CA2102607A1 - Enzyme systems - Google Patents
Enzyme systemsInfo
- Publication number
- CA2102607A1 CA2102607A1 CA002102607A CA2102607A CA2102607A1 CA 2102607 A1 CA2102607 A1 CA 2102607A1 CA 002102607 A CA002102607 A CA 002102607A CA 2102607 A CA2102607 A CA 2102607A CA 2102607 A1 CA2102607 A1 CA 2102607A1
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- CA
- Canada
- Prior art keywords
- enzyme
- product
- group
- agents
- mixtures
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D8/00—Methods for preparing or baking dough
- A21D8/02—Methods for preparing dough; Treating dough prior to baking
- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
- A21D8/047—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with yeasts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/137—Thickening substances
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/20—Compounding polymers with additives, e.g. colouring
- C08J3/201—Pre-melted polymers
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38663—Stabilised liquid enzyme compositions
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38672—Granulated or coated enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C2220/00—Biochemical treatment
- A23C2220/20—Treatment with microorganisms
- A23C2220/204—Use of bacteria which are encapsulated, entrapped or immobilised; Fermentation with these bacteria
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C2260/00—Particular aspects or types of dairy products
- A23C2260/05—Concentrated yoghurt products, e.g. labneh, yoghurt cheese, non-dried non-frozen solid or semi-solid yoghurt products other than spreads; Strained yoghurt; Removal of whey from yoghurt
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/68—Treatment of water, waste water, or sewage by addition of specified substances, e.g. trace elements, for ameliorating potable water
- C02F1/681—Treatment of water, waste water, or sewage by addition of specified substances, e.g. trace elements, for ameliorating potable water by addition of solid materials for removing an oily layer on water
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T428/00—Stock material or miscellaneous articles
- Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
- Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Compositions Of Macromolecular Compounds (AREA)
- Detergent Compositions (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Enzyme products are disclosed. The enzyme products include an enzyme-bearing matrix formed by subjecting a feedstock containing enzyme(s) and carrier materials to conditions which alter the physical and/or chemical structure of the carrier. The matrix suspends the enzyme for protection, delivery, dispersion and activation at the desired time and under selected conditions. Methods of producing the enzyme carrying matrix and enhanced enzyme products are also disclosed.
Description
W092/~032~ PCT/US92/0404~
2102~i07 E~YME SYSTEMS
BACKGROUND OF THE INVENTION
The present application is a Continuation-In-Part of U.S. Patent Application Serial No. 07/702,068 filed on ~ay 17, 1991.
The presant invention relates to new enzyme products. In particular, the invention relates to improved enzyme products such as lea~ening agents, alcohol fermenters, detergent ingredients, degradation agents, diagnostic agents, bioremediation agents, catalases and oxidases.
,::
Enzymes are proteins which catalyze many biological reactions. As a result of their extraordinaxy catalytic power and sp~cificity, enzymes have been used to speed .
up processes that woul~ not otherwise occur~ Many isolated enzymes axe relatively uns~able, often gradually lose activity prior to use, and may be easily inhibited by many factors, ~ O~er the years, a number of en~yme products have been developed for a variety of purposes. For exampls, I foods, qetçrgents, cosmetics and pharmaceuticals ha~e :~ all been enhanced by enzymes~ Many commercially prepared enzyme-based products, however, have certain _, .
: ~3 drawbacks. ~`
,,, ~
WOg2/203~9 PCT/US92/0404~ ` :
2102~i07 E~YME SYSTEMS
BACKGROUND OF THE INVENTION
The present application is a Continuation-In-Part of U.S. Patent Application Serial No. 07/702,068 filed on ~ay 17, 1991.
The presant invention relates to new enzyme products. In particular, the invention relates to improved enzyme products such as lea~ening agents, alcohol fermenters, detergent ingredients, degradation agents, diagnostic agents, bioremediation agents, catalases and oxidases.
,::
Enzymes are proteins which catalyze many biological reactions. As a result of their extraordinaxy catalytic power and sp~cificity, enzymes have been used to speed .
up processes that woul~ not otherwise occur~ Many isolated enzymes axe relatively uns~able, often gradually lose activity prior to use, and may be easily inhibited by many factors, ~ O~er the years, a number of en~yme products have been developed for a variety of purposes. For exampls, I foods, qetçrgents, cosmetics and pharmaceuticals ha~e :~ all been enhanced by enzymes~ Many commercially prepared enzyme-based products, however, have certain _, .
: ~3 drawbacks. ~`
,,, ~
WOg2/203~9 PCT/US92/0404~ ` :
2~ ~ :
1 As an illustration, detergent enzymes, are usually produced in powdered or liquid form. They are difficult to handle, may cause an irritating dust, may be incompatible with other detergent products, and may deteriorate in the presence of moisture. The activity of enzymes in liquid detergents, which contain high levels of water and surfactants, tends to decrease over time. Frequently, the surfactants inactivake the enzymes. Consequently, there is a need to prepare enzyme products suitable eOr detergents which are easy to handle, do not cause irritation to users, and can be ~;
distributed uni~ormly in the detexgent without reduced activity.
:'.
Similarly, it is important to be able to deliver and activate l~avening agents and alcohol fermenters at the desired time and location in a biomass. For "
example, yeast has a tendency to "clumpl' together in aggregates which resisk being dispersed during mixing.
This "clumping" occurs with both dry formulations and paste formulations of~yeast when added to dough or to a ;~ -biomass. Thus, it would be beneficial to be able to
1 As an illustration, detergent enzymes, are usually produced in powdered or liquid form. They are difficult to handle, may cause an irritating dust, may be incompatible with other detergent products, and may deteriorate in the presence of moisture. The activity of enzymes in liquid detergents, which contain high levels of water and surfactants, tends to decrease over time. Frequently, the surfactants inactivake the enzymes. Consequently, there is a need to prepare enzyme products suitable eOr detergents which are easy to handle, do not cause irritation to users, and can be ~;
distributed uni~ormly in the detexgent without reduced activity.
:'.
Similarly, it is important to be able to deliver and activate l~avening agents and alcohol fermenters at the desired time and location in a biomass. For "
example, yeast has a tendency to "clumpl' together in aggregates which resisk being dispersed during mixing.
This "clumping" occurs with both dry formulations and paste formulations of~yeast when added to dough or to a ;~ -biomass. Thus, it would be beneficial to be able to
3 suspend agents, such as yeast, in a medium for delivery and release as desired. This is especially true when~;
the receiviny material is an extensive mass, such as dough in baking and the biomass in fermentation ;~
procedures.
' -~ ' .
,, '"
W092/2032~ 2 1 0 2 ~ 0 7 PCT/US9~/04048 1 Other enzyme-bearing products can benefit from .;
enhanced shelf-life. At room temperature enzymes used as indicators in immunoassays frequently experience short shelf-like. Horseradish peroxidase, lipoprotein lipase, glycerol-3~phosphate oxidase are ordinarily stored as freeze-dried powders at 20C. Co~nonly-used assays are conducted in the range of 20-30~C. It is thus important to provide a matrix which can improve the sheIf life of enzyme~used in immunoassays without impairing their activity.
There have been attempts in the past to deal with the problems associated with the use of enzymes. In United States Patent No.:3,095,358, sorbitol is used to . ..
stabilize aqueous s~lution~ containing papain, proteases and amylases. This method requires large amounts of : ~ stabilizing agen~ and is, therefore, expensi~e.
In U.S. Patent~No. 3,~96,094, partially hydrolyzed ~ and solubilized collagen and glycerol are used to : stabilize aqueous solutions o~ proteolytic enzymes. :
:~ ~ This method requires large quantities of glycerol and, 3 ~ therefore, adds~significantly to the C05t of the enzyme solutidnO ~
; ~ 5 ~ U.S. Patent No. 3~,7~9,671 discloses a method of preparing enzyme-containing prills for use in laundry deter~ents. The disclosed prilling method requires the . .
W092/~0329 P~T/US~2/04~8 :
2~02 ~~ _4_ 1 following steps: (a) heating a normally solid translucent material to a temperature sufficient to melt ~;
the material but insuf~icient to destroy the activity of the enzyme; (b) forming a slurry of the melted material and the enzyme; (c) injecting an inert gas into the slurry to form a uniform dispersion with the gas; ~nd (d) forming prills from the resulting slurry. This method has many steps which require energy, equipment, and manual labor. :
'.~
15~ Although the methods discussed above represent efforts to improve enzyme-containing detergent products, the problems associated with the decreases in enzyme ;.
activity over time and adequate dispersal have not bee~
solved.
,',' '' ~
It is, therefore, an object of this invention to pro~ide an enzyme product which disperses or dissolves uniformly in the target liquid while retaining the enzyme activity for prolonged periods of time pri~r to use.
~-3 It is another object of this invenkion ko provide a ,.
! matrix whlch facilitates mixing an enzyme with a mass so : that the enzy~e can be:dispersed efficiently throughout the mass.
~
"'':
W092/2032~ PCT/US92/0404g ~102607 1 It is yet another object of this invention to provide an enzyme product that exhibits an enhanced shelf 1ife.
~.
Other and further o~jects of the prese~t invention `:
will beco~e apparent the fo11Owing description and its scope will be pointed out with the appended s1aims.
. SUMMARY OF THE INVENTION
15~ The present invention inc1ud2s an enzyme product ~ .
which contains a matrix formed by subjecting a feedstock containing an enzyme and a carrier material to conditions of temperature and shear sufficient to produce the matrix which suspends the enzyme for storage an~ use. The carrier material undergoes transformation :~ during processing in which its physical and/or chemical ; s~ructure is a1tered.
: . .
. -"Enzyme product" in the present invention means a pxoduct which includes one or more enzymes. A
non~imiting list of enzymes which can be suspended in ~-3 the matrix includes amylases, proteases, invertases, :~
g1ucose' oxidases, pectinases, lipases, lactases, and cellulases. The enzymes make up from about 1~ to about 30~ by weight of the matrix, with amounts of from about ...
..
5% to about 25% being preferred an~ the amounts are from a~out l0% to about 20~ being most preferred.
, ~ ' ",.
'~.
