CA2120611C - Process for dust-free enzyme manufacture - Google Patents

Abstract

This invention relates to processes for manufacture of substantially dust-free particles of enzymes by spray-coating solid particulate cores or seeds with fermentation broths containing enzymes.
Fermentation broths containing from 5 % to 100 % total solids as enzyme and 4 % - 55 % total solids content in the broth may be used.
For broths containing 4 % - 31 % total solids, en-zyme and coating agents may be spray-coated so as to achieve a weight gain of 1 % - 24 % w/w aver the initial weight of the cores.
For broths with 31 % - 55 % total solids; a corresponding weight gain of 1 % -100 % may be achieved. The enzyme coated cores may be further coated with coating agents: The total dry weight added to the cores ranges from 1 % to 19 % in the absence of coat-ing agents and 1 % to 24 % in the presence of coating agents when the lower range of total solids content is used. The fatal dry weight added to the cares ranges from 1 % to 100 % when the higher range of total solids content is used. These conditions are especially useful in the granulation of enzymes with very high specific activities, so that effective enzyme activity can be provided by adding low amounts of broth solids to the cores. Reduction in added solids is of significant economic benefit in reducing gran-ulation costs.

Description

W~ 93/07260 ~ ~ ~ ~ ~ ~ ~ ~ P(.'T/~JS91/07675 PROCESS FOR RUST-FREE ENZXME MANUFACTURE
Field of the Invention The present invention relates to a novel procedure for making dry and dust-free enzyme granules fram a fermentation broth containing the enzyme.
Background of the Invention P~i,any commercially useful erezyrnes are produced by microorganisms, particular3.y bactexia, yeast, and ~ilamentous fungie These ea~zymes are especially useful in deterWent and food ~pp~.ications. For exampl;~, enzymes useful a.n e~etergent applications, such as proteas~s, amylases, lipases, and c~llulase~s have b~exr praduc~d i.n a wide variety of microbial hosts. When enzymes are. produced in a microbial host they are usually neither ecreted directly ~:rato the fermentation broth by the rnicr~orc~anism or released into the fermentation broth by l~ysin.g the cel2. The ,enzyme can then be recovered f rom the broth in a soluble ~orrn by a num3aer of techr~~.ques including filtration, centra~f~agation, membrane filtration, chromatography, e~~ ~mo~z6o Pcrous~~~oa67~

and the like. The dissolved enzyme can be converted to a dry farm from a liquid using techniques such as precipitationP
crystallization, or spray-drying. A problem associated with dry enzyme preparations is that there is a high dust level associated with them, which can cause dermatologic distress to the manufacturer, consumer, or any other person handling the enzyme. yt has been a desire in the axt to treat these dry enzymes so as-to reduce the hazard of dusting. To control dusting and increase particle size, dry enzymes are often ' granulated by various means known by those skilled in the art.
Various enzyme formulations and processes for these preparations have been developed in an effort to alleviate the dusting problem. For example, German Patent No. 21 37, 042 discloses a process in which an extrudable enzyme containing formulation is extruded through a die onto the revolving plate of a spherQnizing d~vico to form spherical particles of the enzyme-captaining formulations w7~ich are optionally coated with a matera.al designed to prevent dusting.
7Ln L't.S. Fit. Np. 4,087,368, theme ~.s disclosed an enzyme granule formulation in which rods car spheres ~f an enzyme 3n admixture faith magne~3:um alkyl sulfate an,d ethylene oxide axe provided.
,;
tD:S. Pat: No. 4,01f,090 discloses ~ method for the preparation of free-flowing substantially dust--free, spherical enzyme-containing bead p~epaxed by blending a powdered ,.~'al.l~S'T'~'rl~'~E ~i~~'~"

