U.S. patent application number 11/129953 was filed with the patent office on 2005-09-22 for spray dried enzyme product.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Bach, Poul, Hansen, Kim Uhre, Jacobsen, Carsten, Jorgensen, Anders, Jorgensen, Jorgen Topp, Oftelund, Dan, Sondergaard, Gustav Borup.
Application Number | 20050209122 11/129953 |
Document ID | / |
Family ID | 34799836 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050209122 |
Kind Code |
A1 |
Jorgensen, Jorgen Topp ; et
al. |
September 22, 2005 |
Spray dried enzyme product
Abstract
The invention relates to a particle comprising an enzyme and a
biomass, to a process for preparing a particle comprising spray
drying an enzyme and biomass containing fermentation broth starting
material, to obtain a solid particle comprising an enzyme and a
biomass and to a process for preparing an enzyme containing
particle comprising spray drying an aqueous enzyme containing
liquid starting material to obtain a spray dried first enzyme
containing particle and subsequently subjecting the first dry
particle to a process selected from granulation and coating and
combinations thereof to obtain a second dry enzyme containing
particle.
Inventors: |
Jorgensen, Jorgen Topp;
(Kalundborg, DK) ; Jacobsen, Carsten; (Kobenhavn
V, DK) ; Hansen, Kim Uhre; (Kalundborg, DK) ;
Jorgensen, Anders; (Kalundborg, DK) ; Oftelund,
Dan; (Kalundborg, DK) ; Bach, Poul; (Birkerod,
DK) ; Sondergaard, Gustav Borup; (Hillerod,
DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
34799836 |
Appl. No.: |
11/129953 |
Filed: |
May 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11129953 |
May 16, 2005 |
|
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09675950 |
Sep 29, 2000 |
|
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6924133 |
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60158270 |
Oct 7, 1999 |
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60185206 |
Feb 25, 2000 |
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Current U.S.
Class: |
510/320 ;
435/182 |
Current CPC
Class: |
C11D 11/02 20130101;
C12N 9/2414 20130101; C12N 9/2437 20130101; C11D 3/38609 20130101;
C12Y 302/01004 20130101; A23K 20/189 20160501; C12N 9/2408
20130101; C11D 3/38672 20130101 |
Class at
Publication: |
510/320 ;
435/182 |
International
Class: |
C11D 003/386; C12N
009/88 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 17, 2000 |
DK |
2000 00251 |
Oct 1, 1999 |
DK |
2000 01415 |
Claims
1-31. (canceled)
32. A process for preparing an enzyme containing particle
comprising spray drying an enzyme containing liquid by means of a
Rayleigh Atomizer spray drying device, to obtain a solid particle
comprising an enzyme.
33. The process of claim 32, wherein the particles have a SPAN
value below about 2.5.
34. The process of claim 32, wherein the particles have a SPAN
value below about 1.5.
35. The process of claim 32, wherein the particles have a SPAN
value between 0.9 to 1.3.
36. The process of claim 32, further comprising subjecting the
particles of claim 1 to a process selected from granula-tion and
coating and combinations thereof.
37. The process of claim 36, wherein the granulation process is
selected from mixer granulation, prilling, extrusion, fluid bed and
compacting processes.
38. The process of claim 32, further comprising the step of
additional drying of the particles in a fluid bed dryer.
39. The process of claim 32, wherein the enzyme containing liq-uid
has been subjected to a physical treatment selected from heating,
cooling, radiating, mixing, aerating and ul-tra-sound
treatment.
40. The process of claim 39, wherein the enzyme containing liq-uid
has been heated or cooled to 5-150.degree. C.
41. The process of claim 32, wherein the enzyme containing liquid
has been sterilised.
42. The process of claim 32, wherein the enzyme containing liquid
has been treated to hydrolyse polynucleotides pre-sent in the
starting material.
43. The process of claim 32, wherein the enzyme containing liquid
is a fermentation broth, an enzyme filtrate or an enzyme
concentrate.
44. The process of claim 43, wherein the fermentation broth or the
filtrate contain at least 3 mg active enzyme pro-tein per liter
liquid phase.
45. The process of claim 32, wherein the enzyme containing liquid
has a viscosity of 5-5000 cps.
46. The process of claim 32, wherein additives selected from
inorganic salts, inorganic minerals or clays, carbohy-drates,
coloring pigments, cellulose or derivatives thereof, biocides,
dispersants, anti foaming agents, vis-cosity regulating agents,
acid agents, alkaline agents, enzyme stabilizers, enzyme
inhibitors, binders, other en-zymes and combinations thereof has
been added to the en-zyme containing liquid.
47. The process of claim 32, wherein the enzyme is selected from
oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases
(EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-) and
ligases (EC 6.-.-.-).
48. An enzyme containing particle obtainable by the process of
claim 32.
49. A composition comprising the particles of claim 48.
50. The composition of claim 49, wherein the composition is
selected from cleaning compositions, textile processing
compositions, leather processing compositions, pulp or paper
processing compositions, food and beverage compositions, animal
feed compositions and personal care compositions.
51. The cleaning composition of claim 50, wherein the cleaning
composition is a detergent composition further comprising a
surfactant.
52. A method for cleaning an object with a composition of claim 51.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
09/675,950, filed Sep. 29, 2000, which claims priority or the
benefit under 35 U.S.C. 119 of Danish application nos. PA 2000
00251, filed Feb. 17, 2000, and PA 2000 01415, filed Oct. 1, 1999
and U.S. provisional application Nos. 60/158,270, filed Oct. 7,
1999, and 60/185,206, filed Feb. 25, 2000, the contents of which
are fully incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention relates to enzyme containing particles, to
processes for preparing enzyme containing particles, and to the use
of enzyme containing particles in compositions and applications of
such particles and compositions.
BACKGROUND OF THE INVENTION
[0003] Enzyme products are known and used in a variety of
industries and applications.
[0004] Known art for providing dry particulate enzyme products
includes:
[0005] Spray dried products, as described in Michael S. Showell
(editor); Powdered detergents; Surfactant Science Series; 1998;
vol. 71; page 140-142; Marcel Dekker and GB 1,360,969.
[0006] Layered products, as described in WO 97/23606 and U.S. Pat.
No. 5,814,501
[0007] Improved properties of biological compounds such as enzymes,
desired or required by industry and applications has led to
development of complex particulate enzyme products, a task in which
many challenging problems arise. The products should be cheap with
respect to processes and process components and chemicals but
should also provide the enzyme product with desired properties such
as improved enzyme storage stability, lowered dusting
characteristics, improved particle mechanical strength, desired
color, shape and size.
SUMMARY OF THE INVENTION
[0008] The invention concerns a first aspect a process for
preparing a particle comprising spray drying a fermentation broth
starting material comprising an enzyme and a biomass, to obtain a
solid particle comprising an enzyme and a biomass.
[0009] In a second aspect, the invention provides a process for
preparing an enzyme containing particle comprising spray drying an
aqueous enzyme containing liquid starting material to obtain a
spray dried first enzyme containing particle and subsequently
subjecting the first dry particle to a process selected from
granulation and coating and combinations thereof to obtain a second
dry enzyme containing particle.
[0010] In additional aspects, the invention also provides particles
comprising an enzyme and a biomass, compositions comprising such
particles and methods of using such particles and compositions.
[0011] The present invention provides simple and cost effective
processes for producing dry enzyme particles having good
properties.
DEFINITONS
[0012] The term "fermentation broth" as used in the context of the
present invention is to be understood as an aqueous composition,
comprising both an enzyme and the microbial cells and/or cell
debris thereof, which during a fermentation process has produced
the enzyme.
[0013] The term "cell debris" as used in the context of the present
invention is to be understood as un-dissolved parts of the
fermented microbial cells including construction extending from the
cell, such as mycelium form fungal cells.
