Note: Descriptions are shown in the official language in which they were submitted.
WO 94/26883 ~ ~ ~I d ~ 3 ~ PCT/EP94/01642
PROC'~ FOR DU8T--FRISE: ~N;~ Yl~S MANIJFACTUR~
Field of the Invention
The present invention relates to novel enzyme granules
and a process for the preparation thereof.
Backqround of the Invention
Many commercially useful enzymes are produced by
microorganisms such as bacteria, yeast and fungi. These enzymes
are especially useful in detergents, starch and textile
processing, and in feed and food applications.
Examples of enzymes useful in detergent application
include proteases, amylases, lipases and cellulases. The
enzymes are produced by fermentation and then recovered,
purified and concentrated into a liquid or brought in dry form.
Suitable recovery techniques include filtration, centrifug-
20 ation, membrane filtration, precipitation, crystallisation,
chromatography, spray drying, etc.
Because of dermatologic and other health problems
which might occur in connection with dry enzyme and in
particular dry protease preparations, the dust levels in such
25 preparations should be as low as possible. To achieve this the
dry enzymes are usually brought in granule form, for which
several granulation techniques have been developed. See, for
example, U.S. Patents Nos. 4,009,076, 4,016,040, 4,078,368,
4,242,219, etc.
In order to further reduce the enzyme dust release and
to protect the granules, most of the granules are coated after
their production with a film-forming macromolecular material.
Basically two types of granules can be distinguished.
In the first type the compound of interest is mixed through the
35 granule and in the second type the compound of interest is
located around the core of the granule. It will be clear that
in the latter type the concentration of the compound of
interest (e.g. an enzyme) at the surface of the core is very
high as compared to a granule were the compound is mixed
W094/26883 ~ 3 ~ PCT~4/01642
throughout the granule. Consequently, when the coating is
inadequate or damaged, the compound will be exposed to the
environment at a higher level than in case of a granule with an
uniform distribution of the compound in question.
JP 58-179492A discloses the preparation of enzyme
granules with a protective coating. Here a fluid bed dryer is
used to first spray e.g. a liquid enzyme concentrate onto a
core material and subsequently spray a coating material
containing a cellulose derivative onto the granule, while
10 drying takes place during the whole process.
EP-A-0193829 describes a method for the production of
dust free enzyme containing particles by coating hydratable
core particles with an enzyme and then with a film-forming
material. Coating is carried out by suspending the core
15 particles in a fluidized bed dryer, spraying an aqueous slurry
of enzyme onto the core particles while suspended, and
evaporating water to leave a dried enzyme coat on the
particles. The resultant enzyme-coated particles, while still
suspended in the fluidized bed, are sprayed with a solution or
20 dispersion of the macromolecular material, and dried to remove
solvent to leave a coating of the macromolecular material.
W0 90/09428 discloses a detergent additive granulate
which comprises a core with a primary detergent additive, e.g.
an enzyme, surrounded by a shell comprising a secondary
25 detergent additive, e.g. another enzyme, a binder, and
granulating agents, and optionally a filler, and a protective
coating between the core and the shell, whereby the shell
comprises cellulose or artificial fibres, and whereby the core
optionally may also comprise cellulose or artificial fibres.
30 The detergent additive granulate is said to exhibit a high
physical strength, and the primary and secondary detergent
additives are separated from each other and/or from harmful
environmental factors.
W0 93/07263 discloses a granular enzyme composition
35 which comprises a core, an enzyme layer and an outer coating
layer. The enzyme layer, and optionally the core and coating
layers, contain a vinyl polymer. The granular enzyme
W094/26883 21 4 ~ 5 3 0 PCT~P94/01642
composition is said to have reduced tendencies to form dust and
leave residue, and exhibit improved stability and delayed
release characteristics. The method for making such enzyme-
containing granules is said to have greatly reduced processing
- 5 time.
The present invention provides new enzyme granules
with improved properties. As compared to the prior art cited
above the granules according to the present invention have the
advantage that the compound of interest is not only located at
10 the surface of the granule.
Summary of the Invention
According to the first aspect of the invention enzyme
15 granules are provided comprising porous core material in which
the enzyme in solution is adsorbed.
