete processing of raw vegetables.

While not wanting to be bound to theory, it is believed that when one or more enzyme(s) that are capable of degrading one or more target substrates in a first outer layer of a raw vegetable composition, are applied to the first outer layer of the raw vegetable composition in accordance with the present invention, the enzyme(s) degrade the target substrates of the first outer layer of the raw vegetable composition to form an enzyme-degraded vegetable composition having a compromised first outer layer. Consequently, the use of the aqueous enzyme composition is effective to degrade, tenderize, modify and/or hydrolyze the raw vegetable composition. In addition, the use of the aqueous enzyme composition to degrade the raw vegetable composition reduces the cook time and also permits subsequent in situ modification of the raw vegetable composition by addition of ingredients like vitamins, minerals, or other enzymes that catalyze specific reactions within the raw vegetable composition.

004760952v2.doc 7 As used herein, the term "enzyme" means any complex protein produced by a living cell that is capable of at least catalyzing a specific biochemical reaction on one or more target substrates. The term "enzyme" is also meant to encompass any complex protein capable of catalyzing a specific biochemical reaction that is substantially free of any microorganism. All references to enzyme is also understood as encompassing any synthetically- or geneticallyproduced identical copy of the enzyme that is identical in molecular structure to the enzyme that originated in a living organism.

The enzyme(s) that may be included as part of the aqueous enzyme composition may be generally characterized as lipase(s). As used herein, the term "lipase" means any enzyme that is capable of at least catalyzing hydrolysis of a fat-containing or lipid-containing target substrate. By "hydrolysis" is meant enzymatic degradation of the fat-containing or lipid-containing target substrate that includes triglycerides, diglycerides, monoglycerides, cutin, wax-containing substrates, chemically combined fatty acids and long chain alcohols, phosphatides, cerebrosides, sterols, terpenes, fatty alcohols, solid fat, liquid fat (oils), fatty acids, fat-soluble vitamins, waxes or any combination of any of these.

Furthermore, the term "hydrolysis" is not meant to include the use of microorganisms that produce carbohydrases to hydrolyze and/or degrade raw vegetable compositions in accordance with the present invention. The application of microorganisms that produces carbohydrases and other enzymes to process raw vegetable compositions is commonly referred to as microbial fermentation.

Additionally, although microbial fermentation may involve some degree of hydrolysis, microbial fermentation is known to further transform sugar components like pentoses or hexoses into organic acids that increases the acidity, reduces the pH, and alters the texture and taste of the fermented vegetable composition. In contrast, the present invention uses enzymes that are substantially free of microorganisms to hydrolyze, tenderize, modify and/or degrade the raw vegetable composition. Furthermore, use of the aqueous enzyme composition in accordance with the present invention typically results in a decrease in the acidity of, and/or increase in the pH of the aqueous enzyme composition after degradation.

004760952v2.doc 8 Lipases generally hydrolyze or degrade fat-containing or lipid-containing molecules into free fatty acids, glycerol, mono- and di-glycerides. As an example, suitable lipases for the present invention include lipases that can hydrolyze short, medium or long-chain fatty acids from the 1, 2 or 3 position of triglycerides. In addition, any enzyme that is effective in hydrolyzing waxcontaining substrates or combinations of long chain alcohols and fatty acids to form long chain alcohols and free fatty acids is also suitable for use in the present invention.

Preferably, a lipase that is effective in hydrolyzing at least one long chain fatty acid from a fat-containing or lipid-containing target substrate like a triglyceride is used to practice the present invention. More preferably, a lipase that hydrolyzes at least one long chain fatty acid and/or at least one medium chain fatty acid from a fat-containing or lipid containing target substrate, such as a triglyceride is used in accordance with the present invention.

Still more preferably, a lipase that is effective in hydrolyzing a waxy coat or lipid-containing coat of a raw vegetable compositions and is substantially free of any microorganism is preferably included as part of the aqueous enzyme composition. Lipase may be derived from a number of different sources, such as fungal sources, plant sources, microbial sources, animal sources, or any combination of any of these. As examples, Lipase A "Amano" 12, Lipase AY "Amano" 30, Lipase G "Amano" 50, Lipase M "Amano" 10 that are available from Amano Company of Japan may be used to degrade the waxy coat of raw vegetable compositions when practicing the present invention.

Another enzyme that may be included as part of the aqueous enzyme composition may be generally characterized as carbohydrase(s). As used herein, the term "carbohydrase" means any enzyme that is capable of at least catalyzing hydrolysis of a carbohydrate-containing target substrate. By "hydrolysis" is meant enzymatic degradation of the carbohydrate-containing target substrate that includes complex carbohydrates like cellulose, hemicellulose, pectin, xylan chains of hemicellulose, and/or polymers of other 5-carbon sugars into their sugar components like pentoses or hexoses.

Furthermore, the term "hydrolysis" is not meant to include the use of microorganisms that produce carbohydrases to hydrolyze and/or degrade raw vegetable compositions in accordance with the present invention. The application of microorganisms that produces carbohydrases and other enzymes 004760952v2.doc 9 to process raw vegetable compositions is commonly referred to as microbial fermentation.

Additionally, although microbial fermentation may involve some degree of hydrolysis, microbial fermentation is known to further transform sugar components like pentoses or hexoses into organic acids that increases the acidity, reduces the pH, and alters the texture and taste of the fermented vegetable composition. In contrast, the present invention uses enzymes that are substantially free of microorganisms to hydrolyze, modify, tenderize, and/or degrade the raw vegetable composition.

Preferably, cellulase is one carbohydrase that is also used as part of the aqueous enzyme composition. Still more preferably, cellulase that is substantially free of any microorganism is included as part of the aqueous enzyme composition. Most preferably, cellulase that is substantially free of any microorganism is used to degrade, hydrolyze and/or tenderize the raw vegetable composition when practicing the present invention. Cellulase may be derived from a number of different sources, such as fungal sources, plant sources, microbial sources, animal sources, or any combination of any of these.

Besides cellulase, it is believed that other carbohydrases, such as hemicellulase, alpha-galactosidase, invertase, mannanase, beta-gluconase, betaglucanase, arabanase, polygalacturonase, ferulic acid esterase, xylanase, betagalactosidase, beta-fructofuranosidase, alpha-amylase, beta-amylase, pectinase, pectin depolymerase, pectin methyl esterase, pectin lyase, glucoamylase, oligo- 1,6 glucosidase, lactase, beta-d-glucosidase, or any combination of any of these are suitable additional non-exhaustive examples of carbohydrases that may be used separately or in combination with cellulase in accordance with the present invention.

Preferably, the aqueous enzyme composition includes lipase, cellulase and any combination of hemicellulase, alpha-galactosidase, mannanase, betagluconase, beta-glucanase, arabanase, polygalacturonase, xylanase, betagalactosidase, beta-fructofuranosidase, alpha-amylase, beta-amylase, pectinase, invertase, pectin depolymerase, pectin methyl esterase, pectin lyase, glucoamylase, oligo-1,6 glucosidase, lactase, or beta-d-glucosidase to degrade the raw vegetable composition under normal atmospheric pressures, prior to(l) human consumption or application of traditional processing techniques like cooking, pressure-cooking or the like.

004760952v2.doc More preferably, a blend of lipase, cellulase and hemicellulase is used in the present invention to degrade, tenderize and/or render the raw vegetable composition more absorbent to water, enzymes, additives or the like. Still more preferably, a blend of lipase, cellulase, hemicellulase and pectinase is used in the present invention to degrade the raw vegetable composition so that subsequent processing can be practiced with reduced temperature and/or pressure requirements.

A preferred example of a carbohydrase that may be used as part of the aqueous enzyme composition is Viscozyme®L, available from Novozymes of Franklinton, North Carolina. Alternate examples of carbohydrases that are suitable for use as part of the aqueous enzyme composition is Econase CE, available from Enzyme Development Corporation of New York, New York, Cellulase 4000 or Crystalzyme Cran that is available from Valley Research Inc., of South Bend, Indiana and Multifect GC that is available from Genencor of California.