WO 92/20329 PCr/US92/0404~
2~2SOT'l Carrier materials which can be used for the matrix are saccharides, thermoplastic polymers, biodegradable polymers and water soluble cellulosic materials. The saccharides may be sucrose, lactose, fructose, dextrose, sorbitol, mannitol, maltose and mixtures thereof. The saccharides may also be selected from polydextrins, maltodextrins, and mixtures thereof. Thermoplastic ", ,, polymers include polypropylene, polyst~rene, polyethylene, polyvinylacetate, polyvinylalcohol, poly (methyl methacrylate~, polyacrylic resins, 15~ lactide/glycolide copolymer and mixtures thereof.
Biodegradable polymers include poly(cis-isoprene) aliphatic polyesters, polyurethanes and urea-formaldehyde polymers. The cellulosic materials are ;~
water soluble and include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, ethyl cellulose, ~;
alkali metal alts of carboxy methyl cellulose and mixtures thereof.
As a result of the present invention, Pnzymes can be suspended, protected, dispersed and generally engineered for selective delivery at desired sites under selected conditions. Various enzyme products can be provided which disperse or dissolve uni~ormly in the arget liquid, biomass,; etc. The enzyme products of this invention can also be designed to retain their ~ ~
activity for long~periods of time prior to use. A non- ;
.~.
inclusive list of uses~for the matrix of the invention :~' W092l2~329 P~T/US9~04048 2~026~7 1 includes leavening agents, alcohol fermenters, detergents, digestive aid products, clinical diagnostic agents, bioremediation agents, meat tenderizing products, wound debridement and other therapeuti~al uses.
For a be~ter understan~iny of the present invention, reference is made to the following description and its scope will be pointed out in the appended claims.
, .;
DETAILED DESCRIPTION OF THE INVENTION
In the present invention an enzyme-bearing matrix .
can be formed by subjecting carrier feedstock and an enzyme to conditions of temperature and shear to ~or~
the matrix. This can be accomplished by melt spinning the enzyme with carrier material~ The matrix is ;.;
: included in ~arious enzyme~based products such as :;
leavening agents, alcohol fermenterc, detergents~ .
diagnostic agents, degradation products, petroleum hydrocarbons degraders, digestive aids, t~erapeutic ~
enz~mes, etc. ~:
: 30 The spinning process can be carried out with "cotton ca~dy" fabricating-type ~quipment~ The spinning :~
machine~used in the present invention can be a cotton candy-type machine, such as the Econo Floss model 3017 manufactured by Gold Medal Products Company of :~
'',....
w~92/2032g ~,~ o~6S~ PCl'/US92/0404 l Cincinnati, Ohio. It will be appreciated by those .
skilled in the art that any apparatus or physical process which provides similar forces and temperature '~
gradient conditions can also be used. For simplicity in disclosing and describi.ng this invention, the term 4;
"melt-spinning" will be understood to mean a process :~
which includes a combination of temperature, shear, ;;~
flow, ~low rate, mechanical forces and thermal gradients of the type produced by a cotton candy-type machine. ;~
15~ The apparatus is operated at a temperature and speed which induce flash flow of certain carrier ~eedstocks without deterioration o~ the feedstock and .
enzyme ~5) being processed. The resulting matrix is in the form of a ~loss, fibre, particle, flake, spicule, or ~th~r generally non-descript aggregAte capable of protectively carrying and deli~ering an enzyme.
'';' ~25 Th~ process for producing the matrix includes :
introducing a mixture containing an enzyme and a carrier ~
material simultaneously to conditions of elevated :
temperature and shear created by centrifugally forcing 3 the ingredients th~ough orifices. The extremely short amount of time the lngredients are exposed to the elevated temperature and shear alIows the matrix to be formed without adverse effects.
' :`
~' W092/20329 210 2 6 0 7 PCr/US92/040~8 1 The flash flow phenomena occurs when a solid carrier material mixed with an enzyme is subjected to conditions of melt-spin sufficient to provide internal flow. This condition produces the transformation in ;`~
physical and/or che~ical structure without degradation .~, of the material. Internal flow occurs when the infrastructure of the material breaks down sufficiently ..
, to permit movement of material at a subparticle level, .
and probably at a molecular level. At a molecular level, internal flow contemplates the movement of 15~ molecules relative to each other~ `.;
. .
Internal flow of material is generally associated with melting point or glass transition point. However, it is contemplated that the combined application of heat and external force is suf~icient to produce flow at temperatures below the melting or glass transition point for most compositions~
:25 The enzymes dispersed in the matrix are selected from animal-derived~, plant-derived and microbially- ;:, derived pr~para~ions. ~hese enzymes can be used as part . :~
of a leavening product, an alcohol fermenter, ~
detergent, a clinical diagnostic agent or a bioremedient and pos~ibly mixtures~thereof 7 A nonlimiting list 3 includes amylases, proteases, invertases, oxidases, catalases, pectinases, lipases, lactases, cellulases and mixtures thereof.~
- :.
~;:
W092/20329 ~ 6~ PCT/US92/~4048 1 In one aspect of the present invention, the matrix may be formed by mixing the carrier material with degradation enzymes such as cellulases, cutinases, lipases and pectinases and mixtures thereof. Cellulase sources include ~hose originating in the genexa Trichoderma, Penicillium, Aspergillus, Clostridium, etc.
Additional cellulases can include commercially available products. Such cellulases are capable of degrading the water insoluble cellulose polymer which is part of the surface membrane of fruits and Yegetables~
'';.',.
Cutinase sources include those originating in the genera Pseudomonas, Fusarium, Botrytis, Uloclad.ium, etc.
Additional cutinases can include commercially available ;~
products. Cutinases are capable of degrading water insoluble cutin polymer which m~y be present as part of ;~
the surface membran of fruits or vegetab~es.
,.....
; 25 Lipase sources include tho e originating in the genera iSitaphyl~coccus, Candida, Rhizopus, etc.
Additional lipases can include commercially available products. Such lipases are capable of degrading water -;
insolub1e glycerol components comprising part of the surface membrane of fruits or vegetables.
Pectinase sources incIude those originating in the genera Rhizopu5, Penicillium, Aspergillus, etc.
Additional pectinases can include commercially available : ~ ~ ' '"',`~
~ ' ' 21026~7 l products. Such pectinases are capable of deyradlng the water insoluble pectin components comprising part of the surface membrane of fruits or vegetables.
:
Th~ enzyme bearing matrix of the invention has many uses. For example, a cellulase matrix may be used to increase the permeability of the surface membrane o~
fruits and vegetables. The lncreased water permeability across the surface membrane permits easier delivery o~
substances such as flavorings, sweeteners, stabilizers 15 ~ and preservatives to the interi.or of the fruit or vegetable. Additio~ally, the increased water ~ . .
permeability allows for a more efficient method of dehydration of fruits and vegetables. More important~y, 20 the use of naturally produced degradation enzymes as pe~meability enhancers replaces the use of chemicals :~
such as methanol, chloroform or alkali metal hydroxides, which, if ingested, pose potential harmful side effects .
to consumers o~fruits and~vegetables. ..
Another important use for the enzyme caxrier matrix '~
of this in~ention is in:~the preparation of clinical -: ~ ~iagnostics products. A nonlimiting list of active i~gr di~nts found in cli~ical diagnostic products include:ascorbic ac~d oxidase, ~-glycerophosphate oxidase, lactate oxidase, uriase, cholesterol esterase, : cholesteroI ester hydrolase, creatinine amino hydrolase, ~::
lipase, glycerol kinase, and~mixtures thereo~.
''''''.''''''' ,.. .
W092/~0329 PCT/U~9~/04048 ~r~0~?~ 12- .
1 The clinical enzyme products contemplated herein are particularly well suited for use with the matrix of :~
the invention when it is desired to disperse the dry , 5 powder enzymes in aqueous liquids. It should be readily apparent to the skilled artisan that all of the active . .
ingredients may also be provided in dry or lyophilized form and reconstituted with water prior to use.
Compositions of this type are clearly contemplated by this in~ention. Clinical diagnostic enzymes carried in the matrix of the invention can also ~e incorporated into single-layer or multi-layer analytical elements o~
the types known in the prior art.
In another aspect of this invention, the matrlx may be used to enhance the shelf-life and activity of en~ymes used in i~munoassays. For example, when horseradish peroxidase was spun with the matrix of the invention, the enzyme exhibited a longer shelf-life, an~
became more readily active.
, Another class of enzyme products ascording to the invention are~improved detergent enzymes. ~etergent ~-enzymes are known in the art as enzymes which attack ,;
stains or soiled areas of fabrics. Suitable categories .
of: ative detergent enzymes found in improved detergents ~:
include proteases, lipases, amylases, and mixtures thereof. The preferred detergent enzymes are proteases such as subtilisin and amylases such as those derived ::
W092/20329 ~ 1 0 2 6 0 I PCT/US92/04048 1 from the bacillus species.
The new matrix can be used alone or in combination with other ingredients as a means for dispersing the ; . .
added ingredients throughout the material. For example, particles, chips, flakes, spicules or combinations thereof can be used to disperse enzymes which are otherwise relatively non-dispersable because o~ the physical characteristics of such materials. Thus, the matrix of the invention can be used to carry detergent ,;~
, . enzymes to be dispersed more easily and uni~ormly in ::~
other materials present in ~etergent formulations, such as surfactants, builders, whitening agents, bleaching agents and the like.
In certain embodiments the enzymes are present in the host microorganism such as in fungi, bacteria or , algae. Examples of host microorganisms include yeasts, ~;~
bio-remediation materials and the like.
;~ '.
: In another aspect of the invention, yeasts may be `.~. melt-spun with selected carrier materials to obtain :~
3 enhanced leavening products. Yeasts are single cell microorganisms containing enzymes which are employed in large scale fermentation:processes~ The commercial production of fermented beverages, foods~ production of vitamins, alcoholic fermentation, antibiotic producing fermentations, all require yeasts or their enzymes to . :,' , ~
WO92/2032g PCT/~'S92/0404~ ~
.,6~
1 produce products simpler than the starting material.