The enzyme-containing granules are preferably coated
with a film-forming macromolecular material. Such granules have
an improved mech~n;cal strength.
The invention further provides an efficient method of
preparing the novel enzyme granules.
Detailed Descri~tion and Preferred Embodiments
The granules according to the present invention are
based on core particles having a good pore size and pore size
distribution to allow an enzyme solution to enter into the
particle. Accordingly, the core material comprises the enzyme
in liquid form, thus eliminating the drawback of processing
30 powdered enzymes.
The core materials available up till now do not have
the required characteristics. The pore diameter should not be
too big, since this will inter alia reduce the strength of the
particle, and not too small, since this will prevent the
35 compound of interest, e.g. an enzyme, entering into the pores.
European Patent Application EP-A-0542351 (published on
May 19, 1993, i.e. after the priority date of the present
W094/268~ ~ 0~ 3 0 PCT~4/01642
patent application) discloses a process for the preparation of
salt granulates. The products which are obtainable by this
process are very suitable for use as core material in enzyme
applications. The level of porosity is important with respect
5 to the economics of the process.
In a further aspect of the invention a method is
provided for efficiently preparing the new enzyme granules
which comprises the following steps:
o (a) preparing a non-aqueous enzyme solution
(b) applying said enzyme solution onto porous core
material whereby the enzyme solution is absorbed into the
porous core material
(c) drying the resulting enzyme granules, and,
15 optionally,
(d) coating the enzyme granules with a macromolecular,
film-forming material.
The enzyme solution may be prepared in various ways,
20 mainly depending on the physical characteristics of the enzyme
used. Suitable solvents for the purpose of the invention
include ethylene glycol, propylene glycol, liquid polyethylene
glycols (PEG's) such as PEG 200 and PEG 400, and glycerol. In
certain cases it may be useful to prepare first an aqueous
25 enzyme solution and to add the non-aqueous solvent. The water
is then partly or entirely removed, for example by evaporation.
The "non-aqueous" enzyme solution may contain a certain amount
of water, for example 10-20%, but this should not have an
adverse affect on the various components of the granulate, in
30 particular the porous core material. Instead of an enzyme
solution also an enzyme slurry may be used in certain instances
which will be clear to the man skilled in the art.
The absorption of enzyme into the particle can be done
in various way. Both from an enzyme containing aqueous liquid
S5 or slurry (e.g. a concentrated fermentation broth) as from an
enzyme containing non-aqueous liquid or slurry (e.g. non
ionics, alcohols etc.), or a combination thereof.
W094/268~ ~ ~ PCT~4/01642
Suitable porous core material includes soda, NaCl and
silica and is commercially available. The porous core material
is preferably prepared by the method described in EP-A-0542351.
The enzyme solution can be brought onto the porous
5 core material in various ways which are all known in the art,
for example using ;~;ng devices or a fluidized bed or a mixer-
fluid bed dryer combination. Process steps (b) and (c) may
suitably be combined.
If desired, one or more protective coating layer(s)
10 can be brought onto the core to yield a dust free enzyme
containing particle. Suitable coating materials are frequently
described in the literature, see e.g. JP 58-179492A and EP-A-
0193829. They include cellulose coatings or cellulose based
coatings containing hydroxypropylcellulose, methyl cellulose,
15 hydroxypropyl methyl cellulose and/or hydroxyethyl cellulose.
Also acrylic polymers like EUDRAGITr (Rohm Pharma) can be
applied. The amounts of coating to be applied may vary in
fairly wide ranges but is usually between 0.1 and 25% by
weight, for the cellulose based coatings preferably between 5
20 and 25 wt%.
Coating layers can also be used to add other useful
compounds to the granule. It is even possible to prepare
multilayer granules wherein various coating layers have
different functions, for example to stabilize the compounds
25 which are present, to add colour etc.
In certain applications which will be clear to the
skilled person porous core material is suitably used which
dissolves slowly or which liberates the absorbed enzyme slowly.
This technology in combination with coating technology permits
30 a composition of a granule where several compounds can be
released in a sequence according to demand. An example hereof
is a granule where a protease is absorbed into a porous core, a
lipase is coated on the core and finally a coating layer is
brougth onto the granule.