1~4~ 93/Q7260 PC.°I"1t1~91 /07b75 ~1~~~:~1 concentrate of the enzyme with a binder an molten form and spraying droplets of the blend through a spray nozzle into cool air to solidify the droplets and form the beads.
In U.S. Pat. No. 9,242,21, there is claimed a process for the preparation of enzyme-containing particles prepared by mixing the dry enzyme with a hydrophilic organic cohesive material, a building agent, or a mixture regulating agent and mechanically dividing it into particles of the desired size and shape which are then coated with a water repellent material.
Another type of granular enzyme formulation is described in U.S. Pat. Na 4,009,076: This formulation is prepared by mixing the dry enzyme with a solid nonviab3.e substance and optionally a cohesive organic material as binder to form an enzymatically acti~~e core. An enzyme slurry containing the cohesive organic ma~.erial can be sprayed into, for example, sod~.um tripolyph~spha~.e and he cohesive organic material sprayed onto it ~rith subseq~sent extrusion through a die. The enzyme-cont~3.nxng granule is sprayed with an aqueous solution Containing a plast~cixec~ ozganic resin, then dried.
~ process is described az~ CDR Pat. 0 157 5~~8 in ~rhich sodium tripolyphosgahat~ is sprayed with an ~qu~ous fermentation broth and agglomerated in a cyclone apPara~tus. The agglomerates axe removed from the cyclone ~PFaratus wha.~.e still wet and placed ~1.~'~~°'~1°l~lj't~~ ~~c,~°f W~ X3/07260 PC'f/US91 /07675 in a mechanical blender with a drying detergent formulation and intensively mixed.
In Britzsh Pat. No. 1,483,591,there is described a process for coating water soluble or water dispersible particles, including enzyme particles, using a fluidized-bed reactor. 'his reference involves a dust-free coating technique for enzyme particles which have been granulated by other processes such as prilling or spheronizing.
Tn U.S. Patent 4,689,297 there is described a method for preparing dust-free enzyme involving dissolving ox suspending dry enzyme in solution to make a slurry with 15~ - 3U~ total solids, of which et least 30~ w/w of the solids is enzyme, spraying it on a hydratable core, and then coating it with macromolecular material, so as to increase the: dry weight of the core by between 25 and 55~.
Advances in enzyme engineering and selection have led to the development of enzymes which are ~rery active on a specific weight basis, thereby substantially reducing the percentage of total added enzyme broth solids needed a.n produci~r~ an effective granular pr~duct. Furthermore, proper selection of core and coating materials sign~:ficantly reduces the levels of coati~ag agents required for adequate encapsulation and dust control.
~tt~°T~'~'~ a ~ ~E-~~.'C

1 W~ 93/07260 PCC'f/iJ~91/0757~
2~~~~:~1 It is desirable to be able to produce a dry dust--free product with a relatively low percentage of total weight as enzyme, especially with enzymes of high specific activity.
With these more active enzymes, it is also sometimes desirable to feed the. spray-costar with enzyme solutions of very high solids concentrations. This can occur for several reasons:
(11 proteases of high specific activity are often more subject to autolysis, and the addition of large, percentages of solid stabi3.izer compounds may be needed to achieve high process yields; (2l in general, it is most economical to remove as much water as possible from the enzyme solution prior to spray-coating by methods such as ultrafiltration, thereby reducing the batch cycle time in ~:he sprag-water; (3) it is sometimes.desirable to add processing aids such as binders or powders into the ~nzYme concentrate to reduce product dustiness or help prevent excessive agglomeration of the granules. ~'or any o~ these reasons, it may be desirable to provide a fermentation broth containing between about 31~ and 55~ solids on a dry weight basis, so 3.ong as it is raot too viscous to be pumped thraugh a spray nozzle:
This invention aehieves these and,other desirablQ objectives in a cost-effective, efficient, and safe ;manner.
~ ~3 ~~'~"'~"i'~'~" ~ i~! ~