SWhen enzymes are used during vegetable processing, enzymes may be applied in any form, such as a granular form, or a vapor form, or as part of the aqueous enzyme composition as noted above. The application form that is selected preferably permits the enzyme to contact the vegetable composition being treated, and remain in contact with the vegetable composition being treated for a time that is sufficient to degrade the target substrate. Preferably, the enzyme(s) is applied to the raw vegetable composition as part of the aqueous enzyme composition.

The aqueous enzyme composition may include one or more enzyme component(s), one or more optional catalyst component(s), one or more optional pH-modifying component(s), one or more optional additive(s) or one or more optional solvent component(s). The components of the aqueous enzyme composition may be supplied as individual components, or supplied in various prepared mixtures of two or more components, that are subsequently combined to form the aqueous enzyme composition.

The enzyme component(s) may include only the enzyme(s), the enzyme(s) and water, or may optionally include additional components. The enzyme component(s) may be supplied as individual components, or supplied in various prepared mixtures of two or more components, that are subsequently combined to form the enzyme component(s). Additionally, the enzyme 004760952v2.doc 11 component may be supplied in granular form, vapor form, or as part of an aqueous enzyme component.

The concentration of the enzyme(s) in the enzyme component may generally range from about 0.0001 weight percent to about 100 weight percent, based on the total weight of the enzyme component. The enzyme component may optionally include sucrose, fructose, ash, alcohol, and any other components that are compatible with, and do not interfere with the biochemical rate of catalysis of the enzyme.

Preferably, the concentration of the enzyme component is an amount that is effective to tenderize, hydrolyze, modify and/or degrade the raw vegetable composition. Still more preferably, the concentration of the enzyme component is an amount that is effective to degrade the first outer layer of a raw vegetable composition. Most preferably, the concentration of the enzyme component that is used in accordance with the present invention is an amount that is effective to degrade the first outer layer of a raw vegetable composition, tenderize, hydrolyze, modify and/or degrade the raw vegetable composition, and permit further modification of an inner portion of the raw vegetable composition.

Furthermore, it is to be understood that the concentration of the enzyme component(s) may vary depending on the amount of time that the enzymes remain in contact with the raw vegetable composition. Furthermore, if a short exposure time is employed, then higher concentrations of the enzyme component(s) would be required to achieve the desired degree of degradation, tenderization, hydrolysis and/or modification of the raw vegetable compositions.

Similarly, if longer exposure times are employed, then the concentration of the enzyme component(s) would be reduced to arrive at the desired result.

As an example, when the enzyme component is supplied in the form of a liquid, the enzyme component can be applied at a concentration of less than about 10 weight percent, based on the total weight of the raw vegetable composition to tenderize the raw vegetable compositions like dry edible beans.

More preferably, when the enzyme component is supplied in the form of a liquid, the enzyme component is applied at a concentration of less than about weight percent, based on the total weight of the raw vegetable composition to tenderize raw vegetable compositions.

Similarly, when the enzyme component is supplied in the form of a granular powder, the enzymes can be applied at a concentration of less than 004760952v2.doc 12 about 5 weight percent, based on the total weight of the raw vegetable composition to tenderize, hydrolyze and/or enzymatically modify the raw vegetable composition. More preferably, when the enzyme component is supplied in the form of a granular powder, the enzyme component is applied at a concentration of less than about 1 weight percent, based on the total weight of the raw vegetable composition to tenderize, hydrolyze and/or enzymatically modify the raw vegetable compositions.

The aqueous enzyme composition may optionally include one or more catalyst component(s) in a form that is readily applied to the raw vegetable composition. A catalyst, when included as part of the aqueous enzyme composition, generally enhances the biochemical rate of catalysis of the enzyme component(s). Increasing the biochemical rate of catalysis of the enzyme component(s) may decrease the application time of the aqueous enzyme composition to the raw vegetable composition or the amount of the aqueous enzyme composition applied to the raw vegetable composition.

Alternatively, the catalyst component may be applied separately from the aqueous enzyme composition, either before, during, or after application of the aqueous enzyme composition to the raw vegetable composition. Additionally, the source(s) of the catalyst may be applied in particulate form, as part of an aqueous composition, or in a vapor form so long as the particular form selected results in application to and uptake by the vegetable composition. Some nonexhaustive examples of catalysts that may be included as part of the aqueous enzyme composition are salts that include calcium ions, copper ions, magnesium ions, iron ions, sodium ions, zinc ions, manganese ions, potassium ions, or any combination thereof. The catalyst component(s) may be supplied as individual components, or supplied in various prepared mixtures of two or more components, that are subsequently combined to form the catalyst component(s).

The aqueous enzyme composition may include one or more pHmodifying component(s) that are capable of adjusting the acidity, hereinafter referred to as the pH, of the aqueous enzyme composition. Furthermore, the pH of the aqueous enzyme composition will vary depending on the enzyme(s) present in the aqueous enzyme composition.

Preferably, the pH of the aqueous enzyme composition is about 2.0 to about 7.0. Still more preferably, the pH of the aqueous enzyme composition is about 3.0 to about 7.0 when degrading, tenderizing, hydrolyzing and/or 004760952v2.doc 13 enzymatically modifying the raw vegetable composition. In addition, extremely low pH values of less than about 1.0 are typically effective in deactivating the enzyme component(s) when practicing the present invention. Consequently, human ingestion or addition of strong acids that reduce the pH of the aqueous enzyme composition to below a pH of about 2.0 are believed effective in deactivating the enzyme component(s) of the aqueous enzyme composition.

Some non-exhaustive examples of pH modifying substances include organic acids, such as acetic acid, tartaric acid, malic acid, succinic acid, citric acid, or the like; phosphoric acid; or buffering agents of such organic acids, such as sodium acetate, sodium malate, sodium succinate, sodium citrate, or the like.

Basic compounds like sodium hydroxide or the like may be included as part of the pH-modifying substances that are suitable for use in the present invention.

As lipases and/or cutinases are used to hydrolyze wax- or fat-containing substrates of the raw vegetable composition, an aqueous composition that contains an emulsifier or surfactant may be required in order to attain sufficient contact of the enzyme with the waxy coat of the raw vegetable composition. Fats or waxes are typically hydrophobic while water is required for enzymatic hydrolysis. Therefore, in order to realize an efficient hydrolytic lipase reaction, at least 10 percent water must be available and the lipase and/or cutinase must be in intimate contact. Some non-exhaustive examples of emulsifiers or surfactants include such as mono-glycerides, distilled mono-glycerides, diglycerides, distilled di-glycerides, or lecithin (natural or artificial), vegetable glycerin, glycerol, glycerin, or any combination of any of these. Preferably, the emulsifier and/or surfactant that is used in the present invention is any material that is compatible the process requirements and final product attributes. Still more preferably, colorless vegetable glycerin is used in accordance with the present invention.

Other examples of optional additives that may be included as part of the aqueous enzyme composition include natural and/or artificial flavors; artificial colors; naturally-occurring pigments, such as, for example, chlorophyll, anthocyanin, betalain, betaine, carotenoid, anthoxanthins; herbs; spices; vitamins; minerals; plant extracts; essential oils; sugars such as sucrose, fructose, glucose, or maltose; preservatives; any additive that improves the aqueous enzyme composition application to, uptake by, or subsequent processing of the vegetable composition; or any combination of any of these.

004760952v2.doc 14 The aqueous enzyme composition may also include one or more solvent component(s). The solvent component(s) preferably facilitate homogenous blending of the enzyme component(s), the optional catalyst component(s), the optional pH-modifying component(s), the optional additives, or any combination thereof. The solvent component(s) preferably facilitate aqueous enzyme composition application to, and uptake by the vegetable composition. Some nonexhaustive examples of solvents that may be included in the aqueous enzyme composition include water; oils; alcohol, such as ethanol, methanol, propanol, butanol, or the like; hexane; or any combination thereof. The solvent component(s) may be supplied as individual components, or supplied in various prepared mixtures of two or more components, that are subsequently combined to form the solvent component(s).