[0014] The term "biomass" as used in the context of the present
invention is to be understood as synonym for microbial cells, cell
debris or a combination thereof.
[0015] The term "fermentation filtrate" as used in the context of
the present invention is to be understood as a fermentation broth
from which microbial cells and cell debris has been removed.
[0016] The term "enzyme concentrate" as used in the context of the
present invention is to be understood as a fermentation filtrate
which has been processed to increase the concentration of
enzyme.
[0017] The term "SPAN" as used in the context of the present
invention is to be understood as the width of the particle size
distribution (PSD) and is expressed as:
SPAN=(D90-D10)/D50.
[0018] The PSD can be expressed in terms of the mass mean diameter
of the individual particles. A mean mass diameter of D50 is the
diameter at which 50% of the granules, by mass, have a smaller
diameter, while 50% by mass have a larger diameter. The values D10
and D90 are the diameters at which 10% and 90%, respectively, of
the granules, by mass, have a smaller diameter than the value in
question.
[0019] The term "Rayleigh Atomizer" is to be understood as an
atomizer capable of producing droplets of liquid having a low SPAN
value (usually SPAN values below 1.5 can be obtained such as
between 0.9-1.3), said atomizer characterized by comprising a
spraying member and a surface member comprising at least one bore
hole. In a preferred embodiment the Rayleigh Atomizer is a rotating
atomizing device wherein a liquid is atomized by distributing the
liquid onto the inner surface of a rotating hollow cylinder
comprising bore holes, the liquid forming droplets by passing the
cylinder wall through the bore holes. Such an atomizer is described
in WO 94/21383 claims 9-30 and FIGS. 1-18 and methods for atomizing
in claims 1-8 all incorporated herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Microorganisms
[0021] The microbial cells or micro-organisms, which is fermented
to produce a fermentation broth comprising an enzyme and a biomass
may be any micro-organism suitable for fermentation.
[0022] The micro-organism a unicellular microorganism, e.g., a
prokaryote, or a non-unicellular microorganism, e.g., a
eukaryote.
[0023] Useful unicellular cells are bacterial cells such as gram
positive bacteria including, but not limited to, a Bacillus cell,
e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus
brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,
Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus stearothermophilus, Bacillus subtilis, and
Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces
lividans or Streptomyces murinus, or gram negative bacteria such as
E. coli and Pseudomonas sp. In a preferred embodiment, the
bacterial micro-organism is a Bacillus lentus, Bacillus
licheniformis, Bacillus clausii, Bacillus stearothermophilus, or
Bacillus subtilis cell. In another preferred embodiment, the
Bacillus cell is an alkalophilic Bacillus.
[0024] The micro-organism may also be an eukaryote, such as a
mammalian, insect, plant, or fungal cell. In a preferred
embodiment, the micro-organism is a fungal cell. "Fungi" as used
herein includes the phyla Ascomycota, Basidiomycota,
Chytridiomycota, and Zygomycota (as defined by Hawksworth et al.,
In, Ainsworth and Bisby's Dictionary of The Fungi, 8 th edition,
1995, CAB International, University Press, Cambridge, UK) as well
as the Oomycota (as cited in Hawksworth et al., 1995, supra, page
171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
[0025] In a more preferred embodiment, the fungal micro-organism is
a yeast cell. "Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0026] In an even more preferred embodiment, the yeast
micro-organism is a Candida, Hansenula, Kluyveromyces, Pichia,
Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
[0027] In a most preferred embodiment, the yeast micro-organism is
a Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
In another most preferred embodiment, the yeast micro-organism is a
Kluyveromyces lactis cell. In another most preferred embodiment,
the yeast micro-organism is a Yarrowia lipolytica cell.
[0028] In another more preferred embodiment, the fungal
micro-organism is a filamentous fungal cell. "Filamentous fungi"
include all filamentous forms of the subdivision Eumycota and
Oomycota (as defined by Hawksworth et al., 1995, supra). The
filamentous fungi are characterized by a mycelial wall composed of
chitin, cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongation and
carbon catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of
a unicellular thallus and carbon catabolism may be
fermentative.
[0029] In an even more preferred embodiment, the filamentous fungal
micro-organism is a cell of a species of, but not limited to,
Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora,
Neurospora, Penicillium, Thielavia, Tolypocladium, or
Trichoderma.
[0030] In a most preferred embodiment, the filamentous fungal
micro-organism is an Aspergillus awamori, Aspergillus foetidus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or
Aspergillus oryzae cell. In another most preferred embodiment, the
filamentous fungal micro-organism is a Fusarium bactridioides,
oides, Fusarium cerealis, Fusarium crookwellense, Fusarium
culmorum, Fusarium graminearum, Fusarium graminum, Fusarium
heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium
reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium
sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,
Fusarium torulosum, Fusarium trichothecioides, or Fusarium
venenatum cell.
[0031] In another most preferred embodiment, the filamentous fungal
micro-organism is a Humicola insolens, Humicola lanuginosa, Mucor
miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Thielavia terrestris, Trichoderma harzianum,
Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma
reesei, or Trichoderma viride cell.
[0032] The present invention also encompasses variants of the above
mentioned microorganisms resulting from altering the genetic
material of said microorganism by conventional gene manipulation
methods, such inserting foreign nucleotide sequences encoding an
enzyme into said microorganisms.
[0033] The Enzyme
[0034] The enzyme in the context of the present invention may be
any enzyme or combination of different enzymes obtainable by
fermentation. Accordingly, when reference is made to "an enzyme"
this will in general be understood to include both a single enzyme
and a combination of more than one enzyme.
[0035] It is to be understood that enzyme variants (produced, for
example, by recombinant techniques) are included within the meaning
of the term "enzyme". Examples of such enzyme variants are
disclosed, e.g., in EP 251,446 (Genencor), WO 91/00345 (Novo
Nordisk), EP 525,610 (Solvay) and WO 94/02618 (Gist-Brocades NV).
The enzyme classification employed in the present specification and
claims is in accordance with Recommendations (1992) of the
Nomenclature Committee of the International Union of Biochemistry
and Molecular Biology, Academic Press, Inc., 1992.
[0036] Accordingly the types of enzymes which may appropriately be
incorporated in the enzyme product of the invention include
oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases
(EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-) and
ligases (EC 6.-.-.-).
[0037] Preferred oxidoreductases in the context of the invention
are peroxidases (EC 1.11.1) such as haloperoxidase, laccases (EC
1.10.3.2) and glucose oxidases (EC 1.1.3.4)], while preferred
transferases are transferases in any of the following
subclasses:
[0038] a) Transferases transferring one-carbon groups (EC 2.1);
[0039] b) Transferases transferring aldehyde or ketone residues (EC
2.2); acyltransferases (EC 2.3);
[0040] c) Glycosyltransferases (EC 2.4);
[0041] d) Transferases transferring alkyl or aryl groups, other
than methyl groups (EC 2.5); and
[0042] e) Transferases transferring nitrogenous groups (EC
2.6).
[0043] A most preferred type of transferase in the context of the
invention is a transglutaminase (protein-glutamine
.gamma.-glutamyltransferase; EC 2.3.2.13).
[0044] Further examples of suitable transglutaminases are described
in WO 96/06931 (Novo Nordisk A/S).
[0045] Preferred hydrolases in the context of the invention are:
Carboxylic ester hydrolases (EC 3.1.1.-) such as lipases (EC
3.1.1.3); phytases (EC 3.1.3.-), e.g. 3-phytases (EC 3.1.3.8) and
6-phytases (EC 3.1.3.26); glycosidases (EC 3.2, which fall within a
group denoted herein as "carbohydrases"), such as .alpha.-amylases
(EC 3.2.1.1); peptidases (EC 3.4, also known as proteases); and
other carbonyl hydrolases].