1V~D 93/07260 - ~'~.°T/YJS91 /07675 ~~~~~11 s Summar.y of the Tnvention It has surprisingly been discovered that a dry dust-free enzyme particle can be produced from fermentation broth by the following method:
a) introducing a particulate, hydratable core material Into a flu3.dized-bed spray-coater and maintaining the core particles suspended in the reaction chamber: optionally, producing, building up, ar oth~rwa.se modify~.ng the, core particles in a flui.dined-bed spray-caater end mai~ataining the ce~re particles suspended in the reaction chamber;
b) providing a fermentation broth containa.ng from about 5~
to about 100 w/w of the total solids therein e~f a water salable or dispersible enzyme produced in the fermentat~:c>n broth and a total solids content ref 4 ~- 3'! ~ w/w of the fermentation broth such that the broth has a v3saosity of 10 -~ 5,000 cps at room temperature and:
c) spraying the broth onto the core and evaporating .the liquid to 7:eave the solids coated an the core, such that the fermentation broth solids added ~o the corm provides a total dry weight gain of '~~ - 19$ w/w over the initial weight of the core, and optionally the additional step of:
d) ~p~.a~ing a coating agent over~the product of step (c) and evaporata.ng.the liquid to leave the coating agent over the solids of ('c) such that the total solids co~n~is ing of fermentation brash solids plus coating agent added to the core provides a ~;i"~', ~'T"a'~"~1'1"~ ~~~'i' 'dV0 93/07260 ~ ~ ~ ~ ~ ~ P~GTI'USg1/07675 total dry weight gain of 1~ -~ 24~ w/w over the initial weight of the core.
Also disclosed is a similar process in which the fermentation broth has a total solids content of 31~ to 55~ w/w. In this process the total dry weight gain of the coxe particles is from 1~ to 100 w/w. The enzyme-containing particles prepared by these processes are included in this invention.
Detailed Description of the Invention The method~of the present invention is carried out in a fluidized bed spray-coater.' Typically, such a device comprises a fluidized-bed dryer consista.ng of a conical product chamber that has a porous grid on the bottom and is open on the top to be put up against a cylindrical or conical shaped expansion chamber of a larger diameter than the product chamber; a filter to collect dust and help air flow is placed at the far end of the expansion c3~amber ~,nd one or mare spray nozzles are located within the chamber ~a appl~r the solution to the core. In opex~~ti;on, as the velocity of air passing up through the chamber ~:s ~mcxea~ed, a poant iS reackaed where particles resting on the porous grid ark suspended in the air flow as a fluid, hence the terms'"fluidization" and "fluidized-bed dryer". The particles are lofted by the upward fiorce of the air out of the pr~d~act bhamber into the expansion chamber where the air expands ~t~~~ i 1'~'l~'~~ ~ai~"T

WO 9.3/0760 PCT/U~~l/~7675 .~ ~ ~ ~ ~~a .$. ,.Z.

and the upward farce per unit of area is reduced. This allows the particles to fall back into the product chamber and start the cycle over.
The initial step in the method involves introducing a particulate, hydratable core material into the reaction chamber of the fluidized-bed dryer and suspending the particles therein on a stream of air. The core particles preferab~.y are composed of a highly hydratable material, i.e. a material which is readily dispersible or soluble in water. The core material should either disperse (fall apart by failure to maintain its integrity) ar dissolve by going info a true solution. Clays (bentonite, kaolin), non pareils, and agglomerated potato starch are considered dispersible. Non pareils are spherical particles consa.sting of a stolid nucleus that has been rounded into a spherical shape by binding layers of pr~wder and crystall~.~ed solute, generally starch and sugar, ~o the nucleus in a rotating spherical container and are preferred. Nan pareil particles are often called 'seeds"