Liquid water is the preferred solvent for the aqueous enzyme composition as water is typically required for enzymatic degradation, tenderization and/or hydrolysis. The amount of liquid water included as part of the aqueous enzyme composition depends on an initial concentration of water in the raw vegetable composition, the biochemical rate of catalysis, and/or the desired final product characteristics of the enzyme-degraded raw vegetable composition. Generally, the amount of the aqueous enzyme composition is such that the raw vegetable composition is completely contacted by the aqueous enzyme composition. As an example, when degrading raw edible beans, water is included as part of the aqueous enzyme composition at a range of about 1.25 to about 5 times the weight of raw edible beans. Similarly, when tenderizing raw greens like collards, kale, turnip, or mustard greens, water is included as part of the aqueous enzyme composition at a range of about 1 to about 2 times the weight of the raw greens.

In general, any conventional blending apparatus and technique that is suitable for homogeneously blending the enzyme component(s), the optional catalyst component(s), the optional pH-modifying component(s), the optional additives, the optional solvent component(s), or any combination thereof, such as a mixer, may be used to form the aqueous enzyme composition.

As used herein, the term "application" means to apply the aqueous enzyme composition to the raw vegetable composition by spraying, knifecoating, spreading, soaking, exposing, immersing, slop-coating, dip-coating, roller- coating, dipping, contacting, brush-coating, squirting, submerging, foam 004760952v2.doc padding, leaf-sprinkling, sprinkling, pouring, slop-padding, or any combination thereof.

The temperature of the aqueous enzyme composition depends on the initial temperature of the vegetable composition, the temperature for the optimum biochemical rate of catalysis of the enzyme component(s), and/or the desired characteristics of the enzyme-degraded vegetable composition. The temperature of the aqueous enzyme composition is at the optimum temperature for a maximum biochemical rate of catalysis of the enzyme component(s) of the aqueous enzyme composition.

Generally, the temperature of the aqueous enzyme composition may range from about 30°F to about 250'F. Preferably, the temperature of the aqueous enzyme composition ranges from about 30 0 F to about 250F. Still more preferably, the temperature of the enzyme composition ranges from about to about 200F. Most preferably, the temperature of the aqueous enzyme composition ranges from about 40'F to about 195 0

F.

Although the aqueous enzyme composition may be applied to the raw vegetable composition at a constant temperature, the temperature of the aqueous enzyme composition may be increased at any time during application of the aqueous enzyme composition to the raw vegetable composition. Generally, increasing the temperature increases the biochemical rate of catalysis, and/or water absorption.

However, a negative impact on the texture of the raw vegetable composition may occur if the temperature of the aqueous enzyme composition is too high, such as more than about 250'F, or the temperature of the aqueous enzyme composition is changed too rapidly during application. Furthermore, too high temperatures may inactivate the enzyme component of the aqueous enzyme composition, therefore care is required to avoid premature inactivation of the enzyme component(s) before attaining the desired degree of hydrolysis, tenderization, degradation and/or enzymatic modification when practicing the present invention.

Steam can also be injected into the aqueous enzyme composition to during or after application of the aqueous enzyme composition to the raw vegetable composition to optionally increase the temperature of the aqueous enzyme composition applied to the raw vegetable composition, optionally increase the moisture content of the vegetable composition, optionally 004760952v2.doc 16 gelatinize any starch granules of the vegetable composition, optionally increase the efficacy of the biochemical rate of catalysis of the aqueous enzyme composition, or optionally deactivate the enzyme component in the aqueous enzyme composition. Preferably, steam is injected after the aqueous enzyme composition is applied to the raw vegetable composition if steam is included as part of the vegetable manufacturing process. As noted, inactivation of the enzyme component(s) readily occurs at high temperatures, such as temperatures that occurs with steam application, therefore, care is required to avoid premature inactivation of the enzyme component(s) prior to attaining the desired degree of degradation, tenderization and/or hydrolysis of the raw vegetable composition.

The aqueous enzyme composition is typically applied to the raw vegetable composition at normal atmospheric pressures. By "normal atmospheric pressures" is meant atmospheric pressures of about 14.7 psi.

Furthermore, it is to be understood that "normal atmospheric pressures" also includes atmospheric pressures that occurs even under varying altitudes, temperatures, humidities, or the like.

Additionally, the term "normal atmospheric pressures" is not meant to include application of positive (more than about 14.7 psi) or negative pressures (less than about 14.7 psi or vacuum pressures) to the raw vegetable composition prior to or during application of the aqueous enzyme composition in a manner that facilitates degradation, tenderization, hydration and/or hydrolysis.

As used herein, the term "vegetable" means a plant-based food that originated as a living organism of the Plantae kingdom. All references to "vegetable" are to be understood as encompassing any genetically-altered copy of the plant that originated as a living organism of the Plantae kingdom.

Furthermore, the term "vegetable" encompasses leaves, seeds, roots, tubers, bulbs, flowers, fruits, stems, shoots, nuts, or any combination of any of these that originated as a living organism of the Plantae kingdom.

The raw vegetable composition of the present invention typically contains a first outer layer that substantially covers, overlays, and/or is in adhesive contact with a second inner layer of the raw vegetable composition when practicing the present invention. When the first outer layer is in adhesive contact with the second inner layer, adhesive contact may be accomplished through bonding via cementing substances like pectic substances.

004760952v2.doc 17 The first outer layer of the raw vegetable composition may be characterized as a water-impermeable layer that typically includes a fibrous network of cellulose; xylan chains of hemicellulose; hemicellulose; polysacccharides of five-carbon sugars; lignin; pectic substances, such as protopectin, pectic acid, pectin, or any combination thereof; vitamins; minerals; fats; anti-nutritional components; or any combination of any of these. Some nonexhaustive examples of the first outer layer may include a seed coat of a legume or lentil; a bran layer of a grain; a stem wall of a vegetable; a skin of a root, tuber, and/or bulb vegetable; a peel of a fruit; a testa or a seed wall of a nut.

The second inner layer of the raw vegetable composition generally includes a network of starch granules, fat globules, fiber, proteins, vitamins, minerals, water, phytochemicals, anti-nutritional components, or any combination of any of these. In addition, all references to the second inner layer is also understood to encompass the inner portion of the raw vegetable composition and thus, the second inner layer may also include seeds embedded in the vegetable composition. Some non-exhaustive examples of anti-nutritional components of a vegetable composition include flatulence-causing sugars, such as, for example, raffinose, verbascose and stachyose; lectins; nutrient-binding substances, such as phytic acid; other indigestible polysaccharides; enzyme inhibitors, such as trypsin inhibitor; or toxic compounds, such as goitrogens, solanine, or oxalic acid.

Preferred raw vegetable compositions of the present invention include a tough hard second inner layer. As an example, legumes contain at least one cotyledon that may be characterized as a tough fibrous network of starch, protein, anti-nutritional factors, fat, vitamins, and minerals. Similarly, grains, such as whole wheat or hominy contain an endosperm that may also be characterized as a tough fibrous network of starch, protein, anti-nutritional factors, fat, vitamins and minerals. Furthermore, raw vegetable compositions that include a hard second inner layer typically have less than about 40 weight percent moisture content, and preferably less than about 30 weight percent moisture content. Raw vegetable compositions having a moisture content of less than about 40 weight percent and preferably less than about 30 weight percent can be effectively tenderized when practicing the present invention.

Preferably, the first outer layer is connected or in adhesive contact to the second inner layer or inner portion of raw vegetable compositions when 004760952v2.doc 18 practicing the present invention. By "connected or in adhesive contact" is meant that the raw vegetable composition has a substantial portion of the first outer layer connected to the inner portion or second inner layer of the raw vegetable composition. Additionally, removal of the first outer layer of the raw vegetable composition by peeling, chemicals, grating for example is preferably avoided when practicing the present invention.

As used herein, the term "raw" refers to vegetable composition(s) that are uncooked, un-boiled, dry, edible, as being in a natural condition, or any combination of any of these. Furthermore, it is to be understood that the term "raw" refers to the condition of the first outer layer, the second inner layer or both the first and second layers of the vegetable composition when practicing the present invention.