Regardless of the substrate used or the chosen -microorganism, industrial fermentations require various nutrients for growth including carbohydrates, nitrogen- -containing compounds, growth factors, vitamins and minerals~ In most fermentations, these nutritional re~uirements are met by including, among others, yeast products.
In the production of alcoholic ~everages, cereal , grains are the principal raw material. Another important ingredient is malt that is used to produce amylase. ~mylas~s are organic enzymes that change grain starch into maltose. In fermentation, zymase which is produced by yeasts converts the amylase produced maltose into ethyl alcohol and carbon dioxide. Saccharomyces cervisiae is thP most common type of yeast used in alcoholic fermentation to generate zymase. ;
In fermentation processes desired metabolic changes frequently occur ln a narrow temperature and pH range.
Acc~rdingly, to increase product yields t it is important 3 to deliver yeasts having enzymatic activity in a narrow temperature and pH range. In addition, to optimize product yields, the yeasts must be rapidly and uniformly dispersable in the target liquid. Thus, yeasts suspended in the matrix of the invention are easily dispersable in the nutrient medium and have been found, ` ~
W0~2/20329 PCT/US92/0404~
210~fiO7 l in some cases, to be more readily active.
.: -In another aspect of this invention, the matrix may be used to enhance the shel~ life and activity of enzymes used in yeasts. For example, when Fleischman's ;:
dry yeast was spun with the matrix of the invention, the :.~
:
yeast exhibited a longer shelf~life and became more readily active.
~''"
Another significant use for the enzyme carrier ;~.
15~ matrix of the invention finds application in the biodegradation of petroleum hydrocarbons. Many species of bacteria, fungi and algae have the enzymatic .,,.. ~.
capability to use petroleum hydrocarbons as food. The :.
,, .:,:
bacteria genera most frequently isolated as hydrocarbon ~;
degraders are P-~eudomonas, AcinetobaGter, , ~, F~avobacterium, Brevibacterium, Corynebacterium, Arthrobacter. The fungus genera include Candida, .. ..
25 ~ Cladosporium, Trichosporium and Rhodotorula. These . .
bac~eria and fungi are present in the environment. :.
Genetically engineered bacteria which have the enzymatic capability of degrading several groups of hydrocarbons .
can also be used~as~petroleum biodegraders.
Using the matrix;of the invention, it is possible to dis~erse rapidly and uniformly these biodegrading agents and their nutrients into an otherwise hydrophobic ~`
or: immisoible environment.~ In this manner the microbial ~ ~ ';.;
uro g2t20329 r Pcr/uss2/o4o4s 6~ 1 -16- !
l cleanup of oil spills occurs more rapidly.
, In yet another aspect of this invention, the enzyme carrying matrix may be used in the field of enhanced oil recovery. Microbial products, as well as viable microorganisms, suspended in the matrix may be used as -:~
~timulation agents to enhance oil recovery from petroleum reservoirs. F~r example, a strain of Acinetobacter calcoaceticus produces emulsan, a lipopolysaccharide used to stabilize oil in water 15~ emulsions. Xanthomonas campeskris is a microbial ~.
product producing xant~an, a polysaccharide used as a water flood thickening agent in oil reco~ery. Both these microbial products become easily miscible in petroleum reservoirs when deli~ered with the matrix of the in~ention. The result is enhanced oil recovery.
:
~ The ability of microorganism~ to use petroleum as ,.
food also has detrimental effects. For example, ~ :
~: petroleum fuels cannot become contaminated with water or microorganisms~during storage. Such contamination poses a serious problem~for kerosine based jet aircraft fuels.
3 To diminish this problem, antimicrobials that , :~ concentrate at the oil/water interfaces may be used to ~:~
reduce the rate of~microbial contaminatlon of ;~ hydrocarbons. Ant~imicrobial organisms may be delivered ~:;
: ~ ~ at the oil/water interfaces by using the matrix of the ~ invention.
. :.:
W092/20329 P~T/US92/04~8 l The carriers used in the invention can be any material capable of being processed to form a matrix which can protectively suspend the enzyme for storage and/or selective delivery to the site and/or environment for release and acti~ation~ Carrier materials contemplated for use may be saccharide based, thermoplastic polymers, biodegradable polymers, and/or ~
,~,"
water solu~le cellulosic material a~d mixtures thereof.
A non-limiting list of suitable saccharide carriers 15~ include sucrose, lactose, fructose, dextrose, sorbitol, mannitol, maltose, synthetically-derived saccharide materials such as polydextrose, and the like and ;-mixtures thereo~. Alternative saccharide materials such ~.
as maltodextrins and/or corn syrup solids are al o useful. Please note that for purposes of this invention, applicant refers to maltodextrins and corn ~:
syrup solids (as defined by the FDA) collectively as ~
maltodextrins. ::
,: , Suitable ~hermoplastic polymers can include polypropylene, polystyrene, polyethylene, polyvinyl 3 acetate, polyvinyl alcohol, poly(methacrylate), polyacrylic resins, lactide/glycolide copolymer and .~
mixtures thereo . Suitable water-soluble cellulosic . ;
materials can i~clude methylcellulose, ethylcellulose, hydroxymethyl or ethylcellulose, alkali-metal salts or carboxymethylcelluloses and the like and mixtures .
,5~ ~
1 thereof.
In a preferred embodiment of this invention, maltodextrin has been selected as possessing unique properties as carrier material for the matrix of the invention. Maltodextrins are composed of water-soluble glucose-based polymers obtained from the reaction o~
starch with enzymes or acid in the presence of water.
The hydrolysis reaction produces a carbohydrate mix~ure of saccharides having a dextrose equivalence (D.E.) of 15~ less than 40. In one embodiment of the invention, the D.E. is between 20 and 40. (These maltodextrin ~roducts have been classified by the FDA as corn syrup solids).
In another embodi~ent, the D.E. is between lO and 20.
~O The maltodextrins useful i~ the present invention ~:
include some products sold under the trademark MALTRIN~
by the Grain Processing Corpoxation o~ Muscatine, Iowa ~;~
or "Dri-Sweet" ~ariety of maltodextrins sold by the ~ 25 Hubinger Company of Keokuk, Iowa. Such products are available as powders, granules or the like.
The enzyme and the maltodextrin can be combined by ~.
physically mixing the two ingredients. Ingredients can be~ combined using a blender~or any technique known in the ar~. The~maltodextrin and the enzyme can also be : ~
mixed as a dispersion. The dispersion is formed by contacting the combination of ingredients with an aqueous medium. Dispersion al~lows the combination to be W092/2~329 2 1 ~ 2 6 ~ 7 PCT/U~92/04048 : '.
--19-- ! ' 1 mixed with other materials so that a suhstantially ~.;
homogenous mixture of all ingredients is obtained in the `:
final enzyme product.
EXAMPLES
'~','.
The followin~ examples serve to provide further appreciation of the invention but are not meant in any way to restrict the ef~ective scope of the invention.~.
'.
15~ EXAMPLE 1 ~ :
* '~
A quantity of Columbo~ No Fat ~ogurt was placed in cheese-cloth in a refrigerator for A8 hours permitting the major portion of the water in the yogurt to drain .:.. ;
out. The drained yogurt was then mixed with 35R corn -~
: syrup solids in the ra~io of I:9. This mixture was :-subjected to melt spinning with an Econo Floss~ machine yielding a quanti~y of flakes which were thereafter : ~ maintained unrefrigerated for a period of seven days.
At the end of the seven day period, the flakes were added to skim milk in the ratio of 4 teaspoons of flakes to 1 cup of skim milk. This mixture was then placed in ~-a 110F. environment for 24 hours. -A nice yogurt resulted from which it can be ;
concluded that yogurt can be made in dry form by the subject process which dry form can be stored and .::
;''"'.`
~...
7~ 20-1 subsequently reconstituted.
This example was carried out using packets of "Fleischman's" dry yeast available in any grocery store.
Two packets of the yeast were mixed with 20 grams polypropylene p~wder obtained from Aldrich Chemical Co., Inc. After mixing, the mixture was spun in the floss machine producing a ~ibrous floss.
A series of three 1 pint plastic bottl~s were prepared. Into the first (bottle #1) was placed 10 gm of this floss after first rinsing the floss in tap water. Into the second bottle (bottle #2) was placed an :.;
e~u~l weight of the floss but withou~ rinsing~ Into the third bottle (bottle #3~ was emptied a packet of yeast.
To each bottle was added 3 gm sucrose and one-half pint o~ tap water. over ~he neck oE each bottle was fastened ~:
an elastomeric balloon, and the conditions of the three :~;
balloons were observed and noted over a period of 24 hours.
It was observed that gas was evolved causing ~;
inf~ation of the balLoons to a greater or lesser ext~nt~
Measured Qn a scale of 1 to 5 with 1 beiny minimal and 5 being maximalj the following relative balloon inflations : werP noted. For bottle #1 the inflatio~s were about 3 wo g2/20329 2 1 0 2 6 0 7 PCl`~U~92/OqO4~
1 and 4 after~ respectively, 2 and 24 hours. For bottle ~2 the corresponding inflations were 2 and 4, while for bottle'#3 the corresponding .inflations after 2 and 24 hours were 1 and 5.
From the foregoing it was established that the ~.
floss modlfied yeast was active more rapidly than the original unmodified yeast, that rinsiny the ~loss :~:
accelerated the release of yeast activity from the floss, and that after 24 hours, whether initially rinsed .~:
15~ or not, the floss prc~ ed subs~antially the same amoun~
of total activity. However, the total gas generated due `~
to east activity deri ed from the floss was not quite as ~:
great as that provided by the unmodified yeast. ., ';
The yeast provided in ~he floss material was easily ~
handled and ideal for mixing in a substantial mass, such as a mass of dough in a baking process or a biomass in a ~ormation procedure.
EXAMP~LE 3 -.
"~
Ten grams of Dri-Vac:Lactic culture obtained from Chris Hansen Laboratories containing Streptococcus .:
. .
~hermophi~us and Lactobacillus bulgaricus was mixed with 5 grams of corn oil. 85 grams of Maltrin~ 365 from ~;
Grain Prvcessing Corporation (GPC) were slowly added to ::~
the mixture while mixlng continued until all ingredients , ' ~
W0~2/2~329 PCT/US92/~40 ~ 22-1 were blended thoroughly. One third of the final mixture was saved as an unspun control and two thirds of the final mixture was processed hy flash flow in an Econo Floss~ spinner at 135-145C at 3600 rpm to produce spun flakes.