Salt particles (NaCl crystals, NaC1 pock salt, NaHC03) are considered soluble:- Also suitable are agglomerated tr~sadium citrate, g~an,crystallized NaCI flakes, bentonite granules and pr~lls, bentonit~/kaolin/diatomaceous ear~In disk-pelletixed granules, and sodium citrate crystalss The core particle is of a material which is not dissolved during the subsequent 'W~ 93/Q726t) ~ '~ ~ ~ ~ ~ ~ ~ PCT/~1~91/0?b?5 spraying process and is preferably of a particle size from 150 to 2,000 microns 1100 mesh to 10 mesh on the U.S. Standard Sieve Series) in its longest dimension.
By fermentation broth is meant the liquid in which the enzyme is produced by fermentation of a microorganism. The broth may be modified by deleting or adding material, e.g» filtration of cell solids or addition of binders, salts, pigments, plasticizers, and fragrances, however, it still captains the ,, enzyme. ~t also may be concentrated or purified by removal or substitution of a portion of the 7l~quid material, by such processes as ultrafiltration, extraction, and chromatography.
Enzymes suitab3e for use in this method are those which are soluble or dispersible in the fermentation broth they are produced in and from which the volatile components of the fermentation broth can be reanoved to leave a residual layer of enzyme on the surface, of the core material. Suitable enzymes include, for example, pr~teases tbacter~.al, Fungal, acid, neutral, or alkaline, amylases (alpha end beta), lipases, and ce~:l~aases. T:he enzyme is present in the broth at from about 5~ t~ about 9OO~ xa/w bf t~tal solids, and fexmentation broth solids range from about 0:5$ to about g5.5~ w/w of total s~a~lids in the fermentati.an broth, with any remaining solids comprising added metail2ic silts, sugars, pie~ments, binders, stabilizers, p3ast~.cizexs, and fragrances such that total solids represent i~V~ 33/07260 PCT/1J591/07675 31~ w/w of the fermentation broth. The broth, including .any optional metallic salts, sugars, pigments, binders, stabilizers, plasticizers, and fragrances, must have a viscosity low enough to be pumped and atomized for effective spray-coating (typically 10 to 5,000 cps at room temperature). The broth solids and enzymes are applied to the surface of the core material by fluidizing the core particles_,in a flow of air whereupon a broth containing the enzyme and other solids is then atomized and sprayed into the expansion chamber of the spray-~coater. The atomized droplets contact the surface of the core particles leaving a film of the solids adhering to the surface of the particles when the water and other wolatiles are evaporated.
Airflow is maintained upwards and out the top of the expansion chamber through a.fili~er. The filter may be located inside or outside of the unit, or may be substituted for by a scrubber or cyclone. This filter taps fine drfed particles which contribute to dust. Flui.dized-bed'spray-Boaters that have this filter typically have automatic shakers which shake the filter to prevent excessive restriBtion of the air flow. ~Cxa a preferred embodiment, the shaker snit a,~ turned off during the last 5 minutes of operations, thus r~d~acing the dust content of the product due to release of fines trapped within the filter.
In another preferred embodiment, the fi7.ter is located outside the unit Ar substituted by a scrubber or cyclone.