[0046] In the present context, the term "carbohydrase" is used to
denote not only enzymes capable of breaking down carbohydrate
chains (e.g. starches) of especially five- and six-membered ring
structures (i.e. glycosidases, EC 3.2), but also enzymes capable of
isomerizing carbohydrates, e.g. six-membered ring structures such
as D-glucose to five-membered ring structures such as
D-fructose.
[0047] Carbohydrases of relevance include the following (EC numbers
in parentheses): .alpha.-amylases (3.2.1.1), .beta.-amylases
(3.2.1.2), glucan 1,4-.alpha.-glucosidases (3.2.1.3), cellulases
(3.2.1.4), endo-1,3(4)-.beta.-glucanases (3.2.1.6),
endo-1,4-.beta.-xylanases (3.2.1.8), dextranases (3.2.1.11),
chitinases (3.2.1.14), polygalacturonases (3.2.1.15), lysozymes
(3.2.1.17), .beta.-glucosidases (3.2.1.21), .alpha.-galactosidases
(3.2.1.22), .beta.-galactosidases (3.2.1.23),
amylo-1,6-glucosidases (3.2.1.33), xylan 1,4-.beta.-xylosidases
(3.2.1.37), glucan endo-1,3-.beta.-D-glucosidases (3.2.1.39),
.alpha.-dextrin endo-1,6-.alpha.-glucosidases (3.2.1.41), sucrose
.alpha.-glucosidases (3.2.1.48), glucan endo-1,3-.alpha.-glucosid-
ases (3.2.1.59), glucan 1,4-.beta.-glucosidases (3.2.1.74), glucan
endo-1,6-.beta.-glucosidases (3.2.1.75), arabinan
endo-1,5-.alpha.-L-arab- inosidases (3.2.1.99), lactases
(3.2.1.108), chitosanases (3.2.1.132) and xylose isomerases
(5.3.1.5).
[0048] Examples of commercially available oxidoreductases (EC
1.-.-.-) include Gluzyme.TM. (enzyme available from Novo Nordisk
A/S).
[0049] Examples of commercially available proteases (peptidases)
include Kannase.TM., Everlase.TM., Esperase.TM., Alcalase.TM.,
Neutrase.TM., Durazym.TM., Savinase.TM., Pyrase.TM., Pancreatic
Trypsin NOVO (PTN), Bio-Feed.TM. Pro and Clear-Lens.TM. Pro (all
available from Novo Nordisk A/S, Bagsvaerd, Denmark).
[0050] Other commercially available proteases include Maxatase.TM.,
Maxacal.TM., Maxapem.TM., Opticlean.TM. and Purafect.TM. (available
from Genencor International Inc. or Gist-Brocades).
[0051] Examples of commercially available lipases include
Lipoprime.TM. Lipolase.TM., Lipolase.TM. Ultra, Lipozyme.TM.,
Palatase.TM., Novozym.TM. 435 and Lecitase.TM. (all available from
Novo Nordisk A/S). Other commercially available lipases include
Lumafast.TM. (Pseudomonas mendocina lipase from Genencor
International Inc.); Lipomax.TM. (Ps. pseudoalcaligenes lipase from
Gist-Brocades/Genencor Int. Inc.; and Bacillus sp. lipase from
Solvay enzymes.
[0052] Examples of commercially available carbohydrases include
Alpha-Gal.TM., Bio-Feed.TM. Alpha, Bio-Feed.TM. Beta, Bio-Feed.TM.
Plus, Bio-Feed.TM. Plus, Novozyme.TM. 188, Celluclast.TM.,
Cellusoft.TM., Ceremyl.TM., citrozym.TM., Denimax.TM., Dezyme.TM.,
Dextrozyme.TM., Finizym.TM., Fungamyl.TM., Gamanase.TM.,
Glucanex.TM., Lactozym.TM., Maltogenase.TM., Pentopan.TM.,
Pectinex.TM., Promozyme.TM., Pulpzyme.TM., Novamyl.TM.,
Termamyl.TM., AMG.TM. (Amyloglucosidase Novo), Maltogenase.TM.,
Sweetzyme.TM. and Aquazym.TM. (all available from Novo Nordisk
A/S).
[0053] Combinations of enzymes may be obtained by fermenting two or
more enzymes simultaneously in the same fermentation broth using
this liquid or a processed liquid thereof as a starting material in
a process of the invention. Alternatively combinations of enzymes
may be obtained by fermenting the enzymes separately and using
mixtures of different fermentation broths and/or processed liquids
thereof as a starting material for the process of the
invention.
[0054] Fermentation Broths and Enzyme Containing Liquids
[0055] The starting material for processes of the invention is a
fermentation broth or an enzyme containing liquid depending on the
steps of the process.
[0056] In the first aspect of the invention, supra, the starting
material is a fermentation broth and the process comprises the step
of spray drying said fermentation broth. As defined herein a
fermentation broth in accordance with the invention comprises
microbial cells and/or cell debris thereof (biomass). Some of the
biomass may be removed before spray drying to optimize the broth
properties and suitability for spray drying, e.g. to adjust
viscosity. However, the more biomass is removed the more expensive
the process will be. Accordingly, in a preferred embodiment the
fermentation broth comprises at least 10% of the biomass, more
preferably at least 50%, even more preferably at least 75% and most
preferably at least 90% or at least 95% of the biomass originating
from the fermentation. In another preferred embodiment the broth
contains 0-31% w/w dry matter, preferably 0-20% w/w, more
preferably 0-15% w/w such as 10-15% w/w dry matter, 0% dry matter
being excluded from said ranges. The biomass may constitute up to
90% w/w of the dry matter, preferably up to 75% w/w, more
preferably up to 50% w/w of the dry matter, while the enzyme may
constitute up to 50% w/w of the dry matter, preferably up to 25%
w/w, more preferably up to 10% w/w of the dry matter.
[0057] In another preferred embodiment of the first aspect of the
invention, coarse particles or bodies such as straw, rubble, soy
grits and other non-biomass insolubles are removed from the
fermentation broth starting material before spray drying. Such
coarse particles or bodies may originate from additives such
nutrients added during fermentation. The coarse particles or bodies
may be removed by straining, sedimentation, centrifugation and/or
decanting the broth. This process is called de-sludging.
[0058] In the second aspect of the invention the starting material
is an enzyme containing liquid and the process comprises the step
of spray drying said enzyme containing liquid to obtain a first dry
enzyme containing particle and subsequently subjecting the first
dry particle to a second process step to obtain a second dry enzyme
containing particle.
[0059] The starting material to be dried in accordance with the
second aspect of the invention, is preferably an aqueous liquid,
such as an aqueous solution or dispersion of one or more
enzymes.
[0060] In a preferred embodiment of the second aspect of the
invention, the starting material is a fermentation broth or a
fermentation broth, which have been subjected to one or more
processing steps, such as a fermentation filtrate or an enzyme
concentrate as defined, supra.
[0061] Removing the biomass from a fermentation broth to provide a
fermentation filtrate may be achieved by known methods such as
filtration, centrifugation, flocculation and combinations
thereof.
[0062] An enzyme concentrate may be provided for by processing a
fermentation filtrate. Such processing includes evaporation of
solvents (e.g. water), ultra filtration to reduce contents of water
and low molecular components, extraction of enzyme from the
fermentation filtrate into a second liquid. Also purification
through column chromatography may be used, e.g. by pumping the
fermentation filtrate through a column comprising a resin, allowing
the enzyme to pass at a slower or faster rate than water, salts or
other constituents of the filtrate.
[0063] In both first and second aspect of the invention the
starting material may, before spray drying, be added material which
will improve the properties of the resulting dry enzyme containing
powder or products resulting here from. Useful additives includes
materials selected from salts, inorganic minerals or clays,
carbohydrates, coloring pigments, cellulose or derivatives thereof,
biocides, dispersants, anti foaming agents, viscosity regulating
agents, acid agents, alkaline agents, enzyme stabilizers, enzyme
inhibitors, binders other enzymes and combinations thereof.