In addition, the raw vegetable composition is preferably a whole raw vegetable compositions. By "whole" is meant that the raw vegetable composition has not been subjected to techniques like maceration, pulverization, grating, grinding or the like. For example, dry edible beans that have not been ground to a powder (flour), grated to form flakes, macerated or pulverized are examples of whole raw vegetable compositions. Similarly, green leafy vegetables such as collards, kale or the like that have not been ground, grated, macerated or pulverized are preferred examples of whole raw vegetables that may be used in accordance with the present invention.

In addition, preferred raw vegetable compositions for the present invention include raw vegetable compositions that contain an additional outer layer on top of the first outer layer. Examples of an additional outer layer on top of the first outer layer includes waxy layers (coats) of seeds, grains, legumes or the like. Typically, the waxy coats include cutin or other wax- and/or lipidcontaining molecules.

Example of raw vegetable compositions that may contain a waxy coat/outer layer include beans like pinto beans, light red kidney beans, black-eye peas, lentils, mung beans, pinkie beans, great northern beans, green lima beans, yellow lima beans, garbanzo beans, carob beans, cacao beans, coffee beans, split and/or whole peas, peanuts, yellow peas, green peas, soybeans, black beans, vanilla bean, or any other edible seed from plants of the Leguminosae family; seeds, such as amaranth seeds, watermelon seeds, pomegranate seeds, sunflower seeds, safflower seeds, poppy seeds, sesame seeds, alfalfa seeds, caraway seeds, 004760952v2.doc 19 cardamom seeds, celery seeds, chia seeds, coriander seeds, dill seeds, fennel seeds, fenugreek seeds, flax seeds, milk thistle seeds, nutmeg seeds, mustard seeds, psyllium seed; grains such as barley, buckwheat, hominy, pearl barley, bulghur, corn, millet, oats, rice, rye, triticale, wheat, wild rice, brown rice; or any seed from recognized edible vegetable source.

In addition, raw vegetable compositions having a moisture content of more than about 40 weight percent, as disclosed in pending Application Serial Application No. 10/619,403 may also be effectively tenderized when practicing the present invention. For example, green leafy vegetables that contain an outer layer of a waxy coat were tenderized using a blend of cellulase, hemicellulase, and pectinases. Thus, the inclusion of a lipase was not required. However, when practicing the present invention on grains such as wheat, rice or seeds/vegetable compositions that include the waxy coat, the inclusion of a lipase and/or cutinase is preferred.

Raw vegetable compositions that are generally in the form of a nut may also have less than about 40 weight percent moisture content and may also be included as part of the raw vegetable composition when practicing the present invention. As used herein, a "nut" means a hard shelled dry fruit or seed with a separable first outer layer that substantially encloses an interior kernel.

Some non-exhaustive examples of vegetable compositions in the form of a nut that may be used in accordance with the present invention include an acorn nut, an almond nut, a brazil nut, a butternut, a cashew nut, a chestnut, a coconut, a filbert nut, a hazelnut, a hickory nut, a macadamia nut, a pecan nut, a pine nut, a pistachio nut, a walnut, or any recognized edible nut from a recognized edible vegetable source.

It is also to be understood that the term "raw vegetable composition" is meant to encompass raw vegetable compositions that may have been washed with steam, hot, warm and/or cold water in an attempt to remove dirt and the like from the raw vegetable composition. Cleaning, washing or dirt removal from the vegetable composition may also include the application of food-grade detergents or chemicals using sprinkler-type equipment or soaking equipment.

Such cleaning, washing or dirt removal techniques are believed to not (1) significantly remove the first outer layer of the raw vegetable composition from the second inner layer or inner portions of the raw vegetable compositions, and/or substantially reduce the fibrous components of the raw vegetable 004760952v2.doc composition prior to enzyme application. Therefore, use of such cleaning, washing or dirt removal techniques prior to application of the aqueous enzyme composition are permissible when practicing the present invention.

In addition, physical and/or chemical pretreatment strategies designed to initiate breakdown, improve the porosity of the first outer layer of raw vegetable compositions, remove certain cellulose and hemicellulose fractions or expose degradation sites have been practiced in the widespread belief that enzymatic degradation cannot proceed without such pre-treatment strategies. Physical pretreatment strategies includes application of positive or negative pressure prior to application of the aqueous enzyme composition to vegetable compositions.

Furthermore, chemical pre-treatment strategies include application of strong acid solutions, pre-soaking, boiling or cooking of vegetable compositions that typically increase the moisture content of vegetables prior to application of the aqueous enzyme composition.

The present invention avoids these complicated processing strategies by applying the aqueous enzyme composition to the raw vegetable composition under normal atmospheric pressures without having first subjected the raw vegetable composition to strong acidic solutions, pre-soaking, boiling or cooking prior to application of the aqueous enzyme composition. Such physical and/or chemical treatments are typically reserved, and preferably conducted after the aqueous enzyme composition has degraded the raw vegetable composition to deactivate the enzyme components.

It is also to be understood that the term "whole raw vegetable composition" is meant to encompass broken a raw vegetable composition that has a first outer layer that is in adhesive contact with the second layer, or (2) an exposed second inner layer or inner portion of the raw vegetable composition.

For example, in the manufacture of refried beans, broken portions of whole beans still contain a seed coat and exposed cotyledons. Such broken portions of whole raw beans can be soaked or exposed to the aqueous enzyme composition of the present invention to degrade the seed coat and tenderize the cotyledons prior to human consumption or any subjecting the beans to any other remaining processing steps required for manufacturing refried beans.

Similarly, raw green leafy vegetables may be chopped prior to application of the aqueous enzyme composition to permit enzymatic degradation and subsequent tenderization of the raw green leafy vegetables as chopping is 004760952v2.doc 21 not believed to remove or diminish the fibrous network present in raw green leafy vegetables. Similarly, the common industrial practice of application of heat (wilting) of greens prior to canning is permissible when practicing the present invention as the wilting step is not believed to substantially reduce the fibrous components present in leafy greens. Rather, the wilting step is believed to improve compaction of the leafy greens for subsequent inclusion into cans.

As noted above, the length of time the aqueous enzyme composition is applied to the raw vegetable composition typically depends on the raw vegetable composition, the desired degree of degradation, the concentration of the enzyme component(s) and/or the desired characteristics of the enzyme-degraded vegetable composition. For example, the amount of time required to modify and/or degrade the inner fat, protein or carbohydrate fractions of raw whole vegetables is generally more than about 2 hours. As another example, the time required to enzymatically degrade or hydrolyze protein may range from about 2 hours to about 7 days for cocoa beans. For soybeans and other legumes, the amount of time required to enzymatically degrade or hydrolyze protein is typically 2 hours to 24 hours with 8 to 16 hours being preferred.

While not wanting to be bound to theory, it is believed that when lipase is applied to a raw vegetable composition it degrades the outer waxy layer or lipid-containing layer of the raw vegetable composition by forming a network of degraded sites. Next, cellulases, hemicellulases and pectinases are able to penetrate the network of degraded sites and initiate hydrolysis of carbohydrate containing target substrates of the first outer layer. As hydrolysis of carbohydrate-containing target substrates is also believed to support the formation of degraded sites or holes, any other enzymes that may be present may also enhance degradation so that effective tenderization of the raw vegetable compositions can occur.

In other examples when lipase is not used, the cellulase(s), hemicellulase(s), and/or pectinase(s) is applied to the raw vegetable composition to degrade the first outer layer and form a network of degraded sites or holes.

The holes allow subsequent penetration of other enzymes capable of degrading the interior of the vegetable composition so that effective in situ modification of the raw vegetable composition can occur.

Therefore, the aqueous enzyme composition may generate holes throughout the waxy top layer, the first outer layer and/or the second inner layer 004760952v2.doc 22 of the vegetable composition so that absorption of additives or enzymes and hydrolysis is observed. The aqueous enzyme composition may also target a wide range of substrates within the raw vegetable composition, therefore, the breakdown of these substrates may occur and further aid in the reduction of cook time of the enzyme-degraded raw vegetable composition. Alternatively, degradation of fat, carbohydrate or protein can occur to generate bioactive fraction.