~~.°rrus9'ro767s 'Wt? 93/07260 When recovering fermentation broth enzymes, the broth may be treated in various ways to achieve desired results. For eacample, the broth may be filtered to remove cells and cell debris or to remove microorganisms to yield a sterile product. The broth may be concentrated to achieve the desired total solids concentration of about 4~ to about 31~ w/w of the broth.
Further, as mentioned above, salts, stabilizers, etc.,~ can also be added as desired, and the enzyme may be concentrated or purified as desired: It is further a preferred embodiment of the invention thet the weight gain of the solids in the broth applied tci the core over the initial dry weight of the core is about 1$ to about 19~; w/w.
In another distinct preferred .embodiment, the desired total solids concentration is about 31~ to about 55~ w/w of the broth.
In this embodiment, the weight gain of the solids in the broth applied to the core .over the initial dry weight of the core is about 1~ to about 100 w/w:
H~ndlihg the enzyme in 3.iquid farm in the fermentation broth ha;~ the advantage of lowering the possibility of dermatologic contact due to dusting and produces a,product which is dust-!free and minimizes losses due to any extra step of drying, since the drying process is confined to a single, well-contained reac~e~r:

'W4) 93/072~U PCT/LJ~91/U7~75 iz When sufficient enzyme is applied to the core particles to provide the desired enzyme activity, 'the enzyme coated particles, while still suspended in the reaction chamber of~the caster or later reintroduced therein, are coated with a layer of a water soluble or vaster dispersible coating agent. this is accomplished in a manner similar to that used for application of the enzyme/solids coating. Suitable coating agents include, for example, fatty acid esters, gum arabic and other natural ' gums, alkoxylated alcohols, polyvinyl alcohols, ethoxylated alkylphenols and more specifically, polyethylene glyccrls (PEG) (molecular~~aeight (MW) 300 to 20,000), linear alcohol alkoxylates (MW 1,450 to 2,670), Polyvinyl acetate phthalate (PVAP), polymeric nonylph~nyl ethoxylates (MW 1,975 to 4,315) dinonyl phenyl ethoxylat~e (average MW 6,900), l~ydroxypropylmethyl cellulose, and other modified celluloses. Other coating agents include sugars, Marches, salts, titanium dioxide, and other sealants, stabilizers, release agents, binders or pigments.
the net result of the process is to provide an enzyme coated core particle having a layer, of the coating agent on its surface to provadc the 8esa:red dust-free enzyme-captaining particle.
- ~~n ~ fexmenta~ion br~th with about 4~ to about 31~ w/w total solids cnptent is used, the ~o~tal. weight gain of solids varsus the ~.nitial care is preferably from about 1~ to about 24~ w/w when a coal"ing zs used,. and ~refexabl.y fram about 1 ~ to about lg~ w/w ,~,hcn a coating is not used. When a fermentation broth WO 93/0?260 ~C'~'/US91 /t~?675 2~~~6~ 1 with about 31~ to about 55~ w/w total solids content is used, the total weight gain of solids versus the initial core is about 1~ to about 100 whether a coating is used or not.
The dust-free enzyme particles of the present invention can be used wherever enzymes are needed in a dry system. Thus, they can be used as additives to dry detergent formulations, for removing gelatin coatings on photographic films to aid in silver recovery, in the digestion of wastes Pram food processing ' plants for nitrogen recoWery, in denture cleansers far removing protein bound stains, in food preparation, and as a processing r aid in waste water treatment.
The following examples are representative and not intended to be limiti:~g. One skilled in the art could choose other enzymes, broths, cores, and coating agents based on the proportions of ingredients taught herein.
Pref~xred Embodiments Exam -~ dab--Scale spray-Coating cif FNA Protease Onto Non pare~:ls, With Gum ~rrabic Coating Agent.
A -'W'SG-~5 fl;uidized--bed granulatar was charged with 3800 grams of -35/ø40 mesh n~n-pareil cares or seeds and heated to 60°
C and f luidazed. A two liter adueo~s protease ultrafiltration ~~'~~ ~ j d ~~t~ ~~~~~

~V~ 93/0726U PG7f/'tJ~91/07675 ~~~~~~7.9.~

concentrate containing 3.8~ w/w enzyme and 10.5 w/w total solids (36.2 w/w of total solids were enzyme) was sprayed onto the suspended cores at a 95° C inlet temperature and a 40° -50°
C outlet temperature. The spray rate was about 45 ml/minute (min) and enzyme coating br plating took 37 minutes, resulting in an estimated weight gain o~ 210 grams, or 5.5~ w/w. At this point, 230 grams of material was removed for subseguent analysis, leaving 3780 grams of enzyme coated active cores. A coating agent solution containing 60 grains gum arabic in 600 ml water was applied at 40 rnllmin; the entire granulation or coating lasted 56 minutes. The final product weighed approximately 380 grams, representing a 1.6~ inaxease over the enzyme-plated coxes, and a 7.2$ net increase in weight aver the initial core weight.
Exam le 2 - Lab-Scale SprayaCoating of FNA Protease i7nto Non Pareils, With PEG $000 and Ti02 Coating Agents.
A WSG-5 fluidized-bed grarrulatar was charged with 5000 grams of -20/+90 mesh non pareil cores or seeds which were fluidized.
A 1786 ml aqueous psoteass concentrate containing 3.8~ w/w enzyme and 'D0~5~ w/w total solids (as ire Example 1? was sprayed onto the cores at an inlet temperature e~f about 70° C and an outlet ~emperat~re of 40° - 50° C~ The weight gain due to the protease was 188 groans. ar 3:0~ w/ea: A boating agent solution containing 150 grams PEG 8000 and 250 grams TiO~ in 1 liter of water was sprayed on the enzyme-coated non pareils which were dried.