The entire process for preparing the enzyme granules
according to the invention (absorption, coating with coating
material and other compounds) may be suitably carried out in
W094/26883 21~ ~ ~ 3 ~ PCT~4/01642
one apparatus, for example a mixer or a fluidized bed.
The process according to the invention has several
advantages. When the process is performed in more than one step
the (enzyme) dust formation is reduced during transport of the
5 particles between the apparatus. Besides, the absorption of an
enzyme solution or slurry in a porous particle is much faster
than e.g. spraying an enzyme solution or slurry onto a core in
a fluid bed or than mixing an enzyme with a non-porous carrier.
With the present porous material it is also possible
10 to obtain a sequential or controlled release by means of other
technologies than adjusting the composing or location of the
coating layer(s).
The following examples are offered by way of
illustration and not by way of limitation.
~I'gO~O
W094/268~ PCT~4/01642
EXPERIMENTAL
The protease used in the examples is the high alkaline
protease PB92, see U.S. Patent No. Re 30,602, which is
- s commercially available from Gist-brocades N.V. under the trade
mark MAXACAL~.
The lipase used in the examples is the lipase which is
obtainable from the strain Pseudomonas pseudoalcaliqenes M-1
(CBS 473.85), see e.g. EP-B-0218272, and U.S. Patents Nos.
10 4,933,287 and 5,153,135.
The chymosin used in the examples is produced by a
transformed yeast strain of Kluyveromyces lactis and
commercially available from Gist-brocades N.V. under the trade
mark MAXIREN~. See e.g. EP-B-0096430 and EP-B-0301669 and U.S.
5 Patent No. 4,859,596.
Ex~mple 1
Soda cores with a porosity of 6 (Soda ash Dense), 16
20 (Soda ash Compact~) and 38% (Soda ash Sorbent~), respectively,
were obtained from Akzo N.V. (see also EP-A-0542351). The soda
was sieved to obtain a particle size between 315-710 ~m.
Enzyme liquids were produced by adding ethylene glycol
and propylene glycol, respectively, to a concentrated aqueous
25 protease solution. The water content of the liquid was then
reduced to approximately 10% by evaporation. The protease
concentration in the liquid was 3.7 MADU/g (= 3.7x106 ADU/g).
The liquid was then sprayed onto the soda particles. The
particles were completely filled with the liquid up to the
30 porosity level defined by the particles.
Spraying and absorption were carried out in a rotating
vessel (enzyme liquid inlet temperature 30-50~C). The material
was then coated in a fluid bed dryer essentially according to
the method described in JP 58-179492A or EP-A-0193829) and dust
3~ free enzyme granules were obtained.
The non-aqueous protease solutions with ethylene
glycol, propylene glycol, PEG 400 and glycerol were produced
W094/268~ ~ 3 ~ PCT~4/01642
- 8 -
with a protease activity varying from 3.4 to 3.7 MADU/g. The
liquid absorption process resulted in an enzyme yield of 61% to
98% based on enzyme activity, depending on the water content of
the non-aqueous solution. The tests were carried out with soda
5 cores of 6~, 16 and 38% porosity (see above).
After addition of the protease containing liquid the
particles were coated in a fluidized bed coater (inlet air
temperature 65C, outlet temperature 40C). Good mass yield was
obtained with high retention of enzymatic activity.
EXANPL~ 2
Protease granules were prepared in the same way as
described in Example 1 but using porous NaCl cores instead of
15 soda cores. These NaCl cores were prepared essentially
according to the method described in EP-A-0542351 and had a
porosity of 15%. These cores were filled with a non-aqueous
liquid containing a protease with an activity of 3.4 MADU/g.
This resulted in a granulate which had an activity of 440.000
20 ADU/g (enzyme yield >97%). After coating in a fluidized bed
with a cellulose based coating (20% w/w) under the same
conditions as mentioned above, coated granules were obtained
which had an activity of 397.500 ADU/g. Again a good mass yield
was obtained (>92%).
EXAMPL~ 3
A similar experiment was carried out with chymosin.
Chymosin was dissolved in propylene glycol. The liquid absored
30 well in the porous NaCl, which may absorb 15 % of its weight.