The benefits of the enzyme-degraded raw vegetable composition include an increase in the absorbency of the raw vegetable composition of a component, such as, for example, additives or other enzymes that may be used to further process the raw vegetable composition. In addition, processing the enzymedegraded raw vegetable composition by conventional means, after enzymatic degradation, such as by freezing, hydrating, steaming, freeze-drying, canning, frying, filtration, extraction, boiling, drying, extrusion, cooking, baking, roasting, pulverizing, fermenting, enzyme, pasteurizing, extracting, milling, puffing, steam-pressure cooking, or any combination thereof, is improved since the first outer layer of the raw vegetable composition that typically functions as a barrier during conventional processing has been degraded.

Once sufficient degradation of the raw vegetable composition has occurred to form the enzyme-degraded raw vegetable composition, the enzymedegraded raw vegetable composition may be separated from the aqueous enzyme composition and further subjected to processing steps, such as, for example, blanching, that inactivates any enzyme component(s) remaining in the enzyme-degraded raw vegetable composition. Alternatively, transferring both the raw enzyme-degraded vegetable composition and the aqueous enzyme composition to equipment that permits further processing by freezing, hydrating, steaming, freeze-drying, canning, frying, boiling, drying, filtration, extraction, extrusion, cooking, baking, roasting, pulverizing, fermenting, enzyme, pasteurizing, extracting, milling, puffing, steam-pressure cooking, or any combination thereof, is also effective in deactivating any enzyme component(s) remaining in the enzyme-degraded raw vegetable composition and the aqueous enzyme composition.

The present invention is more particularly described in the following examples that are intended as illustrations only since numerous modifications 004760952v2.doc 23 and variations within the scope of the present invention will be apparent to those skilled in the art.

A method of enzymatically processing a raw vegetable composition Typically application of the aqueous enzyme composition that includes first and second enzyme components occur under normal atmospheric pressures and temperatures that can range from about 40°F to about 250F and preferably from about 40'F to about 195 0 F. In addition, the initial pH values range from about 2.0 to about Alternatively, two or more separate aqueous enzyme compositions may be applied to the raw vegetable composition when practicing the present invention. For example, a first aqueous enzyme composition that includes cellulase and hemicellulase may be applied to a raw vegetable composition having a moisture content of less than about 30 weight percent under normal atmospheric pressures and temperatures to form an enzyme-degraded raw vegetable composition. Next, a second aqueous enzyme composition that contains an enzyme component that is effective to degrade and/or hydrolyze target substrates either in the first outer layer or the second inner layer can be applied to the enzyme-degraded vegetable composition. The second aqueous enzyme composition is able to penetrate the enzyme-degraded vegetable composition and therefore, is capable of degrading and/or hydrolyzing desired target substrates in the enzyme-degraded vegetable composition.

It is believed that the compromised first outer layer of the enzymedegraded raw vegetable composition allows the second enzyme component or the second aqueous enzyme composition to enter and degrade any antinutritional components or other fractions located in the interior of the enzymedegraded raw vegetable composition. Additionally, water included as part of the aqueous enzyme composition(s) may also enter through the degraded first outer layer to hydrate the enzyme-degraded raw vegetable composition. If water is absorbed by the enzyme-degraded raw vegetable composition, the water in the enzyme-degraded raw vegetable composition may help to facilitate degradation of anti-nutritional components or other fractions of the enzyme-degraded raw vegetable composition by the second enzyme component or the second aqueous enzyme composition. Additionally, third, fourth and additional enzymes can be 004760952v2.doc 24 used so that in situ modification, hydrolysis and degradation of the raw vegetable composition can be achieved.

After sufficient enzymatic degradation, an enzyme-processed raw vegetable composition is formed that can be further subjected to other processing steps, such as, for example, blanching, that inactivates any enzyme component(s) remaining in the enzyme-processed vegetable composition.

Preferably, the enzymes that are included as part of the first enzyme component include the above noted enzymes that are effective in degrading the first outer layer of raw vegetable compositions. The enzyme(s) that may be included as part of the second enzyme component or second aqueous enzyme composition are carbohydrases, proteases, lipases or any combination thereof.

Any of the examples of carbohydrases as suitable for use during application of the first enzyme component may be used as part of the second enzyme component in any combination with the first enzyme component, for degradation of any anti-nutritional component of the raw vegetable composition in accordance with the present invention.

As used herein, the term "protease" means any enzyme that is capable of at least catalyzing degradation of a protein-containing target substrate. One particular form of a protease that may be used as part of the second enzyme component in accordance with the present invention is an endoprotease. As used herein, an "endoprotease" means any enzyme that is capable of degrading an internal peptide bond on a target substrate having one or more peptide bonds.

Another particular form of a protease that may be used as part of the second enzyme component in accordance with the present invention is an "exoprotease". As used herein, an "exoprotease" means any enzyme that is capable degrading a peptide bond located at a terminal portion of a target substrate having one or more peptide bonds. Either the endoprotease or the exoprotease may be derived from a number of different sources, such as fungal sources, plant sources, microbial sources, animal sources, or any combination of any of these.

Some non-exhaustive examples of endoproteases and exoproteases include Alcalase Neutrase Esperase®, Protamex, Novozym® FM, Flavourzyme®, and Kojizyme®, all available from Novo Nordisk Biochem North America of Franklinton, North Carolina, and Fungal Acid Proteases, Fungal Proteases, chymotrypsin, trypsin, pepsin, papain, ficin, bromelain, 004760952v2.doc Enzeco® exoprotease that are available from Enzyme Development Corporation of New York, New York.

The enzyme-processed vegetable composition may also be further processed by freezing, hydrating, steaming, freeze-drying, canning, frying, boiling, drying, extrusion, cooking, filtration, extraction, baking, roasting, pulverizing, fermenting, enzyme, pasteurizing, extracting, milling, puffing, steam-pressure cooking, or any combination thereof after enzymatic degradation has occurred. These additional processing steps are also generally effective in deactivating any enzyme component(s) in the enzyme-processed vegetable composition. Partial degradation of the first outer layer or the second inner layer of the vegetable composition may also occur during application of aqueous enzyme compositions that includes first and second enzyme components or the second aqueous enzyme composition.

As a first example, an enzyme-degraded raw vegetable composition that includes flatulence-causing substrates located in an inner portion of the raw vegetable composition is formed by applying a first aqueous enzyme composition that degrades the raw vegetable composition in accordance with the present invention. If a second aqueous enzyme composition that includes any enzyme component capable of degrading any flatulence-causing substrates that cause flatulence in human, such as alpha-galactosidase, beta-fructofuranosidase, beta-galactosidase, invertase, or any combination thereof, is applied to the enzyme-degraded raw vegetable composition either during or after the first aqueous enzyme composition is applied to the raw vegetable composition, degradation of flatulence-causing substrates, such as raffinose, verbascose and stachyose of the enzyme-degraded raw vegetable composition typically occurs.

Generally, the second aqueous enzyme composition is applied to the enzyme-degraded vegetable composition for a time that is sufficient for the second enzyme composition to degrade the flatulence-causing substrates, such as, for example, about 1 minute to about 12 hours. Preferably, the second aqueous enzyme composition remains in contact with the enzyme-degraded raw vegetable composition for about 5 minutes to about 24 hours so that more than about 5 weight percent of flatulence-causing substrates are degraded in the enzyme-degraded raw vegetable composition when practicing the present invention. More preferably, the application time is more than about 2 hours and generally about 4 to about 8 hours when practicing the present invention.

004760952v2.doc 26 As a second example, an enzyme-degraded raw vegetable composition that includes methylxanthine is formed in accordance with the present invention.

As used herein, the term "methylxanthine" refers to the group of compounds used as a stimulant and diuretic typically found in vegetable compositions, such as tea, coffee, kola nuts, mat6 leaves, cacao bean, guarana and the like. It is understood that "methylxanthine" includes substituted forms of methylxanthine, such as, for example, caffeine. If a second aqueous enzyme composition that is capable of degrading methylxanthine, is applied to the enzyme-degraded raw vegetable composition either during or after application of a first aqueous enzyme composition is applied to the raw vegetable composition, degradation of methylxanthine in the enzyme-degraded raw vegetable composition thereby occurs to reduce the level of methylxanthine in the enzyme-degraded raw vegetable composition.