Addition of inorganic salts such as alkali and earth alkali salts
chloride, sulfate, nitrate and carbonate, e.g. Calcium salts may
activate the enzyme. Other salts, especially sodium sulfate or
chloride may be used as a filler material. Addition of
carbohydrates such as sucrose and/or starch may add to particle
strength and improve enzyme stability. Addition of coloring
pigments such as titanium dioxide may provide the broth and the
finished particulate enzyme product with a desired color. Addition
of cellulose may provide strength and elasticity to the enzyme
particles. Addition of stabilizers such as methionin or
thiosulphate may provide protection of the enzyme so, that it is
less liable to inactivation during processes of the invention and
storage of the obtained enzyme products. Addition of minerals and
clays such as zeolites, kaolin, bentonite, talc's, and/or silicates
may provide increased compactness of the dried enzyme particles and
granules there from. Addition of inhibitors such as boric acid
protease inhibitors may improve process yields by lowering protease
digestion of the enzyme or self digestion. Addition of biocides
such as Rodalon.RTM. enables control of the microbial stability and
may reduce odour of the final product. Addition of acids or bases
may be used to control pH to a desired level.
[0064] Also in both first and second aspect of the invention the
starting material may, before spray drying, be subjected to
physical treatments such heating and/or cooling and/or radiating
the broth as well as mixing, aeration, or ultra-sound
treatment.
[0065] In an important embodiment of the invention is that the
starting material, before spray drying, preferably is treated to
kill living microbial cells or treated to hydrolyze or disintegrate
remaining genetic material such as DNA (genomic or plasmidic) or
other poly-nucleotides present in the broth or liquid.
[0066] Sterilization of a broth in the context of the first aspect
of the invention may be achieved by addition of a biocide such as
Rodalon.RTM. (Benzalkonium chloride), heating (pasteurization) or
radiation. Sterilization of a fermentation filtrate or enzyme
concentrate broth in the context of the second aspect of the
invention may be achieved by the same means as for a broth or it
may be achieved by sterile filtration. In all cases disintegration
of genetic material may be achieved using known methods, such as
enzymatic digestion of poly-nucleotides.
[0067] For many applications useful enzyme-containing particles
requires that the particles contains a certain minimum amount of
active enzyme. Accordingly, in a preferred embodiment the starting
material should contain a certain minimum concentration of enzyme
in the liquid and/or the enzyme should constitute a certain minimum
percentage of the solids (i.e. non-volatile components) in the
liquid in order to produce particles by the process of the
invention, which have a useful enzyme contents. We have
surprisingly found it possible to provide fermentations which
directly yields fermentation broths having a sufficiently high
enzyme content, so that the broth may be dried directly or only
minimally refined by removing biomass, sterilization and addition
of additives to obtain a dry powder having a an enzyme content
sufficiently high to give an enzyme product useful in most
applications. Preferably the enzyme content in the fermentation
broth or fermentation filtrate before subjecting it to any further
refinement processes is at least 3 mg active enzyme protein per
liter liquid phase of the fermentation broth, more preferably at
least 20 mg/l, more preferably at least 50 mg/l, most preferably at
least 75 mg/l, such as 80 mg/l or more.
[0068] The starting material for the process of the invention
should further have a viscosity suitable for pumping the liquid,
preferably in the range 5-5000 cps.
[0069] Drying Processes
[0070] Drying of a broth, filtrate or concentrate, in accordance
with the invention is achieved by a spray drying process,
comprising transporting the broth, filtrate or concentrate through
an atomizing device into a drying chamber wherein droplets of
atomized broth, filtrate or concentrate is mixed with a stream of
air in which the volatile parts of the droplets are evaporated and
removed leaving dried enzyme-containing particles.
[0071] In a preferred embodiment of the invention the broth,
filtrate or concentrate is transported by means of a pumping device
and is preferably preheated to a temperature between 5-150.degree.
C., preferably between 50-120.degree. C., more preferably between
90-110.degree. C.
[0072] The atomization device may suitably be selected from high
speed rotating disk atomizers, pressure nozzle atomizers, pneumatic
nozzle atomizers or sonic nozzle atomizers such as described in the
Course Material from the Microencapsulation Seminar, held by Center
for professional advancement on May 9 to May 11, 1990 in
Amsterdam.
[0073] However, a preferred special atomizer, which in use produces
a superior dried enzyme containing particle is a Rayleigh atomizer
with which improved properties of the particles can be obtained,
such as increased mechanical strength, lower dusting, narrow
particle size distribution, i.e. a low SPAN value and improved
spherical shape may be obtained. One such atomizer is described in
WO 94/21383, which is hereby incorporated by reference.
[0074] The spray dried powder which usually will have a water
content of 10 -15% by weight may preferably be further dried to an
even lower moisture contents such as below about 5% w/w by
introducing the spray dried particles into a fluid bed drying
device in which the spray dried particles is kept fluidized by an
upwards stream of preferably heated and dried air evaporating
excess moisture from the fluidized particles.
[0075] The properties of the obtained spray dried solid enzyme
containing particles will depend on the spray drying conditions and
additives added to the starting material. In some spray drying
processes very small particles are formed initially in the drying
step which subsequently agglomerate or glue together to form larger
somewhat fragile agglomerates in the end of the spray drying
process. Such agglomerated particles may preferably have a mean
diameter or size in the range of 150-2000 .mu.m. In other spray
drying processes, such as using the atomizing device of WO 94/21383
smaller non-agglomerated more homogeneous particles may be produced
because of the special design of the atomizer. Such particles
generally have better dusting, strength and SPAN properties and the
mean size preferably fall within the range of 50-300 .mu.m, more
preferable within 100-200 .mu.m.
[0076] Preferred spray drying processes are processes which yields
product have a narrow size distribution, i.e. a low SPAN value.
Preferably the SPAN value is below about 2.5, more preferably below
about 2.0, more preferably below about 1.5, and most preferably
below about 1.0.
[0077] Processing of Dried Particles
[0078] The process of the second aspect of the invention (supra)
requires that the first spray dried particles are subsequently
subjected to a process selected from granulation and coating and
combinations thereof to obtain a second dry enzyme containing
particle. Granulation processes includes mixer granulation,
prilling, extrusion, fluid bed and compacting processes.
[0079] The present invention, however, also encompasses processes,
wherein particles obtained from spray drying a fermentation broth
are subsequently subjected to a process selected from granulation
and coating and combinations thereof.
[0080] Such further processing improves the properties of the
enzyme containing particles.
[0081] Mixer Granulation
[0082] A mixer granulation process includes mixing the spray dried
enzyme-containing particles with water and components selected from
binders, fibers, salts, water insoluble minerals, pigments, enzyme
stabilizers or combinations thereof. The water is added in
sufficient amounts to agglomerate the solid components into
granules of a desired mean size.
[0083] Binders include binders with a high melting point or no
melting point at all and of a non waxy nature e.g. polyvinyl
pyrrolidon, dextrins, polyvinylalkohol, cellulose derivatives, for
example hydroxypropyl cellulose, methyl cellulose or CMC. A
suitable binder is a carbohydrate binder such as Glucidex 21D
available from Roquette Freres, France.