WO 93/0'7260 ~ ~ ~ ~ ~ ~ ~ PCfnJS'9~eo7675 The final prodr~ct weighed 5560 grams, representing an 11.2'k increase over the initial core weight.
Example 3 - Spray-Coating of Lipase Onto Non Pareils, With Hydroxypropylmethylcellulose ~Opadry White) Coating Agent.
A Uni-Glatt laboratory fluidized-bed spray-coater was charged with 960 grams of -20/+40 mesh non pareil cores or seeds which were fluidized. A 1700 ml aqueous lipase concentrate of 6.9 gram/liter lipase with 9.9~ total solids was sprayed onto the non pareil cores at an inlet temperature of 50° - 66° C and an s~utlet temperature of 38° -A2° C and an atomization pressure of 2.5 bar. Enzyme was 19.1 of total solids. The enzyme-coated cores weighed 962 grams, an increase of 5.0~ w/w. After enzyme layering and reactor cleaning, a coating agent solution containing 70 grams Opadry White, a name used in trade for h,ydr~xypropylmethylcellulose, and 400 grams mater was sprayed bnto the enzyme coated active cores, resulting iza coated praduct weight a~f 1032 grams, a net 3.ncr~ase of 12.7 w/w. This product oontained 78~ of the original active ~.ipase a.n the feed concentrate. The pxoduct granules lost no activity ,after incubation at 26.7° C and 80~ relative,humidity ~ver two peeks.
Example ~4 - Spray-Coating of Detergent Cellulase Onto W~p 93/07260 PCI"/lJS91/07675 Non ParPils, Without Coating Agent.
A Uni-Glatt laboratory fluidized-bed spray-costar was charged with 1000 grams of -20/40 mesh non pareil cores or seeds which were fluidized. An 840 ml ar~ueous cellulase concentrate containing 172 gram/liter enzyme and 24.7~k total solids was sprayed at an inlet temperature of 50° - 64°C and an outlet temperature of 38° - 46°C aft a spray-rate of about 10 m1/min.
Enzyme represented 69.6 of total broth; solids. At the end of ~ the run, 1143 grams of product were recovered, representing a '14.3 w/w increase over the non pared cores. Recovery of active enzyme was 98.8 of the ultrafiltration concentrate feed, as measured using bFPU units.
Example 5 - barge-Scale Spray-Coating of FNA Protease Onto Nan Pareils, With PEG 8000 and Ti02 Coating Agents.
A modified Aerom~tic S-8 fluidized-bed granulator was used fox a large-scale spray-coatzng run. The costar was loaded with 681.8 kg 81500 lbs, non pareil seeds of -20/*40 mesh.
The bawl was raised into place and the corps fluidized with 65° C inlet air unti2 the outlet air temperature reached 45°
C: A lOOo4, kg aqueous protease enzyme concentrate containing 5.9~ w/~a active enzyme and 21.3 w/w total s~lids was sprayed onto the cores usang a 1:8 mm multiple-head Schlick nozzle at ~ bar atbmizatioaa a~.r presstre. The outlet air temperature wo 9~~~~zso - ~ ~ ~ ~ ~ .~ 1 i'~f/U~91IU?675 was maintained between 40° and 50°C. Enzyme represented 27.9 w/w of total feed solids. Enzyme coating or layering took about 80 minutes, excluding a 1S minute shutdown to examine the product in the bowl for coating uniformity. The application rate was about 1.2 liters per minute. Ey calculation, this represented a weight gain of 3.1~ w/w over the cores. The bowl was temporarily removed and the machine was cleaned. Overcoating was then applied to the enzyme granules in the same dryer/coatex unit. The coating agent solution consisted of 123.6 kg of tap water heated to 50° C, in which 67.4 kg PEC 8000 and 33.7 kg Ti02 were dissolved or suspended to give a 150 liter solution.
The enzyme coated granules were fluidized and heated to 45°
C as before. Atamizatian air pressure was held at 4 bar and outlet air temperature varied between 40° C end 50° C. The total coating run time was 136 minutes with an application rate of about 1.1 liter per minute. after coating, the product was dried for four minutes until, the outlet temperature reached A8°C, then cooled for 1~1 more minutes until cutlet temperature reached 32°C. The bowl was removed and the product sieved to a --16/+40 dut. Tine mass balance is shown in Table 1. The ffinal product repx~sented a net increase of 13.0~s w/w over the core weight using the actual product weight or a net increase of 17.9 w/w ~v~r the core weight using the theoretical,sum of lingr'edient weights. Froduct dust was e~ctremely low. The product granules produced 0.3 milligrams total dust and 2.42 micrograms protease dust per 60 gram sample when subjected to a 40 minute ~Ps.~~~~'3'ITI~'~'~ ~~°I~~"I.

~lt~ 93!07260 PC.'I'/US91/07fi75 standard elutriation test.

Ory Percent Pure Percent Weight , of Ex~zyme of Raw Materials (k ? Feed (k ) Feed 681.8 8~.8 -- __ Cores lution 21.9 2.7 5.96 100.0 S

o Enzyme (5.94 ~ w/w enzyme) PEG sooo 6'~.~ s.~ -! -_ TiO2 s 33.7 4.2 .-- __ 809.3 100.0 ~ 5.96 100.0 TOTAL

End Products ( 0 . 68 ~ w/w ~n,zym~e ) .