Preferably, the second aqueous enzyme composition is applied to the enzyme-degraded vegetable composition for a time that is sufficient to degrade methylxanthine, such as, for example, about 1 minute to no more than about 7 days. For example, the enzyme application time may range from about 2 hours to about 24 hours. In another example, the enzyme application time may range from about 24 hours to about 7 days. The second enzyme composition that is capable of degrading methylxanthine generally remains in contact with the enzyme-degraded raw vegetable composition for a time that is sufficient to degrade more than about 5 weight percent methylxanthine of the raw whole vegetable composition when practicing the present invention.

In a third example, raw coffee beans or other caffeine containing raw vegetable compositions that have not been fermented and/or roasted may be degraded using cellulases, pectinases, and/or hemicellulases in an effort to decaffeinate raw coffee beans. In this example, the raw vegetable composition is enzymatically degraded for about 2 hours to about 24 hours prior to draining, drying, roasting and/or grinding.

After enzymatic degradation, the enzyme-processed vegetable composition having reduced levels of methylxanthine can then be blanched to inactivate any remaining enzymes and/or further processed by pulverizing, grinding, milling, heating, filtration, extraction, roasting, freezing, drying, freeze-drying, or any combination thereof. Subsequent additional processing by pulverizing, blending, grinding, paste-forming, roasting, freezing, drying, 004760952v2.doc 27 freeze-drying, extraction, or any combination thereof, may also deactivate the enzyme(s).

The benefits of processing vegetable compositions that include methylxanthine in accordance with the present invention include reducing the need for expensive solvent extraction equipment and chemicals that are traditionally required to decaffeinate raw vegetable compositions, such as, for example coffee bean. Additionally, the present embodiment may improve the flavor of decaffeinated coffee beans which may result in an increase in market share for a decaffeinated coffee manufacturer selling the enzyme-processed coffee beans.

As a third example, bitter flavor notes that characterize green unfermented cocoa beans may be reduced in accordance with the present invention. Cocoa beans can be divided into four categories, according to their color: fully fermented, predominantly a brown hue; purple/brown; purple; and slaty, in which slaty beans represent unfermented or green cocoa beans.

Purple/brown cocoa beans include all beans showing any blue, purple or violet color on an exposed surface, whether suffused or as a patch of the cocoa beans.

Purple cocoa beans should include all cocoa beans showing a completely blue, purple or violet color over the whole exposed surface.

An enzyme-degraded green unfermented cacao bean is formed in accordance with the present invention after application of an aqueous enzyme composition that includes cellulase, hemicelluase and pectinase degrades the first outer layer of green, unfermented or slaty cocoa beans. As used herein, a "green or unfermented cacao bean" includes cacao beans that do not have a sufficient quantity of amino acids and peptides required to form an acceptable cocoa flavor during subsequent roasting. Furthermore, "green or unfermented cacao beans" generally include beans that have not been subjected to a fermentation step or a drying step. Some non-exhaustive examples of green unfermented cacao bean that may be used in accordance with the present invention include beans derived from Theobroma sp., such as Ghanian cacao beans, Amelonado sp., Criollo, Forastero, or Trinitario that have been removed from the cocoa pod. Unfermented cocoa beans that have been degraded with cellulases, hemicellulases, and/or pectinases can then be subjected to normal fermentation and/or drying steps of about 1 to about 7 days that inactivates the enzymes and completes the fermentation process.

004760952v2.doc 28 If a second aqueous enzyme composition that includes an endoprotease, an exoprotease, or any combination thereof, is absorbed by the enzymedegraded green, slaty or unfermented cacao bean, degradation of protein in the enzyme-degraded green unfermented cacao bean generally occurs. Preferably, the endoprotease, the exoprotease, or any combination thereof, is capable of degrading protein that include hydrophobic amino acids and peptides that typically contribute to the bitter flavor notes that characterize green or unfermented cacao beans. Still more preferably, the endoprotease, the exoprotease, or any combination thereof, is applied to the green or unfermented enzyme-degraded cacao bean for a time, temperature, pH and moisture content of the green unfermented enzyme-degraded cacao bean that is sufficient to degrade the bitter flavor notes in the green unfermented cacao bean.

In practice, unfermented cocoa beans are enzymatically degraded for at least about 2 hours to about 7 days. After enzyme application, the unfermented cocoa beans are subjected to conditions that promote normal fermentation and/or drying of the enzyme-degraded cocoa beans. For example, enzymatic degradation may be allowed to proceed for about 8 hours to about 24 hours and then subjected to normal fermentation and/or drying of the enzyme-degraded cocoa beans. In another example, enzymes are applied for about 24 hours to about 5 to about 7 days and then subjected to normal fermentation and/or drying steps. Fresh enzyme composition may also be applied during degradation at timed intervals to facilitate hydrolysis and/or modification.

While not wanting to be bound to theory, it is believed that the enzymedegraded cacao bean includes sites through which the endoprotease, exoprotease, or any combination thereof, may be absorbed. Furthermore, the sites in the enzyme-degraded green unfermented cacao bean may facilitate subsequent natural fermentation of the green unfermented cacao bean by enhancing the capability of indigenous microflora of the natural fermentation process to colonize and digest the green unfermented cacao bean at the sites during the fermentation process.

The benefit of processing green unfermented cacao beans in accordance with the present invention include obtaining a less bitter cacao bean after fermentation and roasting steps. A less bitter cacao bean requires little flavor modification required during manufacture of cocoa containing products.

004760952v4.doc 29 In an example of practicing the method of reducing the flatulencecausing substrates in a raw vegetable composition, about 740 grams of water was added to about 7.5 grams of vinegar and brought up to a temperature of about 150F. About 2.5 milliliters of Viscozyme®L, and 2.5 milliliters of Alpha- Gal TM 600L, supplied by Novo Nordisk Biochem North America Inc., of Franklinton, North Carolina, were added to the vinegar and water mixture to form an aqueous enzyme composition with an initial pH of about 5.0. About 250 grams of raw collard greens were added to the aqueous enzyme composition and allowed to soak for about 30 minutes since greens have a much higher moisture content than grains or legumes. The raw collard greens were then cooked for about 30 minutes at about 200'F in the soak water. After cooking, the collard greens were drained and evaluated. Little, if any, flatulence was experienced after consumption of about 100 grams of collards even after 4 hours from the time of consumption of the collard greens.

In another example of practicing the method of reducing the flatulencecausing sugars in a raw vegetable composition, about 740 grams of water was added to about 7.5 grams of vinegar and brought up to a temperature of about 119 0 F to about 123°F. About 2.5 milliliters of Viscozyme®L, and 1.25 milliliters of Alpha-Gal T M 600L, supplied by Novo Nordisk Biochem North America Inc., of Franklinton, North Carolina, were added to the vinegar and water mixture to form an aqueous enzyme composition with an initial pH of about 5.0. About 250 grams of raw great northern beans were added to the aqueous enzyme composition and allowed to soak for about 120 to 240 minutes. The great northern beans were then blanched for about 5 minutes at about 200'F to deactivate the enzymes and cooked until palatable. The great northern beans did not result in any observable flatulence after human consumption.

Other examples are listed in Tables 1-6 below: 004760952v4.doc TABLE 1 Conditions Raw Raw Raw Raw Raw Raw Raw whole whole whole whole whole whole whole soybean soybean soybean soybean soybean soybean soybean Initial Wt 300 300 300 300 300 300 300 Lipase 0 0 0 0 0 0 1 0 Viscozyme 0.75 0.75 0.75 0.75 0.75 0.75 1.50 1 fungal acid 0 0.075 0 0.037 0 0.035 0 proteases Protease 0.075 0 0 0 0.038 0.042 0 Papain fungal 0 0 0.078 0.038 0.038 0 0.30 proteases Water 451 450 450 450 450 450 892 Initial Temp 112.7 111.9 113.6 109.2 107.6 112.7 125.9 Initial pH 4.17 4.17 4.17 4.44 4.44 4.44 4.25 Final Temp 92.4 90.6 91.0 89.3 91.3 89.0 94.7 Final pH 5.67 5.67 5.65 5.64 5.67 5.72 5.44 Application 122 121 130 121 130 135 133 Time (min) Final Wt 600 601 603 604 603 604 632 means this step was not performed or the variable was not measured.