This resulted in the same good enzyme yields and loading of the
porous material.
,~
Ex~mple
Porous core material (Soda ash Compact~, 600 g) with a
particle size of 300-710 ~m was introduced in a fluid bed
W094/26883 ~o~o PCT~4/01642
_ g _, . .
dryer. A solution of ethylene glycol containing a lipase and a
non-ionic (Triton X114~) was introduced into the fluid bed
dryer at a temperature of 38~C and sprayed onto the porous
particles. The water was evaporated and the non-ionic and the
- 5 lipase were absorbed into the particles. A coating layer was
then sprayed onto the particles and dust-free granules were
obtained.
BXA~PLE 5
In a similar way as described in Example 4 protease
granules were made. The protease containing solutions of
ethylene glycol and propylene glycol, respectively, were
sprayed into the fluid bed directly on top of the porous
15 material, which was fluidized (particle size between 400 and
600 ~m). Both when the protease was dissolved in ethylene
glycol and propylene glycol, the liquid was absorbed well into
the material.
After applying an additional cellulose based coating
20 (SEPIFILM~ of Seppic) in the fluidized bed coater, and sieving
over 300 and 600 ~m sieves, the dust levels were further
reduced.
Four samples of protease granules which were prepared
in a similar way as described above and coated with cellulose
25 based coating material (20% w/w), were tested in the Heubach
attrition test on dust formation (see WO 93/07263~. In this
test, enzyme dust levels below 0.5 mg/20 g are considered
extremely low. Total dust figures below lo mg/20 g are equally
considered extremely low. The results are given in Table 1.
W094/26883 PCT~P94/01642
3~ - lo -
Table 1
Heubach attrition levels of 4 coated protease granulate samples
Heubach attrition levels
Sample # 1 2 3 4
Enzyme dust [mg/20 g 0.45 0.31 0.14 0.20
of sample, based on
300.000 ADU/g]
Total dust [mg/20 g7.3 5.6 7.6 5.9
of sample]
It appeared that the uncoated granules were smeared
under the test conditions. In contrast, the coated particles
15 apart from their lower dust level were strong enough to
withstand the crushing forces of the steel balls of the Heubach
attrition test.
W094/268~ ~ PCT~4/01642
Example 6
Various porous core materials (soda, NaCl and silica)
were filled with non-aqueous protease solutions. Ethylene
5 glycol and polyethylene glycol were used as the solvents.
Soda ash Sorbent~ (see Example 1) is able to absorb a
liquid upto 37% by weight (based on the soda), the NaCl used
may absorb 15% of its weight, while the silica may absorb 100%.
Table 2 shows the residual enzyme activity after
10 absorption of various enzyme containing liquids into various
core materials.
Table 2
Protease Yields measured after partial and complete
loadinq of various Porous materials
product wt% of liquid absorbed
20 25 30 37 100
residual Soda ash 100 102 90 93 93 92 94
20 enzyme Sorbent~
activity
NaCl 95 101 97
[%]
silica 102
Example 7
The obtained material was tested for its protease
stability at 7C (at ambient relative humidity) using various
non-aqueous liquids. Table 3 shows the results of the stability
tests of the protease which is absorbed into the soda.
SUBSTITUTE SHE~T (RVLE 26J
WOg4/26883 PCT~4/01642
_ 12 -
Table 3
StabilitY of the absorbed Protease in the time
sliquid in storage time
which the (months)
protease
is 2 3
dissolved)
residual EG 100 92 85
activity
PG 100 103 102
[%]
PEG lO0 102 98
10 ~): EG = ethylene glycol, PG = propylene glycol,
PEG = polyethylene glycol 400
All publications (including patents and patent
applications) mentioned in this specification are indicative to
15 the level of skill of those skilled in the art to which this
invention pertains. All publications are herein incorporated by
reference to the same extent as if each individual publication
was specifically and individually indicated to be incorporated
by reference.
Although the foregoing invention has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be apparent to one of
ordinary skill in the art that many changes and modifications
can be made thereto without departing from the spirit or scope
25 of the appended claims.
SUBSTITUTE SHEET (RULE 26)