[0084] Fibers include pure and/or or impure cellulose in fibrous
form such as sawdust, pure fibrous cellulose, cotton, or other
forms of pure or impure fibrous cellulose. Also, filter aids based
on fibrous cellulose can be used. Several brands of cellulose in
fibrous form are on the market, e.g. CEPO and ARBOCELL. In a
publication from Svenska Trmjolsfabrikerna AB, "Cepo Cellulose
Powder" it is stated that for Cepo S/20 cellulose the approximate
maximum fiber length is 500 .mu.m, the approximate average fibre
length is 160 .mu.m, the approximate maximum fibre width is 50
.mu.m and the approximate average fibre width is 30 .mu.m. Also, it
is stated that CEPO SS/200 cellulose has an approximate maximum
fibre length of 150 .mu.m, an approximate average fibre length of
50 .mu.m, an approximate maximum fiber width of 45 .mu.m and an
approximate average fiber width of 25 .mu.m. Cellulose fibers with
these dimensions are very well suited for the purpose of the
invention. The words "Cepo" and "Arbocel" are Trade marks. A
preferred fibrous cellulose is Arbocel.TM. BFC200. Also synthetic
fibres may be used as described in EP 304331 B1 and typical fibres
may be made of polyethylene, polypropylene, polyester, especially
nylon, polyvinylformat, poly(meth)acrylic compounds.
[0085] Salts include water soluble and/or insoluble salts such as
alkali and/or earth alkali salts of sulfate, chloride, carbonate
and phosphate.
[0086] Water insoluble minerals include zeolites, clays like kaolin
and bentonite, talcs, and/or silicates.
[0087] Pigments include titaniumdioxide.
[0088] Enzyme stabilizers include alkaline or neutral materials,
reducing agents, antioxidants and/or salts of first transition
series metal ions. Each of these may be used in conjunction with
other protective agents of the same or different categories.
Examples of alkaline stabilizers agents are alkali metal silicates,
-carbonates or bicarbonates which provide a chemical scavenging
effect by actively neutralizing e.g. oxidants. Examples of reducing
protective agents are salts of sulfite, thiosulfite or thiosulfate,
while examples of antioxidants are methionine, butylated
hydroxytoluene (BHT) or butylated hydroxyanisol (BHA). Most
preferred agents are salts of thiosulfates, e.g. sodium
thiosulfate. Mixer granulation processes are known to the art e.g.
from U.S. Pat. No. 4,106,991 (NOVO NORDISK) and related documents
EP 170360 B1 (NOVO NORDISK), EP 304332 B1 (NOVO NORDISK), EP 304331
(NOVO NORDISK), WO 90/09440 (NOVO NORDISK) and WO 90/09428 (NOVO
NORDISK) in which the addition of a liquid enzyme concentrate in
the context of this invention is replaced by addition of water to
the spray dried enzyme containing particles. Mixer granulation
processes also includes so called "marumerizer" processes as
described in U.S. Pat. No. 4,661,452 incorporated by reference.
[0089] In a preferred embodiment the enzyme containing mixture to
be processed in a mixer granulation process may also comprise a
particulate component having a diameter less than the diameter of
the finished granule. By adding such a particulate component to the
process the size and/or size distribution may be better controlled
as described in Danish patent application PA 1999 01000
(unpublished at the filing date of the present application) hereby
incorporated by reference. The particulate component may be an
agglomerate made from inorganic or organic starting materials which
are capable of maintaining the particulate integrity (i.e. it does
not disintegrate during the mixer granulation process). An
inorganic particulate component may be e.g. an agglomerated silica
and/or salt. An organic particulate compound may be a natural
compound such as agglomerated carbohydrates, e.g. sugars, starches
dextrins or it may be an agglomerated artificial compound or
polymeric compound. A referred particulate compound is however a
vegetable flour. The term "vegetable flour" here encompasses,
within the scope of the invention, all powdered grained products,
which have been obtained by size reduction (grinding) of solid
vegetable materials of natural origin (the flour source). It is
expedient in the method according to the invention to use vegetable
flours that are obtained by grinding of cereal grains, legumes
and/or fruits of the Malvaceae family (e.g., cottonseed). The
cereals that can serve as flour sources within the scope of the
invention are especially wheat or rye, but barley, oats, rice, and
maize, as well as sorghum and other types of millet can also be
used. Although buckwheat itself is not a cereal (it is a knot
grass), its beechnut-like flour-yielding parts can likewise be used
as flour source within the scope of the invention. In a particular
variation of the invention legumes may serve as a flour source.
Legumes here are to be understood as vegetable foodstuffs (legumes)
belonging to the fruits and vegetables. The fruits of leguminous
species such as Pisum (pea), Cajamus (pigeon pea), Cicer (chick
pea); lens (lentils); Phaseolus (kidney bean), Vigna (cow pea);
Dolchius (lablab bean); Cassavalia (sword bean), Vicia (horse-bean
or vetch); Peluschken [maple peal; Arachis (peanut); lupins;
lucerne; soybeans as well as lima beans and, if applicable, other
legumes and other Malvaceae fruits (e.g., of the genus Gossipium,
cotton); potato or yams may be considered as flour sources within
the scope of the invention. Especially preferred are peas and in
particular soybeans. The particulate compound of the invention may
also be a combination of the above mentioned flours and/or
agglomerates. A preferred particulate compound is a wheat based
flour such the commercially available product Farigel (Farigel de
Ble F1100, WestHove, France). The vegetable flour of the invention
has preferably been subjected to a steam treatment e.g. with dry
superheated steam with a temperature of about 100.degree. C. to
about 110.degree. C. at nearly normal pressure to low over pressure
(e.g., 0.8 to 1.2 bar over pressure) and a treatment time
(residence time in the superheated steam treatment apparatus
described below) of up to about 1 hour. Dry superheated steam is a
superheated and unsaturated steam, which can be obtained in the
conventional way by superheating and removal of possible water
condensate or by expansion of steam from high pressure. The
particulate component of the invention is distinguished by the
steam treatment being done after grinding the vegetable flour
source, ie. on the prepared particulate component ready to be used
in the mixer granulation process. The advantages of using a steam
treated particulate component is of course that that it lowers the
number of bacteria or fungus present in the particulate component
which may cause microbial growth in the product, but more important
the particulate component will be fully or partly gelatinised.
Gelatinising improves the integrity of the particles so they do not
disintegrate, dissolves or becomes dispersed in the granulation
process, but keep their particulate characteristics.
[0090] The preferred mean particle size of the particulate
component is at least 40 .mu.m, more preferred at least 60 .mu.m
such as at least 80 .mu.m, e.g. at least 100 .mu.m. Some useful
particulate components may be even larger e.g. have a mean particle
size of at least 140 .mu.m or even at least 200 .mu.m.
[0091] The mixing equipment can be a batch mixer or a continuous
mixer, such as a convective mixer [see, e.g., Harnby et al., Mixing
in the Process Industries, pp. 39-53 (ISBN 0-408-11574-2)].
Non-convective mixing equipment, e.g. rotating drum mixers or
so-called pan-granulators, may also be employed.
[0092] Prilling Process
[0093] In a prilling process the spray dried enzyme particles is
suspended in molten wax and the suspension is spray cooled, e.g.
through an atomizing device, into a cooling chamber where the
droplets wax comprising enzyme particles solidify. This process is
known from Michael S. Showell (editor); Powdered detergents;
Surfactant Science Series; 1998; vol. 71; page 140-142; Marcel
Dekker and DK-PA 1999 01358 both hereby incorporated by reference.
The atomizer used in the prilling process is suitably the same as
described for spray drying, supra, preferably a Rayleigh
atomiser.