All weights are in grams unless indicated otherwise.

004760952v4.doc 31 TABLE 2 Conditions Raw Raw raw Raw Raw Raw Raw whole whole whole whole whole whole whole soybean soybean soybean soybean soybean soybean Initial Wt 300 300 300 300 300 300 300 gr__ Lipase gr 0 0 0 0 0 0.30 0.30 Viscozyme 1.50 1.50 1.50 1.50 1.50 1.50 1.50 120L Cellulase 0 0 0 0.15 0 0 0 4000 Multifect 0 0 0 0 0 0 0 fungal acid 0.30 0 0.15 0 0.15 0 0 proteases Papain gr 0 0.30 0.15 0.15 0 0.30 0.30 Fungal 0 0 0 0.15 0.30 0 0 proteases Water (gr) 900 897 899 903 900 900 901 Initial 122.4 127.9 127 128.6 127.7 110-125 110-125 Temp Initial pH 4.29 4.21 4.26 4.29 4.26 4 to 5 4 to Final Temip 93.6 94.3 98.0 97.2 96.1-- F Final pHI 5.35 5.44 5.60 5.67 5.48-- Application 2.10 2.10 2.20 2.10 2.10 47---8r Time hrs Final Wt 627 631 636 637 642 All weights are in grams unless otherwise indicated.

'not measured or not performed.

#fresh enzyme and/or enzyme solution added after 2 hours 004760952v4.doc 32 TABLE 3 Conditions raw raw raw Raw Raw Raw Raw navy dark black pinto whole whole whole red soybean soybean soybean kidney Initial Wt 300 300 300 300 300 300 300 Lipase 0 0 0 0 0.30 0.30 0.30 Viscozyme 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1 Chymotrypsin, 0.30 0 0.30 0 0 0 0 trypsin Bromelain, 0 0.30 0 0 0.30 0.30 0.30 papain Protease 0 0 0 0 0 0 0 fuingal proteases Water 900 900 900 900 900 900 901 Initial Temp F 110 to 110 to 110 to 110 to 110 to 110 to 110 to 125 125 125 125 125 125 125 Initial pH 3 to 5 3 to 5 3 to 5 3 to 5 3 to 5 3 to 5 3 to Application 2.10 2.10 2.20 2.25 2.25 4#8 Time All weights are in grams unless indicted otherwise.

4 fresh enzyme and/or enzyme solution was added after 2 hours of soak time 004760952v4.doc 33 TABLE 4 Conditions Raw Raw Cocoa Cocoa Cocoa Cocoa whole whole beans beans beans beans beans beans InitialWt 300 300 2to5Kg 2to5Kg 2to5Kg Lipase (gr) 0 0.30 0 0 0 0 Carbohydrase 1.50 1.50 0.5% 0.5% 0.5% fungal acid 0 0 0.10% 0.10% 0.10% 0.10% proteases papain 0.30 0.30 0.05% 0.05% 0.05% 0.05% fungal 0 0 0.10% 0.10% 0.10% 0.10% proteases Water 900 900 2 to 5Kg 2 to 5Kg 2 to 5Kg 2 to Initial Temp 110 to 110 to 110 to 110 to 110 to 110 to 125 125 125 125 125 125 Initial pH 4 to 5 4 to 5 4 to 5 4 to 5 4 to 5 4 to Application 16 hrs 16 hrs 24hrs" 48hrs 72hrs' 4 days soybeans, dark red kidney, black, pinto, navy, brown rice, corn, (8) hominy, grain wheat All weights are in grams unless indicated otherwise.

fresh enzyme and/or enzyme solution added after 2 hours.

fresh enzyme solution added after 8 and 16 hrs.

fresh enzyme solution added to certain treatment after 24 hrs.

fresh enzyme solution added to certain treatments after every 24 hrs.

enzyme is percent by weight of raw vegetable composition 004760952v4.doc 34 TABLE Conditions Cocoa Cocoa Cocoa Cocoa Cocoa Cocoa beans beans beans beans beans beans Initial Wt 2to5Kg 2to5Kg 2to5Kg 2to5Kg 2to5Kg Lipase 0 0.3% 0.05% 0.05% 0.05% 0.05% Viscozyme 0.50% 0.5% 0.5% 0.5% 0.5% 1 Fungal acid 0.25% 0.25% 0.10% 0.10% 0.10% 0.10% proteases Papain 0 0 0.05% 0.05% 0.05% 0.05% Fungal 0 0 0.10% 0.10% 0.10 0.10% proteases______ Water 2to5Kg 2to5Kg 2 to 5Kg 2 to 5Kg 2 to 5Kg 2 to Initial Temp 110 to 110 to 110 to 110 to 110 to 110 to 125 125 125 125 125 125 Initial pH 3 to 4 3 to 4 4 to 5 4 to5 4 to 5 4 to Application 4dy Tdays days" days"+- 7days" 7 days"+ Time #*fresh enzyme solution added to certain treatment after 24 hrs.

fresh enzyme solution added to certain treatments after every 24 hrs.

indicates fresh enzyme solution added to certain treatments after the Pd and 5t" day.

enzyme is percent by weight of raw vegetable composition TABLE 6 Conditions Cocoa Cocoa Cocoa Cocoa Cocoa Cocoa beans beans beans beans beans beans Initial Wt 2to5Kg 2to5Kg 2to5Kg 2to5Kg 2to5Kg Lipase 0 0% 0 0 0.3% 0.3% Viscozyme 0.50% 0.5% 0.5% 1.5% 1.0% 1 20L__ Water 2to5Kg 2to5Kg 2 to 5Kg 2 to 5Kg 2 to 5Kg 2 to Initial Temp 110 to 110 to 110 to 110 to 110 to 110 to 125 125 125 125 125 125 Initial pH 3 to 4 3 to 4 4to 5 4to 5 4 to5 4 to Application 4days'" days'* days" daysm*+ 7days" 5 days'* Time fresh enzyme solution added to certain treatments after every 24 hrs.

indicates fresh enzyme solution added to certain treatments after the Pd and 5 t day.

enzyme is percent by weight of raw vegetable composition.

004760952v4.doc A method of modifying a vegetable composition As noted, the enzyme-degraded raw vegetable composition of the present invention is capable of absorbing additives to form a modified vegetable composition. The method of modifying a vegetable composition in accordance with the present invention is a significant improvement in the art of vegetable processing. Typically, a vegetable composition is mechanically modified, such as by peeling, grinding, pulverizing, prior to inclusion of an additive, followed by subsequent processing by conventional means. The added step of mechanical modification, along with any safety and health hazards that accompany using equipment involved in the mechanical modification of vegetables, may be eliminated when practicing the present invention. Additionally, the present invention accomplishes in situ modification of a raw vegetable composition.

As an example of the present embodiment that modifies raw vegetable composition, about 740 grams of water was added to about 7.5 milliliters of vinegar and brought up to a temperature of about 119 0 F to about 123 0 F. About milliliters of Viscozyme®L, supplied by Novo Nordisk Biochem North America Inc., of Franklinton, North Carolina, was added to the vinegar and water mixture to form an aqueous enzyme composition with an initial pH of about 4.8. About 250 grams of raw pinto beans were added to the aqueous enzyme composition and allowed to soak for about 135 minutes. The change in pH, indicating the absorption of vinegar, is presented in Table 7 below: TABLE 7 TIME (minute) TEMPERATURE pH 0 123.1 4.78 118.5 5.15 117.7 5.47 118.9 5.56 135 119.2 5.72 Conclusion In view of the foregoing disclosure and embodiments, it is believed that processing a raw vegetable composition in accordance with the present 004760952v4.doc 36 invention represents a significant improvement in the art of vegetable processing. The development of an effective process that reduces the complexity and costs associated with vegetable production, by reducing the first outer layer of a vegetable composition that typically hinders processing, creates a vegetable product with enhanced processing characteristics.