The following culture samples were prepared:
A. 180 grams o~ sterilized whole milk with 2.5 grams o~ the above spun flakes;
B. 180 grams of sterilized whole milk with 2.5 15~ grams of the unspun control mixture; and C. 180 yrarns o:E sterilized whole milk with 0. 25 grams of the Dri Vac Lactic culture. `~
The samples were cultured in a 40C water bath overnight. Sample A resulted in a smooth, firm and intact mass o~ yogurt which had a velvety smooth texture when separated into pieces with a spoon. Samples B and C produced a yogurt which had a coarse, porous texture.
The mass of samples B and C was not as firm as that of Sample A. The texture of Sample A had much better mouthfeel than 5amples~B and C. .
The ~ddition of a proven amount of culture t~ the .
sterilized milk is much~easier to obtain with the flakes than with the original lactic culture. Thus, the : ~
: present invention enables the artisan to prepare a yogurt product more efficiently and with predictable ,.
~, ~, .
~o g2/2~329 2 1 0 2 fi 0 7 P~T/US92/0404~
-23- ;
l results.
. . , This example is carried out using packets of Fleischman's active dry~yeast available in grocery ~ ,.;
stores~ The yeast was finely ground in a ceramic mortar and pestle and sie~ed through 60 and 80 mesh screens.
Five grams of the sieved yeast were mixed with 2.5 grams ~ ;
of corn oil. The mixture was then added to 4~.5 grams f Maltrin~ 365 brand ~altodextrin obtained from GPC and mixed until a homogenous yeast mixture was ob~ained.
The yeast mixture was proce~sed by flash ~low ak 135-140 at 3600 r.p.m. in an Econo Floss~ spinning machine producing yeast bearing flakes. ~:
:, TWO one-p~int plastic bo~tles were prepared. Into the first (bottle #1) was placed 10 grams of yeast- ~;
bearing flakes. One gram of the sieved yeast was placed . , :
into the second bottle (bottle ~2). To each bottle was ~ ~ .
added 15 grams sucrose and sne-half pint of tap water.
Over the neck of each bottle was fastened an elastomeric balloon, the conditions of the three balloons were observed and noted over a period of 24 hours.
, Observing the inflation of the balloons, it appears that the bottle with the flakes inflated the balloon to ',,;~
W092/20329 PCT/US92/04~ .
Q1 -24- ::
l approximately the same extent as the bottle with the sieved yeast. However, the rate of inflation for bottle #1 was less than that for bottle #2.
::
The flakes produced in the present example provided :~:
a suitable medium for handling and mixing yeast in large masses such as dough for baking or a biomass undergoing . ' fermentation.
EXAMPLE 5 .::
95 gr. of Maltrin~ 365 obtained from GPC and 0.1 gram of Horseradish Peroxidose obtained from ~enzyme Diagnostics were mixed thorollghIy by geometric dilution.
Five grams of mineral oil was then added slowly while ~:
: mixing until a uniform mixture was obtained. ~-, ~
The enzyme mixture was processed by flash flow at 135-140C at 3600 r.p.m. on an Econo Floss spinnîng unit , resulting in light pink flakes.
The enzymatic activity of processed and unproceissed enzyme was determined by the method entitled Peroxidase. :.
This method was supplied by Genzyme Diagnostics. The ,~,~.
~; principle of this method is the oxidation of Pyrogallol to Purpurogallin by Peroxidase. Reactivity is .
determined by tlme course ultraviolet (W) ~:
: : ~ spectrophotometry.~The reaction rate is determined by : . .;
......
W092/20329 2 1 0 2 6 0 7 PCT/US92/0404~
-25~
1 the slope between 20 and 30 seconds. :;
The enzyme in the flakes remained acti~e after the flash flow processing. Samples of the processed and unprocessed enzymes were held at 135C for one hour and analyzed for activity. The enzyme in the flake retained the same level of activity as before incubation while the unprocessed enzymes had lost about 20% of its activity. Thus, the present invention significantly enhanced the stability of the enzyme.
..
A 100 ~ram mix~ure o~ Maltrin~ 365 from GPC and 10%
w/w of the am~lase enzyme Termamyl from Novo Nordisk ..
was obtained by tho~oughly mixing in a mortar and pestle :~
assembly. The mixture was processed by flash flow at 3600 rpm and 135-140C us~ing an Econo Floss~ spinning :;
unit. The processed material was stored at 5C until it ;~-was analyzed for enzymatic activity.
.
: : Thereafter, a sample of the processed flakes and the unprocessed enzyme~were equilibrated in an oven at ~!
100C t~ determine stability. After four hours the , enzyme~in the flakes had retalned subs~antially all its original activity while ~he unprocessed sample had lost --over half ~f .ts original activity.
,, '~
W092/~0329 ~6~ PC~/US92/04~8 -26- !
1 The enzymatic activity was determined by the method ~-entitled "Manual Procedure for Determination of Alpha-Amylase Activity in Enzyme Preparations and Detergents".
This method was provided by Novo Nordisk Bioindustrials, Inc. The principle of the method is to allow the alpha-amylase to degrade a starch polymer substrate. Phadebas tablets (Phadebas~ Amylase Test, supplied by Pharmacia Diagnostics) are used. This material is a cross-linked water insoluble blue colored starch polymer. The tahlet also contains bovine serum albumin and a buffer ., 15 ~ substance. After the tablet is suspended in water, the starch is hydrolysed by the alpha-amylase, giving soluble blue fragments. The absorbance of the resulting blue solution measured at 620 nm (W spectrophotometry) ~;
is a function of the alpha-amylase acti~ity.
Thus, th0 present invention produced an enzyme-: bearing flake which remained active under equilibrated :~ 2:5 conditions set forth above for a longer period of time than the untreated enzyme~ ~:
~:
: EXAMPLE 7 ~30 A ZOO gram~mixture of the Maltrin~ 365 from GPC and ~;~
: lV~ wlw of the protease:enzyme Alcalase from Novo Nordisk was obtalned;by thoroughly mixing in a mortar and pestle assembly. ~The mixture was processed by flash ~ "
: flow at 3600 rpm~and 135-140C using an Econo ~loss , ' , , WO ~2/20~i29 P~r/US92/04048 2:102`~7 spirlning unit . The processed material was stored at 5 C
until it was analyzed or enzymatic activity.
Therea~ter, ~ sample of the spun enzyme and the :~.
unspun enzyme were equilibrated in an oven at 57C for 21 hours to determine stability. After 21 hours, both samples retained substantially the same activity as was ~:~
pr~sent in the original spun and unspun sample. -~
The proteolytic activity was determined by the 15~ method entitled "Determination of Proteolytlc ~ctivity .~:
Using Azocasein as a Substrate". This method was ,~ , provided by Novo Nordisk Bioindustrials, Inc. The principle of the method is to allow the proteolytic ~.
enzyme to hydrolyze azocasein for 30 minutes at 40C.
Undi~ested protein i5 precipitated with tri~hloroa~etic acid and the quantity of digested product is determined by ultraviolet (W~ spectrophotometry.
The protease enzyme remained active a~ter flash flow processing for the same period of time as the -untreated enzyme.
The products and process of the present invention have shown dramatic improvement in enzyme handlin~ and ::~
use artq :
..' WO~2/2032g P~T/US92/04048 c~6~
1 Moreover, while there have been described what are presently believed to be the preferred embodiments of the preferred invention, those skilled in the art will realize that changes in modification may be made thereto without departing from the spirit of the invention, and it is intended to claim also changes and modifications as forward in the true scope of the invention.
.
.~ :
;.;.
,~
. ~
~'' ,''~, ;~''.;
:-, :
. .. .
~ . "`;
: :.
,, '',,:
.'..
the receiviny material is an extensive mass, such as dough in baking and the biomass in fermentation ;~
procedures.
' -~ ' .
,, '"
W092/2032~ 2 1 0 2 ~ 0 7 PCT/US9~/04048 1 Other enzyme-bearing products can benefit from .;
enhanced shelf-life. At room temperature enzymes used as indicators in immunoassays frequently experience short shelf-like. Horseradish peroxidase, lipoprotein lipase, glycerol-3~phosphate oxidase are ordinarily stored as freeze-dried powders at 20C. Co~nonly-used assays are conducted in the range of 20-30~C. It is thus important to provide a matrix which can improve the sheIf life of enzyme~used in immunoassays without impairing their activity.
There have been attempts in the past to deal with the problems associated with the use of enzymes. In United States Patent No.:3,095,358, sorbitol is used to . ..
stabilize aqueous s~lution~ containing papain, proteases and amylases. This method requires large amounts of : ~ stabilizing agen~ and is, therefore, expensi~e.
In U.S. Patent~No. 3,~96,094, partially hydrolyzed ~ and solubilized collagen and glycerol are used to : stabilize aqueous solutions o~ proteolytic enzymes. :
:~ ~ This method requires large quantities of glycerol and, 3 ~ therefore, adds~significantly to the C05t of the enzyme solutidnO ~
; ~ 5 ~ U.S. Patent No. 3~,7~9,671 discloses a method of preparing enzyme-containing prills for use in laundry deter~ents. The disclosed prilling method requires the . .
W092/~0329 P~T/US~2/04~8 :
2~02 ~~ _4_ 1 following steps: (a) heating a normally solid translucent material to a temperature sufficient to melt ~;
the material but insuf~icient to destroy the activity of the enzyme; (b) forming a slurry of the melted material and the enzyme; (c) injecting an inert gas into the slurry to form a uniform dispersion with the gas; ~nd (d) forming prills from the resulting slurry. This method has many steps which require energy, equipment, and manual labor. :
'.~
15~ Although the methods discussed above represent efforts to improve enzyme-containing detergent products, the problems associated with the decreases in enzyme ;.
activity over time and adequate dispersal have not bee~
solved.