[0094] Prilled particles may be made to have a desired mean size
and a uniform density and further the enzyme is protected within
the wax matrix. The term "wax" as used herein, is to be construed a
compound having a melting point between 25-150.degree. C. Preferred
waxes are organic compounds or salts of organic compounds having a
melting point in the said range or mixtures thereof. By applying
mixtures of different waxes, optionally in combination with heavy
and/or light solids, enzyme particles of a desired true density can
be obtained. Also, an important feature of the wax or mixture of
waxes is that the wax should be water soluble or water dispersible,
preferably in neutral and alkaline solution, so that the wax matrix
of the enzyme particle may disintegrate and/or dissolve in an
aqueous solution. Examples of water soluble waxes are poly ethylene
glycols (PEG's) Accordingly amongst water soluble waxes the
solubility of wax in water should preferably be up to 75 parts wax
to 25 parts water, such as for PEG 1000. Amongst water insoluble
waxes which are dispersible in an aqueous solution are
triglycerides and oils. Further a useful wax do not dissolve or
disintegrate in a substantially nonaqueous liquid phase. The term
"substantially nonaqueous" in this context may be defined as the
liquid phase containing little (e.g. below 5% w/w or below 3% w/w)
or no water (non-aqueous). The wax should also be compatible with
the enzyme, i.e. it should not inactivate the enzyme, e.g. by
reacting with the enzyme or permanently altering structures, such
as foldings, helical portions, sheeted portions, prosthetic groups
and the like necessary for the enzyme to retain the activity. Still
further the wax should be mixable with the enzyme, i.e. the enzyme
may be dissolved in the (molten) wax and/or the enzyme may be
dispersed in the (molten) wax in an amorphous or crystalline form
as enzyme protein particles. A suitable wax is in a solid state at
room temperature (25.degree. C.), and accordingly is has a melting
point or a melting range (polymer waxes tend to melt over a range
of temperatures) above this temperature. A preferred wax has a
melting point or range between about 35.degree. C. to about
120.degree. C. The lower limit is preferred to set a reasonable
distance between the temperature at which the wax melts to the
temperature at which liquid detergents comprising the enzyme
particles are usually stored (20-30.degree. C). Also, difficulties
is contemplated, in the manufacture of the enzyme particles when
the melting point of the wax is below 35 .degree. C. The upper
temperature limit is set as the maximum temperature usually
applicable for enzymes without experiencing significant losses of
enzyme activity, due to heat denaturation. A more preferred melting
point or range is between about 40.degree. C. to about 100.degree.
C., such as between about 50.degree. C. to about 80.degree. C. In a
further preferred embodiment the wax have a molecular weight
between about 150 Daltons to about 10.000 Daltons. The wax may be
chemically synthesized or it may equally well be a wax isolated
from a natural source or a derivative thereof. Accordingly in the
wax of the invention is preferably selected from the following non
limiting list of waxes.
[0095] Poly ethylene glycols, abbreviated PEG, type of wax.
Different PEG waxes are commercially available having different
molecular sizes, wherein PEG's with low molecular sizes also have
the lowest melting points. Examples of suitable PEG's are PEG 1500,
PEG 3000, PEG 4000, PEG 6000, PEG 9000 e.g. from BASF--Germany. To
meet the desired properties of true density and melting point for
the wax and/or the enzyme particle, it also contemplated that
mixtures of waxes with low melting point with waxes of a high
melting point is a very useful embodiment of the invention.
[0096] polypropylens or polyethylens or mixtures thereof.
[0097] Nonionic tensides which are solid at room temperature such
as ethoxylated fatty alcohols having a high level of ethoxy groups
such as Lutensol AT80 from BASF having 80 units of ehtyleneoxide
per molecule. Alternatively polymers of ethyleneoxide,
propyleneoxide or copolymers thereof are useful, such as in block
polymers, e.g. Pluronic PE 6800 from BASF Germany.
[0098] Waxes isolated from a natural source, such as Carnauba wax
(melting point between 80-88.degree. C.), Candelilla wax (melting
point between 68-70.degree. C.) and bees wax. Other natural waxes
or derivatives thereof are waxes derived from animals or plants,
e.g. of marine origin. Examples of such waxes are hydrogenated ox
tallow, hydrogenated palm oil, hydrogenated cotton seeds and/or
hydrogenated soy bean oil, wherein the term "hydrogenated" as used
herein is to be construed as saturation of unsaturated carbohydrate
chains, e.g. in triglycerides, wherein carbon=carbon double bonds
are converted to carbon-carbon single bonds. An example
hydrogenated palm oil is commercially available e.g. from Hobum
Oele und Fette GmbH--Germany or Deutche Cargill GmbH--Germany.
[0099] Fatty acid alcohols, such as the linear long chain fatty
acid alcohol NAFOL 1822 (C.sub.18, 20, 22) from Condea Chemie
GMBH--Germany, having a melting point between 55-60.degree. C. and
having a true density of about 0.96 g/cm.sup.3.
[0100] Mono-glycerider and/or di-glycerider, such as glyceryl
stearate, wherein stearate is a mixture of stearic and palmitic
acid are useful waxes. An example of this is Dimo-dan PM--from
Danisco Ingredients, Denmark--having a melting point of about 1
g/cm.sup.3
[0101] Fatty acids, such as hydrogenated linear long chained fatty
acids.
[0102] Paraffines, i.e. solid hydrocarbons.
[0103] Micro-crystalline wax.
[0104] In further embodiments waxes which are useful in the
invention can be found in C. M. McTaggart et. al., Int. J. Pharm.
19, 139 (1984) or Flanders et.al., Drug Dev. Ind. Pharm. 13, 1001
(1987) both incorporated herein by reference.
[0105] Extrusion Processes
[0106] In an extrusion or pelletizing process moisture is added to
the spray dried enzyme containing particles, either alone or mixed
with additive such as described for mixer granulation, to provide
an enzyme containing paste. This paste is then pressed to pellets
or under pressure extruded through a small opening and cut into
larger particles which is subsequently dried. Extrusion is known
from Michael S. Showell (editor); Powdered detergents; Surfactant
Science Series; 1998; vol. 71; page 140-142; Marcel Dekker and U.S.
Pat No. 4,661,452 both incorporated by reference.
[0107] Fluid Bed Processes
[0108] A fluid bed process in the context of the invention
comprises fluidizing the spray dried enzyme containing particles in
a fluid bed and atomizing a solution comprising a binder to the
fluidized powder so as to bind the particles of the enzyme
containing powder together to form larger and stronger
particles.
[0109] The spray dried enzyme containing particles are less
suitable use in processes for producing layered granules, herein
defined as a process wherein the enzyme is applied as a layer
around an inert particulate core material e.g. in a fluid bed
device wherein the inert core is fluidized and the enzyme layer is
applied by spraying onto the core an enzyme containing solution.
Such a process requires re-dissolution or resuspension of the dried
enzyme-containing powder in a liquid in order apply the enzyme
containing layer around the inert core, thus introducing a more
energy consuming step than for the processes, supra. Accordingly,
in a preferred embodiment the subsequent processing of the dried
enzyme containing particles is not a layering process.
[0110] Coating Processes
[0111] Spray dried particles or granules obtained there from may
preferably be coated with one or more coating layers to provide
further improved properties of the granule. Conventional coatings
and methods as known to the art may suitably be used, such as the
coatings described in WO 89/08694, WO 89/08695, 270 608 B1 and/or
WO 00/01793. Other examples of conventional coating materials may
be found in U.S. Pat. No. 4,106,991, EP 170360, EP 304332, EP
304331, EP 458849, EP 458845, WO 97/39116, WO 92/12645A, WO
89/08695, WO 89/08694, WO 87/07292, WO 91/06638, WO 92/13030, WO
93/07260, WO 93/07263, WO 96/38527, WO 96/16151, WO 97/23606, U.S.
Pat. No. 5,324,649, U.S. Pat No. 4,689,297, EP 206417, EP 193829,
DE 4344215, DE 4322229 A, DD 263790, JP 61162185 A and/or JP
58179492.
[0112] The coating may comprise materials selected from binders,
fibers, salts, water insoluble minerals, pigments, enzyme
stabilizers or combinations thereof as described, supra, in the
mixer granulation section.