Furthermore, the development of an in situ method of processing and modifying a raw vegetable composition greatly enhances the ability of a food manufacturer to produce vegetable products that offer a wide variety of nutritional characteristics to consumers. For example, in situ modification of the carbohydrate component of raw vegetables is possible when cellulases, hemicellulases, pectinases, alpha-galactosidases, or the like are applied for more than about 2 hours. Similarly, in situ hydrolysis of the protein fraction of raw vegetable compositions generally occurs when a combination of carbohydrases and proteases are applied for more than about 2 hours. Additionally, in situ modification of lipids of raw vegetable compositions generally occurs when a combination of carbohydrases and lipases are applied for more than about 2 hours.

Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be ascertained, understood and regarded as relevant by a person skilled in the art.

Claims

1. A method of enzymatically degrading a raw vegetable composition for subsequent human consumption, the method comprising: providing a raw vegetable composition having a moisture content of less than about 30 weight percent; applying an aqueous enzyme composition comprising water and a cellulase to the raw vegetable composition under normal atmospheric pressures for more than about 2 hours, wherein the aqueous enzyme composition is at an initial pH of between about 2.0 and 7.0; and deactivating the aqueous enzyme composition.

2. The method of claim 1 wherein the aqueous enzyme composition is effective to degrade a first outer layer of the raw vegetable composition.

3. The method of claim 1 wherein the aqueous enzyme composition is effective to tenderize the raw vegetable composition.

4. The method of claim 1 further including applying a second aqueous enzyme composition to the raw vegetable composition, wherein the second aqueous enzyme composition is selected from the group consisting of alpha- galactosidase, mannanase, beta-giuconase, beta-gluconase, arabinase, xylanase, beta-galactosidase, invertase, beta-fructofuranosidase, alpha-amylase, beta- amylase, pectinase, pectin depolymerase, pectin methyl esterase, pectin lyase, glucoamylase, oligo-1,6 glucosidase, lactase, beta-rf-glucosidase, and any combination thereof. A method of enzymatically processing a vegetable composition for subsequent human consumption, the method comprising: providing a raw vegetable composition having a moisture content of less than about 30 weight percent; applying a first enzyme composition comprising water and a cellulase to the raw vegetable composition under normal atmospheric pressures for about 2 hours to about 24 hours, wherein the first enzyme composition is at an initial pH of between about 2.0 and 004760952v4.doc 38 applying a second enzyme composition comprising water and a carbohydrase to the enzyme-degraded raw vegetable composition; and deactivating the first enzyme composition and the second enzyme composition.

6. The method of claim 5 wherein the second enzyme composition is selected from the group consisting of hemicellulase, alpha-galactosidase, mannanase, beta-gluconase, beta-gluconase, arabinase, xylanase, beta- galactosidase, invertase, beta-fructofuranosidase, alpha-amylase, beta-amylase, pectinase, pectin depolymerase., pectin methyl esterase, pectin lyase, glucoamylase. oligo-1,6 glucosidase, lactase, beta-c/-glucosidase, and any combination thereof.

7. A method of processing a vegetable composition for subsequent human consumption, the method comprising: providing a raw vegetable composition having a moisture content of less than about 40 weight percent; applying an enzyme composition having a pH of between about 2.0 and to the raw vegetable composition under normal atmospheric pressures for 2 0 more than about 2 hours, wherein the enzyme composition includes water, a first enzyme component, and a second enzyme component, wherein the first enzyme component includes a cellulase that degrades the raw vegetable composition, and wherein the second enzyme component includes a protease that degrades a protein or a peptide; and deactivating the enzyme composition.

8. The method of claim 7 wherein the raw vegetable composition is a legume, a soybean, grain, an edible seed, a green or unfermented cocoa bean, or any combination thereof.

9. The method of claim 7 wherein the protease degrades a hydrophobic amino acid containing protein, a hydrophobic amino acid-containing peptide, or any combination thereof. 004760952v4.doc 39 The method of claim 7 wherein the enzyme composition is applied for less than 7 days.

11. The method of claim 7 wherein the protease is effective to hydrolyze the protein of the vegetable composition.

12. A method of processing a vegetable composition for subsequent human consumption, the method comprising: providing a raw vegetable composition having a moisture content of less than about 30 weight percent; applying an enzyme composition having an initial pH of between about and 7.0 to the raw vegetable composition for about 2 hours to about 24 hours, wherein the enzyme composition includes water, a first enzyme component, and a second enzyme component, wherein the first enzyme component includes a cellulase that is effective to degrade the raw vegetable composition, wherein the second enzyme component is effective to degrade methylxanthine; and deactivating the enzyme composition.

13. The method of claim 12 wherein the raw vegetable composition is a green coffee bean, a cacao bean, a guarana, a cola nut, or any combination thereof.

14. A method of tenderizing a vegetable composition for subsequent human consumption, the method comprising: applying an enzyme composition that includes water and a cellulase to a raw vegetable composition under normal atmospheric pressures for more than about 2 hours, wherein the enzyme composition has an initial pH of between about 2.0 and 7.0, and wherein the enzyme composition is effective to tenderize the raw vegetable composition; and deactivating the enzyme composition. The method of claim 14 wherein the enzyme composition is effective to reduce a cook time of the bean. 004760952v4.doc

16. A method of modifying a raw vegetable composition comprising: applying an enzyme composition that includes water, at least one protease, and at least one carbohydrase to a raw vegetable composition under normal atmospheric pressure for more than about 2 hours, wherein the enzyme composition has a pH of between about 2.0 and 7.0, and wherein the enzyme composition is effective to hydrolyze the protein content of the raw vegetable composition; and deactivating the enzyme composition.

17. The method of claim 16 wherein the enzyme composition is applied for about 2 hours to about 24 hours.

18. The method of claim 16 wherein the enzyme composition is applied for at least about 24 hours.

19. The method of claim 16 wherein the enzyme composition is applied for no more than 7 days. A method of modifying a raw vegetable composition comprising: applying an enzyme composition that includes water, at least one lipase, and at least one carbohydrase to a raw vegetable composition under normal atmospheric pressures for more than about 2 hours, wherein the enzyme composition has a pH of between about 2.0 and 7.0, and wherein the enzyme composition is effective to hydrolyze the lipid content of the raw vegetable composition; and deactivating the enzyme composition.

21. The method of claim 20 wherein the enzyme composition is applied for about 2 hours to about 24 hours.

22. The method of claim 20 wherein the enzyme composition is applied for at least about 24 hours.

23. The method of claim 20 wherein the enzyme composition is applied for no more than 7 days. 004760952v4.doc 41

24. An enzyme-degraded vegetable composition comprising a raw vegetable composition degraded by an enzyme composition, comprising water, at least one carbohydrase and at least one protease, for more than about 2 hours at an initial pH of about 2 to about 7. An enzyme-degraded vegetable composition comprising a raw vegetable composition degraded by an enzyme composition, comprising water, at least one carbohydrase and at least one lipase, for more than about 2 hours at an initial pH of about 2 to about 7.

26. An enzyme-degraded vegetable composition comprising a raw vegetable composition degraded by an enzyme composition, comprising water and cellulose, for more than about 2 hours at an initial pH of about 2 to about 7.

27. A method of modifying a raw vegetable composition comprising: applying an enzyme composition that includes water, at least one lipase, at least one protease, and at least one carbohydrase to a raw vegetable composition under normal atmospheric pressures for more than about 2 hours, wherein the enzyme composition has a pH of between about 2.0 and wherein the enzyme composition is effective to hydrolyze the lipid content of the raw vegetable composition; and deactivating the enzyme composition.

28. The method of claim 27 wherein the enzyme composition is applied for about 2 hours to about 24 hours.

29. The method of claim 27 wherein the enzyme composition is applied for at least about 24 hours. The method of claim 27 wherein the enzyme composition is applied for no more than 7 days.

31. A method according to any one of claims 1, 5, 7, 12, 14, 16, 20 or 27, substantially as described herein with reference to any one of the examples. 004760952v4.doc 42

32. An enzyme-degraded vegetable composition according to any one of claims 24, 25 or 26 substantially as described herein with reference to any one of the examples.