,',' '' ~
It is, therefore, an object of this invention to pro~ide an enzyme product which disperses or dissolves uniformly in the target liquid while retaining the enzyme activity for prolonged periods of time pri~r to use.
~-3 It is another object of this invenkion ko provide a ,.
! matrix whlch facilitates mixing an enzyme with a mass so : that the enzy~e can be:dispersed efficiently throughout the mass.
~
"'':
W092/2032~ PCT/US92/0404g ~102607 1 It is yet another object of this invention to provide an enzyme product that exhibits an enhanced shelf 1ife.
~.
Other and further o~jects of the prese~t invention `:
will beco~e apparent the fo11Owing description and its scope will be pointed out with the appended s1aims.
. SUMMARY OF THE INVENTION
15~ The present invention inc1ud2s an enzyme product ~ .
which contains a matrix formed by subjecting a feedstock containing an enzyme and a carrier material to conditions of temperature and shear sufficient to produce the matrix which suspends the enzyme for storage an~ use. The carrier material undergoes transformation :~ during processing in which its physical and/or chemical ; s~ructure is a1tered.
: . .
. -"Enzyme product" in the present invention means a pxoduct which includes one or more enzymes. A
non~imiting list of enzymes which can be suspended in ~-3 the matrix includes amylases, proteases, invertases, :~
g1ucose' oxidases, pectinases, lipases, lactases, and cellulases. The enzymes make up from about 1~ to about 30~ by weight of the matrix, with amounts of from about ...
..
5% to about 25% being preferred an~ the amounts are from a~out l0% to about 20~ being most preferred.
, ~ ' ",.
'~.
WO 92/20329 PCr/US92/0404~
2~2SOT'l Carrier materials which can be used for the matrix are saccharides, thermoplastic polymers, biodegradable polymers and water soluble cellulosic materials. The saccharides may be sucrose, lactose, fructose, dextrose, sorbitol, mannitol, maltose and mixtures thereof. The saccharides may also be selected from polydextrins, maltodextrins, and mixtures thereof. Thermoplastic ", ,, polymers include polypropylene, polyst~rene, polyethylene, polyvinylacetate, polyvinylalcohol, poly (methyl methacrylate~, polyacrylic resins, 15~ lactide/glycolide copolymer and mixtures thereof.
Biodegradable polymers include poly(cis-isoprene) aliphatic polyesters, polyurethanes and urea-formaldehyde polymers. The cellulosic materials are ;~
water soluble and include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, ethyl cellulose, ~;
alkali metal alts of carboxy methyl cellulose and mixtures thereof.
As a result of the present invention, Pnzymes can be suspended, protected, dispersed and generally engineered for selective delivery at desired sites under selected conditions. Various enzyme products can be provided which disperse or dissolve uni~ormly in the arget liquid, biomass,; etc. The enzyme products of this invention can also be designed to retain their ~ ~
activity for long~periods of time prior to use. A non- ;
.~.
inclusive list of uses~for the matrix of the invention :~' W092l2~329 P~T/US9~04048 2~026~7 1 includes leavening agents, alcohol fermenters, detergents, digestive aid products, clinical diagnostic agents, bioremediation agents, meat tenderizing products, wound debridement and other therapeuti~al uses.
For a be~ter understan~iny of the present invention, reference is made to the following description and its scope will be pointed out in the appended claims.
, .;
DETAILED DESCRIPTION OF THE INVENTION
In the present invention an enzyme-bearing matrix .
can be formed by subjecting carrier feedstock and an enzyme to conditions of temperature and shear to ~or~
the matrix. This can be accomplished by melt spinning the enzyme with carrier material~ The matrix is ;.;
: included in ~arious enzyme~based products such as :;
leavening agents, alcohol fermenterc, detergents~ .
diagnostic agents, degradation products, petroleum hydrocarbons degraders, digestive aids, t~erapeutic ~
enz~mes, etc. ~:
: 30 The spinning process can be carried out with "cotton ca~dy" fabricating-type ~quipment~ The spinning :~
machine~used in the present invention can be a cotton candy-type machine, such as the Econo Floss model 3017 manufactured by Gold Medal Products Company of :~
'',....
w~92/2032g ~,~ o~6S~ PCl'/US92/0404 l Cincinnati, Ohio. It will be appreciated by those .
skilled in the art that any apparatus or physical process which provides similar forces and temperature '~
gradient conditions can also be used. For simplicity in disclosing and describi.ng this invention, the term 4;
"melt-spinning" will be understood to mean a process :~
which includes a combination of temperature, shear, ;;~
flow, ~low rate, mechanical forces and thermal gradients of the type produced by a cotton candy-type machine. ;~
15~ The apparatus is operated at a temperature and speed which induce flash flow of certain carrier ~eedstocks without deterioration o~ the feedstock and .
enzyme ~5) being processed. The resulting matrix is in the form of a ~loss, fibre, particle, flake, spicule, or ~th~r generally non-descript aggregAte capable of protectively carrying and deli~ering an enzyme.
'';' ~25 Th~ process for producing the matrix includes :
introducing a mixture containing an enzyme and a carrier ~
material simultaneously to conditions of elevated :
temperature and shear created by centrifugally forcing 3 the ingredients th~ough orifices. The extremely short amount of time the lngredients are exposed to the elevated temperature and shear alIows the matrix to be formed without adverse effects.
' :`
~' W092/20329 210 2 6 0 7 PCr/US92/040~8 1 The flash flow phenomena occurs when a solid carrier material mixed with an enzyme is subjected to conditions of melt-spin sufficient to provide internal flow. This condition produces the transformation in ;`~
physical and/or che~ical structure without degradation .~, of the material. Internal flow occurs when the infrastructure of the material breaks down sufficiently ..
, to permit movement of material at a subparticle level, .
and probably at a molecular level. At a molecular level, internal flow contemplates the movement of 15~ molecules relative to each other~ `.;
. .
Internal flow of material is generally associated with melting point or glass transition point. However, it is contemplated that the combined application of heat and external force is suf~icient to produce flow at temperatures below the melting or glass transition point for most compositions~
:25 The enzymes dispersed in the matrix are selected from animal-derived~, plant-derived and microbially- ;:, derived pr~para~ions. ~hese enzymes can be used as part . :~
of a leavening product, an alcohol fermenter, ~
detergent, a clinical diagnostic agent or a bioremedient and pos~ibly mixtures~thereof 7 A nonlimiting list 3 includes amylases, proteases, invertases, oxidases, catalases, pectinases, lipases, lactases, cellulases and mixtures thereof.~
- :.
~;:
W092/20329 ~ 6~ PCT/US92/~4048 1 In one aspect of the present invention, the matrix may be formed by mixing the carrier material with degradation enzymes such as cellulases, cutinases, lipases and pectinases and mixtures thereof. Cellulase sources include ~hose originating in the genexa Trichoderma, Penicillium, Aspergillus, Clostridium, etc.
Additional cellulases can include commercially available products. Such cellulases are capable of degrading the water insoluble cellulose polymer which is part of the surface membrane of fruits and Yegetables~
'';.',.
Cutinase sources include those originating in the genera Pseudomonas, Fusarium, Botrytis, Uloclad.ium, etc.
Additional cutinases can include commercially available ;~
products. Cutinases are capable of degrading water insoluble cutin polymer which m~y be present as part of ;~
the surface membran of fruits or vegetab~es.
,.....
; 25 Lipase sources include tho e originating in the genera iSitaphyl~coccus, Candida, Rhizopus, etc.
Additional lipases can include commercially available products. Such lipases are capable of degrading water -;
insolub1e glycerol components comprising part of the surface membrane of fruits or vegetables.
Pectinase sources incIude those originating in the genera Rhizopu5, Penicillium, Aspergillus, etc.
Additional pectinases can include commercially available : ~ ~ ' '"',`~
~ ' ' 21026~7 l products. Such pectinases are capable of deyradlng the water insoluble pectin components comprising part of the surface membrane of fruits or vegetables.
:
Th~ enzyme bearing matrix of the invention has many uses. For example, a cellulase matrix may be used to increase the permeability of the surface membrane o~
fruits and vegetables. The lncreased water permeability across the surface membrane permits easier delivery o~
substances such as flavorings, sweeteners, stabilizers 15 ~ and preservatives to the interi.or of the fruit or vegetable. Additio~ally, the increased water ~ . .
permeability allows for a more efficient method of dehydration of fruits and vegetables. More important~y, 20 the use of naturally produced degradation enzymes as pe~meability enhancers replaces the use of chemicals :~
such as methanol, chloroform or alkali metal hydroxides, which, if ingested, pose potential harmful side effects .
to consumers o~fruits and~vegetables. ..
Another important use for the enzyme caxrier matrix '~
of this in~ention is in:~the preparation of clinical -: ~ ~iagnostics products. A nonlimiting list of active i~gr di~nts found in cli~ical diagnostic products include:ascorbic ac~d oxidase, ~-glycerophosphate oxidase, lactate oxidase, uriase, cholesterol esterase, : cholesteroI ester hydrolase, creatinine amino hydrolase, ~::
lipase, glycerol kinase, and~mixtures thereo~.
''''''.''''''' ,.. .
W092/~0329 PCT/U~9~/04048 ~r~0~?~ 12- .
1 The clinical enzyme products contemplated herein are particularly well suited for use with the matrix of :~
the invention when it is desired to disperse the dry , 5 powder enzymes in aqueous liquids. It should be readily apparent to the skilled artisan that all of the active . .
ingredients may also be provided in dry or lyophilized form and reconstituted with water prior to use.
Compositions of this type are clearly contemplated by this in~ention. Clinical diagnostic enzymes carried in the matrix of the invention can also ~e incorporated into single-layer or multi-layer analytical elements o~
the types known in the prior art.
In another aspect of this invention, the matrlx may be used to enhance the shelf-life and activity of en~ymes used in i~munoassays. For example, when horseradish peroxidase was spun with the matrix of the invention, the enzyme exhibited a longer shelf-life, an~
became more readily active.
, Another class of enzyme products ascording to the invention are~improved detergent enzymes. ~etergent ~-enzymes are known in the art as enzymes which attack ,;
stains or soiled areas of fabrics. Suitable categories .
of: ative detergent enzymes found in improved detergents ~:
include proteases, lipases, amylases, and mixtures thereof. The preferred detergent enzymes are proteases such as subtilisin and amylases such as those derived ::
W092/20329 ~ 1 0 2 6 0 I PCT/US92/04048 1 from the bacillus species.