[0113] In a particular embodiment the coating may comprise minor
amounts of a protective agent capable of reacting with a component
capable of inactivating (being hostile to) the enzyme entering the
particle or granule from a surrounding matrix, i.e. before the
component come into contact and inactivate the enzyme. The
protective agent may thus e.g. be capable of neutralizing, reducing
or otherwise reacting with the component rendering it harmless to
the enzyme. Typical components capable of inactivating the enzyme
are oxidants such as perborates, percarbonates, organic peracids
and the like.
[0114] Protective agents may fall into several categories: alkaline
or neutral materials, reducing agents, antioxidants and/or salts of
first transition series metal ions. Each of these may be used in
conjunction with other protective agents of the same or different
categories. Examples of alkaline protective agents are alkali metal
silicates, carbonates or bicarbonates which provide a chemical
scavenging effect by actively neutralizing e.g. oxidants. Examples
of reducing protective agents are salts of sulfite, thiosulfite or
thiosulfate, while examples of antioxidants are methionine,
butylated hydroxytoluene (BHT) or butylated hydroxyanisol (BHA).
Most preferred agents are salts of thiosulfates, e.g. sodium
thiosulfate. The amounts of protective agent in the coating may be
5-40% w/w of the coating, preferably 5-30%, e.g. 10-20%.
[0115] The coating should encapsulate the enzyme containing
particle or granule by forming a substantially continuous
homogenous layer.
[0116] The coating may perform any of a number of functions in the
particle or granule, depending on the intended use. Thus, for
example, a coating may achieve one or more of the following
effects:
[0117] (i) further reduction of the dust-formation tendency of an
enzyme particle or granule;
[0118] (ii) further protection of enzyme(s) in the enzyme
particle/granule against oxidation by bleaching substances/systems
(e.g. perborates, percarbonates, organic peracids and the
like);
[0119] (iii) dissolution at a desired rate upon introduction of the
particle/granule into a liquid medium (such as an aqueous
medium);
[0120] (iv) provide a better physical strength of the enzyme
particle/granule.
[0121] The coating may further comprise one or more of the
following: anti-oxidants, chlorine scavengers, plasticizers,
pigments, lubricants (such as surfactants or antistatic agents)
additional enzymes and fragrances.
[0122] Plasticizers useful in coating layers in the context of the
present invention include, for example: polyols such as sugars,
sugar alcohols, or polyethylene glycols (PEGs) having a molecular
weight less than 1000; urea, phthalate esters such as dibutyl or
dimethyl phthalate; and water.
[0123] Suitable pigments include, but are not limited to, finely
divided whiteners, such as titanium dioxide or kaolin, coloured
pigments, water soluble colorants, as well as combinations of one
or more pigments and water soluble colorants.
[0124] As used in the present context, the term "lubricant" refers
to any agent which reduces surface friction, lubricates the surface
of the granule, decreases tendency to build-up of static
electricity, and/or reduces friability of the granules. Lubricants
can also play a related role in improving the coating process, by
reducing the tackiness of binders in the coating. Thus, lubricants
can serve as anti-agglomeration agents and wetting agents.
[0125] Examples of suitable lubricants are polyethylene glycols
(PEGs) and ethoxylated fatty alcohols.
[0126] In a preferred embodiment of the invention the spray dried
particle or subsequently processed granule of the invention is
coated with a protective coating having a high constant humidity
such as described in the Danish patent application WO 00/01793
pages 5-9 and given examples which was unpublished at the date of
filing this application and which is hereby incorporated by
reference.
[0127] Milling/Grinding and Screening
[0128] It is to be understood that the processes as described above
may suitably be supplemented with milling/grinding and/or screening
processes at any stage of the processes. For example it may be
desirable to grind the spray dried enzyme containing particles
prior to subsequent processing steps and to screen the final
product to obtain the a desired size fraction.
[0129] Particles Comprising Enzymes and Biomass
[0130] The invention encompasses particles comprising an enzyme and
a biomass such as particles obtained by the second first aspect of
the invention, i.e. particles obtained from a process selected from
spray drying a fermentation broth, granulating a spray dried
fermentation broth, coating a spray dried fermentation broth and
granulating and coating a spray dried fermentation broth.
[0131] Applications
[0132] The particles of the invention is useful in a vide range of
compositions and applications. Examples of useful compositions are
cleaning compositions, textile processing compositions, leather
processing compositions, pulp or paper processing compositions,
food and beverage compositions, animal feed compositions and
personal care compositions. Cleaning compositions includes such as
detergents and anti-microbial compositions. Textile processing
compositions includes compositions for enzymatic bleach and/or
stone washing of textiles, such as denim. Food and beverage
compositions includes enzymatic compositions used in industries
producing wine, oils and fats, citrus and juice products, starch
and sugar products, alcohols and/or brewed products, soy products
and baking flour or dough.
[0133] Accordingly, the present invention encompasses compositions
comprising particles comprising an enzyme and a biomass, preferably
obtained from a process selected from spray drying a fermentation
broth, granulating a spray dried fermentation broth, coating a
spray dried fermentation broth and granulating and coating a spray
dried fermentation broth.
[0134] The present invention also encompasses the use of particles
comprising enzyme and biomass and compositions comprising such
particles. Especially use for treatment of textile, leather, pulp,
paper, food, beverage, hard surfaces and the human or animal body.
The particles comprising enzyme and biomass and compositions
comprising such particles may also be used in the manufacture of a
medicament for treatment of the human or animal body.
[0135] Detergent Compositions
[0136] A preferred composition is a detergent composition
comprising a surfactant and an enzyme and biomass containing
particle obtained from spray drying a fermentation broth or a
granule obtained from such spray dried particle.
[0137] The detergent composition of the invention may for example
be formulated as a hand or machine laundry detergent composition
including a laundry additive composition suitable for pretreatment
of stained fabrics and a rinse added fabric softener composition,
or be formulated as a detergent composition for use in general
household hard surface cleaning operations, or be formulated for
hand or machine dishwashing operations.
[0138] In a specific aspect, the invention provides a detergent
additive comprising the enzyme and biomass containing spray dried
particle or granule of the invention. The detergent additive as
well as the detergent composition may comprise one or more other
enzymes such as a protease, a lipase, a cutinase, an amylase, a
carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase,
a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a
peroxidase.
[0139] In general the properties of the chosen enzyme(s) should be
compatible with the selected detergent, (i.e. pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective
amounts.
[0140] Proteases: Suitable proteases include those of animal,
vegetable or microbial origin. Microbial origin is preferred.
Chemically modified or protein engineered mutants are included. The
protease may be a serine protease or a metalprotease, preferably an
alkaline microbial protease or a trypsin-like protease. Examples of
alkaline proteases are subtilisins, especially those derived from
Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin
309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
Examples of trypsin-like proteases are trypsin (e.g. of porcine or
bovine origin) and the Fusarium protease described in WO 89/06270
and WO 94/25583.
[0141] Examples of useful proteases are the variants described in
WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially
the variants with substitutions in one or more of the following
positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170,
194, 206, 218, 222, 224, 235 and 274.
[0142] Preferred commercially available protease enzymes include
Alcalase.TM., Savinase.TM., Primase.TM., Duralase.TM.,
Esperase.TM., and Kannase.TM. (Novo Nordisk A/S), Maxatase.TM.,
Maxacal.TM., Maxapem.TM., Properase.TM., Purafect.TM., Purafect
OxP.TM., FN2.TM., and FN3.TM. (Genencor International Inc.).
[0143] Lipases: Suitable lipases include those of bacterial or
fungal origin. Chemically modified or protein engineered mutants
are included. Examples of useful lipases include lipases from
Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T.
lanuginosus) as described in EP 258 068 and EP 305 216 or from H.
insolens as described in WO 96/13580, a Pseudomonas lipase, e.g.
from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.
cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,
Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.
wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B.
subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,
1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus
(WO 91/16422).