The new matrix can be used alone or in combination with other ingredients as a means for dispersing the ; . .
added ingredients throughout the material. For example, particles, chips, flakes, spicules or combinations thereof can be used to disperse enzymes which are otherwise relatively non-dispersable because o~ the physical characteristics of such materials. Thus, the matrix of the invention can be used to carry detergent ,;~
, . enzymes to be dispersed more easily and uni~ormly in ::~
other materials present in ~etergent formulations, such as surfactants, builders, whitening agents, bleaching agents and the like.
In certain embodiments the enzymes are present in the host microorganism such as in fungi, bacteria or , algae. Examples of host microorganisms include yeasts, ~;~
bio-remediation materials and the like.
;~ '.
: In another aspect of the invention, yeasts may be `.~. melt-spun with selected carrier materials to obtain :~
3 enhanced leavening products. Yeasts are single cell microorganisms containing enzymes which are employed in large scale fermentation:processes~ The commercial production of fermented beverages, foods~ production of vitamins, alcoholic fermentation, antibiotic producing fermentations, all require yeasts or their enzymes to . :,' , ~
WO92/2032g PCT/~'S92/0404~ ~
.,6~
1 produce products simpler than the starting material.
Regardless of the substrate used or the chosen -microorganism, industrial fermentations require various nutrients for growth including carbohydrates, nitrogen- -containing compounds, growth factors, vitamins and minerals~ In most fermentations, these nutritional re~uirements are met by including, among others, yeast products.
In the production of alcoholic ~everages, cereal , grains are the principal raw material. Another important ingredient is malt that is used to produce amylase. ~mylas~s are organic enzymes that change grain starch into maltose. In fermentation, zymase which is produced by yeasts converts the amylase produced maltose into ethyl alcohol and carbon dioxide. Saccharomyces cervisiae is thP most common type of yeast used in alcoholic fermentation to generate zymase. ;
In fermentation processes desired metabolic changes frequently occur ln a narrow temperature and pH range.
Acc~rdingly, to increase product yields t it is important 3 to deliver yeasts having enzymatic activity in a narrow temperature and pH range. In addition, to optimize product yields, the yeasts must be rapidly and uniformly dispersable in the target liquid. Thus, yeasts suspended in the matrix of the invention are easily dispersable in the nutrient medium and have been found, ` ~
W0~2/20329 PCT/US92/0404~
210~fiO7 l in some cases, to be more readily active.
.: -In another aspect of this invention, the matrix may be used to enhance the shel~ life and activity of enzymes used in yeasts. For example, when Fleischman's ;:
dry yeast was spun with the matrix of the invention, the :.~
:
yeast exhibited a longer shelf~life and became more readily active.
~''"
Another significant use for the enzyme carrier ;~.
15~ matrix of the invention finds application in the biodegradation of petroleum hydrocarbons. Many species of bacteria, fungi and algae have the enzymatic .,,.. ~.
capability to use petroleum hydrocarbons as food. The :.
,, .:,:
bacteria genera most frequently isolated as hydrocarbon ~;
degraders are P-~eudomonas, AcinetobaGter, , ~, F~avobacterium, Brevibacterium, Corynebacterium, Arthrobacter. The fungus genera include Candida, .. ..
25 ~ Cladosporium, Trichosporium and Rhodotorula. These . .
bac~eria and fungi are present in the environment. :.
Genetically engineered bacteria which have the enzymatic capability of degrading several groups of hydrocarbons .
can also be used~as~petroleum biodegraders.
Using the matrix;of the invention, it is possible to dis~erse rapidly and uniformly these biodegrading agents and their nutrients into an otherwise hydrophobic ~`
or: immisoible environment.~ In this manner the microbial ~ ~ ';.;
uro g2t20329 r Pcr/uss2/o4o4s 6~ 1 -16- !
l cleanup of oil spills occurs more rapidly.
, In yet another aspect of this invention, the enzyme carrying matrix may be used in the field of enhanced oil recovery. Microbial products, as well as viable microorganisms, suspended in the matrix may be used as -:~
~timulation agents to enhance oil recovery from petroleum reservoirs. F~r example, a strain of Acinetobacter calcoaceticus produces emulsan, a lipopolysaccharide used to stabilize oil in water 15~ emulsions. Xanthomonas campeskris is a microbial ~.
product producing xant~an, a polysaccharide used as a water flood thickening agent in oil reco~ery. Both these microbial products become easily miscible in petroleum reservoirs when deli~ered with the matrix of the in~ention. The result is enhanced oil recovery.
:
~ The ability of microorganism~ to use petroleum as ,.
food also has detrimental effects. For example, ~ :
~: petroleum fuels cannot become contaminated with water or microorganisms~during storage. Such contamination poses a serious problem~for kerosine based jet aircraft fuels.
3 To diminish this problem, antimicrobials that , :~ concentrate at the oil/water interfaces may be used to ~:~
reduce the rate of~microbial contaminatlon of ;~ hydrocarbons. Ant~imicrobial organisms may be delivered ~:;
: ~ ~ at the oil/water interfaces by using the matrix of the ~ invention.
. :.:
W092/20329 P~T/US92/04~8 l The carriers used in the invention can be any material capable of being processed to form a matrix which can protectively suspend the enzyme for storage and/or selective delivery to the site and/or environment for release and acti~ation~ Carrier materials contemplated for use may be saccharide based, thermoplastic polymers, biodegradable polymers, and/or ~
,~,"
water solu~le cellulosic material a~d mixtures thereof.
A non-limiting list of suitable saccharide carriers 15~ include sucrose, lactose, fructose, dextrose, sorbitol, mannitol, maltose, synthetically-derived saccharide materials such as polydextrose, and the like and ;-mixtures thereo~. Alternative saccharide materials such ~.
as maltodextrins and/or corn syrup solids are al o useful. Please note that for purposes of this invention, applicant refers to maltodextrins and corn ~:
syrup solids (as defined by the FDA) collectively as ~
maltodextrins. ::
,: , Suitable ~hermoplastic polymers can include polypropylene, polystyrene, polyethylene, polyvinyl 3 acetate, polyvinyl alcohol, poly(methacrylate), polyacrylic resins, lactide/glycolide copolymer and .~
mixtures thereo . Suitable water-soluble cellulosic . ;
materials can i~clude methylcellulose, ethylcellulose, hydroxymethyl or ethylcellulose, alkali-metal salts or carboxymethylcelluloses and the like and mixtures .
,5~ ~
1 thereof.
In a preferred embodiment of this invention, maltodextrin has been selected as possessing unique properties as carrier material for the matrix of the invention. Maltodextrins are composed of water-soluble glucose-based polymers obtained from the reaction o~
starch with enzymes or acid in the presence of water.
The hydrolysis reaction produces a carbohydrate mix~ure of saccharides having a dextrose equivalence (D.E.) of 15~ less than 40. In one embodiment of the invention, the D.E. is between 20 and 40. (These maltodextrin ~roducts have been classified by the FDA as corn syrup solids).
In another embodi~ent, the D.E. is between lO and 20.
~O The maltodextrins useful i~ the present invention ~:
include some products sold under the trademark MALTRIN~
by the Grain Processing Corpoxation o~ Muscatine, Iowa ~;~
or "Dri-Sweet" ~ariety of maltodextrins sold by the ~ 25 Hubinger Company of Keokuk, Iowa. Such products are available as powders, granules or the like.
The enzyme and the maltodextrin can be combined by ~.
physically mixing the two ingredients. Ingredients can be~ combined using a blender~or any technique known in the ar~. The~maltodextrin and the enzyme can also be : ~
mixed as a dispersion. The dispersion is formed by contacting the combination of ingredients with an aqueous medium. Dispersion al~lows the combination to be W092/2~329 2 1 ~ 2 6 ~ 7 PCT/U~92/04048 : '.
--19-- ! ' 1 mixed with other materials so that a suhstantially ~.;
homogenous mixture of all ingredients is obtained in the `:
final enzyme product.
EXAMPLES
'~','.
The followin~ examples serve to provide further appreciation of the invention but are not meant in any way to restrict the ef~ective scope of the invention.~.
'.
15~ EXAMPLE 1 ~ :
* '~
A quantity of Columbo~ No Fat ~ogurt was placed in cheese-cloth in a refrigerator for A8 hours permitting the major portion of the water in the yogurt to drain .:.. ;
out. The drained yogurt was then mixed with 35R corn -~
: syrup solids in the ra~io of I:9. This mixture was :-subjected to melt spinning with an Econo Floss~ machine yielding a quanti~y of flakes which were thereafter : ~ maintained unrefrigerated for a period of seven days.
At the end of the seven day period, the flakes were added to skim milk in the ratio of 4 teaspoons of flakes to 1 cup of skim milk. This mixture was then placed in ~-a 110F. environment for 24 hours. -A nice yogurt resulted from which it can be ;
concluded that yogurt can be made in dry form by the subject process which dry form can be stored and .::
;''"'.`
~...
7~ 20-1 subsequently reconstituted.
This example was carried out using packets of "Fleischman's" dry yeast available in any grocery store.
Two packets of the yeast were mixed with 20 grams polypropylene p~wder obtained from Aldrich Chemical Co., Inc. After mixing, the mixture was spun in the floss machine producing a ~ibrous floss.
A series of three 1 pint plastic bottl~s were prepared. Into the first (bottle #1) was placed 10 gm of this floss after first rinsing the floss in tap water. Into the second bottle (bottle #2) was placed an :.;
e~u~l weight of the floss but withou~ rinsing~ Into the third bottle (bottle #3~ was emptied a packet of yeast.
To each bottle was added 3 gm sucrose and one-half pint o~ tap water. over ~he neck oE each bottle was fastened ~:
an elastomeric balloon, and the conditions of the three :~;
balloons were observed and noted over a period of 24 hours.