[0144] Other examples are lipase variants such as those described
in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381,
WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO
97/04079 and WO 97/07202.
[0145] Preferred commercially available lipase enzymes include
Lipolase.TM. and Lipolase Ultra.TM. (Novo Nordisk A/S).
[0146] Amylases: Suitable amylases (.alpha.and/or .beta.) include
those of bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Amylases include, for example,
.alpha.-amylases obtained from Bacillus, e.g. a special strain of
B. licheniformis, described in more detail in GB 1,296,839.
[0147] Examples of useful amylases are the variants described in WO
94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the
variants with substitutions in one or more of the following
positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188,
190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.
[0148] Commercially available amylases are Duramyl.TM.,
Termamyl.TM., Fungamyl.TM. and BAN.TM. (Novo Nordisk A/S),
Rapidase.TM. and Purastar.TM. (from Genencor International
Inc.).
[0149] Cellulases: Suitable cellulases include those of bacterial
or fungal origin. Chemically modified or protein engineered mutants
are included. Suitable cellulases include cellulases from the
genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,
Acremonium, e.g. the fungal cellulases produced from Humicola
insolens, Myceliophthora thermophila and Fusarium oxysporum
disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S.
Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.
[0150] Especially suitable cellulases are the alkaline or neutral
cellulases having colour care benefits. Examples of such cellulases
are cellulases described in EP 0 495 257, EP 0 531 372, WO
96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase
variants such as those described in WO 94/07998, EP 0 531 315, U.S.
Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No.
5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
[0151] Commercially available cellulases include Celluzyme.TM., and
Carezyme.TM. (Novo Nordisk A/S), Clazinase.TM., and Puradax HA.TM.
(Genencor International Inc.), and KAC-500(B) .TM. (Kao
Corporation).
[0152] Peroxidases/Oxidases: Suitable peroxidases/oxidases include
those of plant, bacterial or fungal origin. Chemically modified or
protein engineered mutants are included. Examples of useful
peroxidases include peroxidases from Coprinus, e.g. from C.
cinereus, and variants thereof as those described in WO 93/24618,
WO 95/10602, and WO 98/15257.
[0153] Commercially available peroxidases include Guardzyme.TM.
(Novo Nordisk A/S).
[0154] The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive of the invention, i.e. a separate
additive or a combined additive, is formulated so as to contain one
or more of the enzyme granules of the invention.
[0155] The detergent composition of the invention may be in any
convenient form, e.g., a bar, a tablet, a powder, a granule, a
paste or a liquid. A liquid detergent may be aqueous, typically
containing up to 70% water and 0-30 % organic solvent, or
non-aqueous.
[0156] The detergent composition comprises one or more surfactants,
which may be non-ionic including semi-polar and/or anionic and/or
cationic and/or zwitterionic. The surfactants are typically present
at a level of from 0.1% to 60% by weight.
[0157] When included therein the detergent will usually contain
from about 1% to about 40% of an anionic surfactant such as linear
alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty
alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid
or soap.
[0158] When included therein the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl N-alkyl derivatives of glucosamine ("glucamides").
[0159] The detergent may contain 0-65% of a detergent builder or
complexing agent such as zeolite, diphosphate, triphosphate,
phosphonate, carbonate, citrate, nitrilotriacetic acid,
ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic
acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered
silicates (e.g. SKS-6 from Hoechst).
[0160] The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene
glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates such as polyacrylates,
maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid
copolymers.
[0161] The detergent may contain a bleaching system which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
Alternatively, the bleaching system may comprise peroxyacids of
e.g. the amide, imide, or sulfone type.
[0162] The enzyme(s) of the detergent composition of the invention
may be stabilized using conventional stabilizing agents, e.g., a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, boric acid, or a boric acid derivative, e:g.,
an aromatic borate ester, or a phenyl boronic acid derivative such
as 4-formylphenyl boronic acid, and the composition may be
formulated as described in e.g. WO 92/19709 and WO 92/19708.
[0163] The detergent may also contain other conventional detergent
ingredients such as e.g. fabric conditioners including clays, foam
boosters, suds suppressors, anti-corrosion agents, soil-suspending
agents, anti-soil redeposition agents, dyes, bactericides, optical
brighteners, hydrotropes, tarnish inhibitors, or perfumes.
[0164] It is at present contemplated that in the detergent
compositions any enzyme, in particular the enzyme of the invention,
may be added in an amount corresponding to 0.01-100 mg of enzyme
protein per liter of wash liqour, preferably 0.05-5 mg of enzyme
protein per liter of wash liqour, in particular 0.1-1 mg of enzyme
protein per liter of wash liqour.
[0165] The enzyme of the invention may additionally be incorporated
in the detergent formulations disclosed in WO 97/07202 which is
hereby incorporated as reference.
[0166] MATERIALS AND METHODS
[0167] The unit for protease activity used herein is Kilo Novo
Protease. KNPU of a sample is determined relatively to a
Savinase.RTM. standard in a standard assay by measuring for a given
amount of sample the formation rate (pmol/minute) of free amino
groups liberated from digestion of di-methyl-casein (DMC.) in
solution by the enzyme in the sample. The formation rate is
monitored by recording the linear development of absorbance at 420
nm of the simultaneous reaction between the formed free amino
groups and added 2,4,6-tri-nitro-benzene-sulfonic acid (TNBS). The
digestion of DMC and the colour reaction is carried out at
50.degree. C. in a pH 8.3 boric acid buffer with a 9 min. reaction
time followed by a 3 min. measuring time.
EXAMPLE 1
[0168] Spray-drying of a high product titre protease fermentation
broth.
[0169] A production size batch of protease containing fermentation
broth with an dry matter content of 13% w/w was sieved through a
rotary brush strainer to remove large solid particulates. The
fermented micro-organism was killed by adding 0.5% Rodalon (50%
Benzalkoniumchlorid) to the broth while maintaining agitation.
Formic acid was then added to reduce pH to 5.5 and the broth left
for an hour or more to allow the Rodalon to be effective. The broth
was heated to an inlet temperature of 140.degree. C. and introduced
to a drying chamber (Niro Atomizer, SD-200-R spray drying tower)
via a rotating spray nozzle. The exit temperature being 55.degree.
C. The resulting spray dried powder had an activity of 41 KNPU/g
and a water content of 4.86%
EXAMPLE 2
[0170] Production-scale spray-drying of a high product titre
cellulase fermentation broth.
[0171] A production size batch of cellulase containing fermentation
broth is sieved through a rotary brush strainer to remove large
solid particulates. To kill the production strain, 0.5% Rodalon
(50% Benzalkoniumchlorid) is added while the broth is well agitated
and the broth is left for at least one hour to allow the Rodalon to
be effective. The broth introduced to a spray tower (Niro Atomizer,
SD-200-R) via a rotating spray nozzle at an inlet temperature of
140.degree. C. The exit temperature should be around 55.degree. C.
The resulting spray dried powder have an estimated activity of
20000 ECU/g and a water content estimated below 5.0% w/w.
EXAMPLE 3
[0172] Production-scale spray-drying of a high product titre
amylase fermentation broth.
[0173] A production size batch of amylase containing fermentation
broth is sieved through a rotary brush strainer to remove large
solid particulates. To kill the production strain, 0.5% Rodalon
(50% Benzalkonium-chlorid) is added while the broth is well
agitated and the broth is left for at least one hour to allow the
Rodalon to be effective. The broth is introduced to the spray tower
(Niro Atomizer, SD-200-R) via a rotating spray nozzle at an inlet
temperature of 140.degree. C. The exit temperature should be around
55.degree. C. The resulting spray dried powder have an estimated
activity of 960 KNU/g and a water content estimated below 5.0%
w/w.
* * * * *