It was observed that gas was evolved causing ~;
inf~ation of the balLoons to a greater or lesser ext~nt~
Measured Qn a scale of 1 to 5 with 1 beiny minimal and 5 being maximalj the following relative balloon inflations : werP noted. For bottle #1 the inflatio~s were about 3 wo g2/20329 2 1 0 2 6 0 7 PCl`~U~92/OqO4~
1 and 4 after~ respectively, 2 and 24 hours. For bottle ~2 the corresponding inflations were 2 and 4, while for bottle'#3 the corresponding .inflations after 2 and 24 hours were 1 and 5.
From the foregoing it was established that the ~.
floss modlfied yeast was active more rapidly than the original unmodified yeast, that rinsiny the ~loss :~:
accelerated the release of yeast activity from the floss, and that after 24 hours, whether initially rinsed .~:
15~ or not, the floss prc~ ed subs~antially the same amoun~
of total activity. However, the total gas generated due `~
to east activity deri ed from the floss was not quite as ~:
great as that provided by the unmodified yeast. ., ';
The yeast provided in ~he floss material was easily ~
handled and ideal for mixing in a substantial mass, such as a mass of dough in a baking process or a biomass in a ~ormation procedure.
EXAMP~LE 3 -.
"~
Ten grams of Dri-Vac:Lactic culture obtained from Chris Hansen Laboratories containing Streptococcus .:
. .
~hermophi~us and Lactobacillus bulgaricus was mixed with 5 grams of corn oil. 85 grams of Maltrin~ 365 from ~;
Grain Prvcessing Corporation (GPC) were slowly added to ::~
the mixture while mixlng continued until all ingredients , ' ~
W0~2/2~329 PCT/US92/~40 ~ 22-1 were blended thoroughly. One third of the final mixture was saved as an unspun control and two thirds of the final mixture was processed hy flash flow in an Econo Floss~ spinner at 135-145C at 3600 rpm to produce spun flakes.
The following culture samples were prepared:
A. 180 grams o~ sterilized whole milk with 2.5 grams o~ the above spun flakes;
B. 180 grams of sterilized whole milk with 2.5 15~ grams of the unspun control mixture; and C. 180 yrarns o:E sterilized whole milk with 0. 25 grams of the Dri Vac Lactic culture. `~
The samples were cultured in a 40C water bath overnight. Sample A resulted in a smooth, firm and intact mass o~ yogurt which had a velvety smooth texture when separated into pieces with a spoon. Samples B and C produced a yogurt which had a coarse, porous texture.
The mass of samples B and C was not as firm as that of Sample A. The texture of Sample A had much better mouthfeel than 5amples~B and C. .
The ~ddition of a proven amount of culture t~ the .
sterilized milk is much~easier to obtain with the flakes than with the original lactic culture. Thus, the : ~
: present invention enables the artisan to prepare a yogurt product more efficiently and with predictable ,.
~, ~, .
~o g2/2~329 2 1 0 2 fi 0 7 P~T/US92/0404~
-23- ;
l results.
. . , This example is carried out using packets of Fleischman's active dry~yeast available in grocery ~ ,.;
stores~ The yeast was finely ground in a ceramic mortar and pestle and sie~ed through 60 and 80 mesh screens.
Five grams of the sieved yeast were mixed with 2.5 grams ~ ;
of corn oil. The mixture was then added to 4~.5 grams f Maltrin~ 365 brand ~altodextrin obtained from GPC and mixed until a homogenous yeast mixture was ob~ained.
The yeast mixture was proce~sed by flash ~low ak 135-140 at 3600 r.p.m. in an Econo Floss~ spinning machine producing yeast bearing flakes. ~:
:, TWO one-p~int plastic bo~tles were prepared. Into the first (bottle #1) was placed 10 grams of yeast- ~;
bearing flakes. One gram of the sieved yeast was placed . , :
into the second bottle (bottle ~2). To each bottle was ~ ~ .
added 15 grams sucrose and sne-half pint of tap water.
Over the neck of each bottle was fastened an elastomeric balloon, the conditions of the three balloons were observed and noted over a period of 24 hours.
, Observing the inflation of the balloons, it appears that the bottle with the flakes inflated the balloon to ',,;~
W092/20329 PCT/US92/04~ .
Q1 -24- ::
l approximately the same extent as the bottle with the sieved yeast. However, the rate of inflation for bottle #1 was less than that for bottle #2.
::
The flakes produced in the present example provided :~:
a suitable medium for handling and mixing yeast in large masses such as dough for baking or a biomass undergoing . ' fermentation.
EXAMPLE 5 .::
95 gr. of Maltrin~ 365 obtained from GPC and 0.1 gram of Horseradish Peroxidose obtained from ~enzyme Diagnostics were mixed thorollghIy by geometric dilution.
Five grams of mineral oil was then added slowly while ~:
: mixing until a uniform mixture was obtained. ~-, ~
The enzyme mixture was processed by flash flow at 135-140C at 3600 r.p.m. on an Econo Floss spinnîng unit , resulting in light pink flakes.
The enzymatic activity of processed and unproceissed enzyme was determined by the method entitled Peroxidase. :.
This method was supplied by Genzyme Diagnostics. The ,~,~.
~; principle of this method is the oxidation of Pyrogallol to Purpurogallin by Peroxidase. Reactivity is .
determined by tlme course ultraviolet (W) ~:
: : ~ spectrophotometry.~The reaction rate is determined by : . .;
......
W092/20329 2 1 0 2 6 0 7 PCT/US92/0404~
-25~
1 the slope between 20 and 30 seconds. :;
The enzyme in the flakes remained acti~e after the flash flow processing. Samples of the processed and unprocessed enzymes were held at 135C for one hour and analyzed for activity. The enzyme in the flake retained the same level of activity as before incubation while the unprocessed enzymes had lost about 20% of its activity. Thus, the present invention significantly enhanced the stability of the enzyme.
..
A 100 ~ram mix~ure o~ Maltrin~ 365 from GPC and 10%
w/w of the am~lase enzyme Termamyl from Novo Nordisk ..
was obtained by tho~oughly mixing in a mortar and pestle :~
assembly. The mixture was processed by flash flow at 3600 rpm and 135-140C us~ing an Econo Floss~ spinning :;
unit. The processed material was stored at 5C until it ;~-was analyzed for enzymatic activity.
.
: : Thereafter, a sample of the processed flakes and the unprocessed enzyme~were equilibrated in an oven at ~!
100C t~ determine stability. After four hours the , enzyme~in the flakes had retalned subs~antially all its original activity while ~he unprocessed sample had lost --over half ~f .ts original activity.
,, '~
W092/~0329 ~6~ PC~/US92/04~8 -26- !
1 The enzymatic activity was determined by the method ~-entitled "Manual Procedure for Determination of Alpha-Amylase Activity in Enzyme Preparations and Detergents".
This method was provided by Novo Nordisk Bioindustrials, Inc. The principle of the method is to allow the alpha-amylase to degrade a starch polymer substrate. Phadebas tablets (Phadebas~ Amylase Test, supplied by Pharmacia Diagnostics) are used. This material is a cross-linked water insoluble blue colored starch polymer. The tahlet also contains bovine serum albumin and a buffer ., 15 ~ substance. After the tablet is suspended in water, the starch is hydrolysed by the alpha-amylase, giving soluble blue fragments. The absorbance of the resulting blue solution measured at 620 nm (W spectrophotometry) ~;
is a function of the alpha-amylase acti~ity.
Thus, th0 present invention produced an enzyme-: bearing flake which remained active under equilibrated :~ 2:5 conditions set forth above for a longer period of time than the untreated enzyme~ ~:
~:
: EXAMPLE 7 ~30 A ZOO gram~mixture of the Maltrin~ 365 from GPC and ~;~
: lV~ wlw of the protease:enzyme Alcalase from Novo Nordisk was obtalned;by thoroughly mixing in a mortar and pestle assembly. ~The mixture was processed by flash ~ "
: flow at 3600 rpm~and 135-140C using an Econo ~loss , ' , , WO ~2/20~i29 P~r/US92/04048 2:102`~7 spirlning unit . The processed material was stored at 5 C
until it was analyzed or enzymatic activity.
Therea~ter, ~ sample of the spun enzyme and the :~.
unspun enzyme were equilibrated in an oven at 57C for 21 hours to determine stability. After 21 hours, both samples retained substantially the same activity as was ~:~
pr~sent in the original spun and unspun sample. -~
The proteolytic activity was determined by the 15~ method entitled "Determination of Proteolytlc ~ctivity .~:
Using Azocasein as a Substrate". This method was ,~ , provided by Novo Nordisk Bioindustrials, Inc. The principle of the method is to allow the proteolytic ~.
enzyme to hydrolyze azocasein for 30 minutes at 40C.
Undi~ested protein i5 precipitated with tri~hloroa~etic acid and the quantity of digested product is determined by ultraviolet (W~ spectrophotometry.
The protease enzyme remained active a~ter flash flow processing for the same period of time as the -untreated enzyme.
The products and process of the present invention have shown dramatic improvement in enzyme handlin~ and ::~
use artq :
..' WO~2/2032g P~T/US92/04048 c~6~
1 Moreover, while there have been described what are presently believed to be the preferred embodiments of the preferred invention, those skilled in the art will realize that changes in modification may be made thereto without departing from the spirit of the invention, and it is intended to claim also changes and modifications as forward in the true scope of the invention.
.
.~ :
;.;.
,~
. ~
~'' ,''~, ;~''.;
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. .. .
~ . "`;
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,, '',,:
.'..
Claims (33)
1. An enzyme product comprising an enzyme-bearing matrix formed by subjecting a feedstock comprising said enzyme and a carrier material to conditions which alter the physical and/or chemical structure of said carrier to form said enzyme-bearing matrix for delivery of said enzyme as desired for said product.
2. The enzyme product of Claim 1, wherein said conditions comprise subjecting said mixture simultaneously to flash heating and applied physical force.
3. The enzyme product of Claim 2, wherein said conditions are created by melt-spinning said feedstock.
4. The enzyme product of Claim 1, wherein said carrier material is selected from the group consisting of saccharides, thermoplastic polymers, biodegradable polymers, and water-soluble cellulosic materials.
5. The enzyme product of Claim 4, wherein said saccharides are selected from the group consisting of polydextrose, maldodextrins, sucrose, lactose, dextrose, mannitol, sorbitol, glucose, maltose and mixtures thereof.
6. The enzyme product of Claim 4, wherein said thermoplastic polymers are selected from the group consisting of polypropylene, polystyrene, polyethylene, polyvinyl acetate, polyvinyl alcohol, poly(methyl methacrylate), polyacrylic resins, lactide/glycolide copolymer and mixtures thereof.
7. The enzyme product of Claim 4, wherein said biodegradable polymers are selected from the group consisting of poly(cis-isoprene), aliphatic polyesters, polyurethanes and urea-formaldehyde polymers.
8. The enzyme product of Claim 4, wherein said cellulosic materials are selected from the group consisting of methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, ethyl cellulose, alkali metal salts of carboxy methyl cellulose and mixtures thereof.
9. The enzyme product of Claim 1, wherein said enzyme is selected from the group consisting of amylases, proteases, invertases, oxidases, catalases, pectinases, lipases, lactases, cellulases and mixtures thereof.
10. The enzyme product of Claim 9, wherein said enzyme is present in an amount from about 2% to about 40% by weight of the matrix.
11. The enzyme product of Claim 10, wherein said enzyme is present in an amount from about 10% to about 30%.
12. The enzyme product of Claim 11, wherein said enzyme is present in an amount from about 15% to about 22%.
13. The enzyme product of Claim 1, wherein said enzyme is present in an amount from about 1% to about 10% by weight of said product and said enzyme is a protease.
14. The enzyme product of Claim 1, wherein said enzyme is selected from the group consisting of leavening agents, fermentation agents, biodegradation products, detergent agents, immunoassay agents, clinical diagnostic agents, food digestive aids and therapeutic agents.
15. A baking dough comprising the enzyme product of Claim 1, wherein said enzyme is a leavening agent.
16. A fermentation biomass comprising the enzyme product of Claim 1, wherein said enzyme is a fermentation agent.
-32- ,
-32- ,
17. A yogurt product comprising the enzyme product of Claim 1, wherein said enzyme is contained in a yogurt culture.
18. A detergent formulation comprising the enzyme product of Claim 1.
19. The detergent formulation of Claim 18, wherein said enzyme is subtilisin.
20. A method of preparing an enzyme product comprising:
providing an enzyme-bearing matrix formed by subjecting a feedstock comprising said enzyme and a carrier material to conditions which alter the physical and/or chemical structure of said carrier to form said enzyme-bearing matrix for delivery of said enzyme as desired for said product.
providing an enzyme-bearing matrix formed by subjecting a feedstock comprising said enzyme and a carrier material to conditions which alter the physical and/or chemical structure of said carrier to form said enzyme-bearing matrix for delivery of said enzyme as desired for said product.
21. The method of Claim 20, wherein said conditions comprise subjecting said feedstock simultaneously to flash heating and applied physical force.
22. The method of Claim 21, wherein said conditions are created by melt spinning said feedstock.
23. The method of Claim 20, wherein said carrier material is selected from the group consisting of saccharides, thermoplastic polymers, biodegradable polymers and water soluble cellulosic materials.
24. The method of Claim 23, wherein said saccharides are selected from the group consisting of polydextrose, maltodextrins, sucrose, lactose, dextrose, mannitol, sorbitol, glucose, maltose, and mixtures thereof.
25. The method of Claim 23, wherein said thermoplastic polymers are selected from the group consisting of polypropylene, polystyrene, polyethylene, polyvinyl acetate, polyvinyl alcohol, poly (methyl methacrylate), polyacrylic resins, lactide/glycolide copolymer and mixtures thereof.
26. The method of Claim 23, wherein said biodegradable polymers are selected from the group consisting of poly(sis-isoprene), aliphatic polyesters, polyurethenes and urea formaldehyde polymers.
27. The method of Claim 23, wherein said cellulosic materials are selected from the group consisting of methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, ethyl cellulose, alkali metal salts of carboxy methyl cellulose and mixtures thereof.
28. The method of Claim 20, wherein said enzyme is selected from the group consisting of amylases, proteases, invertases, oxidases, catalases, pectinases, lipases, lactases, cellulases and mixtures thereof.
29. The method of Claim 28, wherein said enzyme is present in an amount from about 1% to about 30% by weight of the matrix.
30. The method of Claim 29, wherein said enzyme is present in an amount from about 5% to about 25%.
31. The method of Claim 30, wherein said enzyme is present in an amount from about 10% to about 20%.
32. The method of preparing an enzyme product according to Claim 20, wherein said enzyme is selected from the group consisting of leavening agents, fermentation agents, biodegradation products, detergent enzymes, immunoassay agents, clinical diagnostic agents and food digestive aids.
33. A method of preparing a detergent comprising combining an enzyme-bearing matrix formed by subjecting a feedstock comprising an enzyme and a carrier material to conditions which alter the physical and/or chemical structure of said carrier to form said enzyme-bearing matrix for delivery of said enzyme as desired with detergent ingredients to provide said detergent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US70206891A | 1991-05-17 | 1991-05-17 | |
| US702,068 | 1991-05-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2102607A1 true CA2102607A1 (en) | 1992-11-18 |
Family
ID=24819742
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002109622A Abandoned CA2109622A1 (en) | 1991-05-17 | 1992-05-13 | New thermoplastic polymeric material and process for making same |
| CA002102607A Abandoned CA2102607A1 (en) | 1991-05-17 | 1992-05-13 | Enzyme systems |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002109622A Abandoned CA2109622A1 (en) | 1991-05-17 | 1992-05-13 | New thermoplastic polymeric material and process for making same |
Country Status (10)
| Country | Link |
|---|---|
| US (2) | US6129926A (en) |
| EP (2) | EP0584228B1 (en) |
| JP (2) | JPH07500242A (en) |
| KR (1) | KR100241403B1 (en) |
| AU (1) | AU653040B2 (en) |
| CA (2) | CA2109622A1 (en) |
| DE (2) | DE69231247T2 (en) |
| DK (1) | DK0584228T3 (en) |
| HU (2) | HUT68509A (en) |
| WO (2) | WO1992020329A1 (en) |
Families Citing this family (34)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5387431A (en) * | 1991-10-25 | 1995-02-07 | Fuisz Technologies Ltd. | Saccharide-based matrix |
| US5518730A (en) * | 1992-06-03 | 1996-05-21 | Fuisz Technologies Ltd. | Biodegradable controlled release flash flow melt-spun delivery system |
| US5843922A (en) * | 1994-07-29 | 1998-12-01 | Fuisz Technologies Ltd. | Preparation of oligosaccharides and products therefrom |
| EP0835302A1 (en) * | 1995-06-28 | 1998-04-15 | Novo Nordisk A/S | A cellulase with reduced mobility |
| US5858952A (en) * | 1995-12-22 | 1999-01-12 | Kao Corporation | Enzyme-containing granulated product method of preparation and compositions containing the granulated product |
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-
1992
- 1992-05-13 WO PCT/US1992/004048 patent/WO1992020329A1/en active IP Right Grant
- 1992-05-13 HU HU9303094A patent/HUT68509A/en unknown
- 1992-05-13 DE DE69231247T patent/DE69231247T2/en not_active Expired - Fee Related
- 1992-05-13 CA CA002109622A patent/CA2109622A1/en not_active Abandoned
- 1992-05-13 US US08/074,863 patent/US6129926A/en not_active Expired - Fee Related
- 1992-05-13 WO PCT/US1992/004053 patent/WO1992020330A1/en active IP Right Grant
- 1992-05-13 DK DK92912148T patent/DK0584228T3/en active
- 1992-05-13 HU HU9303093A patent/HU214736B/en not_active IP Right Cessation
- 1992-05-13 AU AU20164/92A patent/AU653040B2/en not_active Ceased
- 1992-05-13 KR KR1019930703477A patent/KR100241403B1/en not_active IP Right Cessation
- 1992-05-13 EP EP92912148A patent/EP0584228B1/en not_active Expired - Lifetime
- 1992-05-13 JP JP5500191A patent/JPH07500242A/en active Pending
- 1992-05-13 EP EP92912612A patent/EP0584245B1/en not_active Expired - Lifetime
- 1992-05-13 CA CA002102607A patent/CA2102607A1/en not_active Abandoned
- 1992-05-13 US US08/150,045 patent/US5624684A/en not_active Expired - Fee Related
- 1992-05-13 JP JP5500194A patent/JPH07502050A/en active Pending
- 1992-05-13 DE DE69232721T patent/DE69232721T2/en not_active Expired - Lifetime
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|---|---|
| HUT67171A (en) | 1995-02-28 |
| AU653040B2 (en) | 1994-09-15 |
| EP0584228A1 (en) | 1994-03-02 |
| HUT68509A (en) | 1995-06-28 |
| JPH07502050A (en) | 1995-03-02 |
| US5624684A (en) | 1997-04-29 |
| DE69231247T2 (en) | 2000-11-23 |
| HU9303093D0 (en) | 1994-01-28 |
| DE69232721T2 (en) | 2002-12-05 |
| WO1992020329A1 (en) | 1992-11-26 |
| CA2109622A1 (en) | 1992-11-26 |
| AU667312B2 (en) | 1996-03-21 |
| DE69231247D1 (en) | 2000-08-17 |
| EP0584245B1 (en) | 2002-08-07 |
| EP0584245A1 (en) | 1994-03-02 |
| DK0584228T3 (en) | 2000-09-18 |
| EP0584228A4 (en) | 1995-02-08 |
| HU9303094D0 (en) | 1994-02-28 |
| AU2016492A (en) | 1992-12-30 |
| JPH07500242A (en) | 1995-01-12 |
| KR100241403B1 (en) | 2000-02-01 |
| AU2140192A (en) | 1992-12-30 |
| DE69232721D1 (en) | 2002-09-12 |
| US6129926A (en) | 2000-10-10 |
| EP0584245A4 (en) | 1995-02-08 |
| WO1992020330A1 (en) | 1992-11-26 |
| EP0584228B1 (en) | 2000-07-12 |
| HU214736B (en) | 1998-08-28 |
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