Prod~xct (+40 mesh) 7f9.7 95.7 5.24 87.9 Fines (--40 mesh) ~.0 0.2 0.01 0.2 771 :7 9.5, 9 5. 25 88.1 TOT:~L

~~ ~~~~~~ ~~~

WC) 93/Q7260 ~ ~ ~ ~ ~ '~ ~ P(.'T/US91/07675 Exam le 6 - Large-Scale Spray-Coating of Alkaline Protease Onto Non Pareils, With ~iydroxypropylmethylcellulose and Ti02 Coating Agents.
A WSG 300 granulator is charged with 700 kg of non parei2 seeds or cores of -ZO/+40 mesh and the cores are fluidized.
A 3fi kg concentrated aqueous solution containing 10~ w/w protease and 29~ w/w total solids, with enzyme 34.5 w/w of total solids, is sprayed onto the fluidized cores. Spraying is continued until the total charge weighs "710.4 kg, which represents a 1.49' w/w gain over the initial core weight. A coating agent solution consist~.ng~of 3.5 kg hydroxypropylmethy1ce21ulose 3.5 kg Ti02, and 30 kg water is sprayed on the fluidized enzyme coated cores.
The total product raeight is 717.4 kg, which represents a total weight gain of 2.49 ~a/w.
Exam le 7 - LargerScale Spray-Coating of Cellulose Onto Non Pareils, ~~.th PEG 20, 000 and Ti02 Coating A.gerits.
A ~nISG 300 granulator is charged with 700 kg non pareil seeds of -20/40 mesh and the seeds are fluidized. A 334 kg aqueous concentrated cellulose solution dontaining ~26~ w/w cellulose and 39~,w/w tonal solids, ~rith enzyme 66.7 w/w of total ~olads, His sprayed onto the fluidized cores: Spraying continues until the total charge we~.ghs 530: kg, Which represents a gain of 18.6 w/w over the initial core 'weight. A coating agent solution dVfl93/O?2b0 ~ IPCT/~JS91/~D7675 a consisting of 18.5 kg PEG 20,000, 18.5 kg Ti02, and 100, kg water is sprayed on the fluidized enzyme coated seeds. The total product weight as 867.3 kg, which represents a total weight gain of 23.9 w/w.
ExamQle 8 - Spray-Coating of High Solids Protease Onto Non Pareils, With PEG 8000 and Ti02 Coating Agents.
A Glatt GPCG-S fluidized-bed granulator is charged with 10.0 kg non pareil seeds. A 4766 gram arlueous Eacillus lichenformis subtilisin ultrafiltration concentrate containing 20~ ia/w enzyme solids and 32~ w/w total solids, with enzyme 63~ w/w of total solids, is sprayed onto the fluidized cares at a rate of 85 g/minute end an atomization air pressure of 2.S bar. The inlet air temperature is about 70° C and the outlet temperature is about 48° C. A ca~~ang solution of 2000 grams PEG 8000 and S00 grams Ti02, suspended in 10 liters of water, is then sprayed onto the ~nzyme-coated cores under the same conditions. The final coated produet weighs 14.0 kg, a weight gain of 40~ w/w.
Exampl.~ 9 - Spray-Costing o~ Hagri Solids Cellulose Concentrate Onto Ikon Paxeils, With PEG 8000 and Ti.OZ Coating ~ Ag~ntS:
A Uni-Glatt laborat~ry spray-COat~r is charged with 600 grams non pareil seeds. At an inlet temperature of 60° C, an outlet CVO 93/07260 ~ ~ ~ ~ ~ .~ ~ PCf/YJS91/07675 temperature of 42° C, and an atomization air' pressure of 3 bar, an aqueous cellulose concentrate of 455 grams containing 30.3 w/w enzyme and 39.5 w/w total solids, with enzyme 77~ w/w of total solids, is sprayed onto the fluidized cores, increasing the weight of the cores to 780 grams, a 29.7 w/w increase.
A coating solution containing X00 grams PEG 800 and 200 grams Ti02 in 1.5 liters of water is sprayed on the cores under the same conditions. The harvested product weighs 1180 grams, a net increase in weight of 97$ over the original cores.
Example 10 - Spray-Coating of Protease Concentrate With Added Salts Onta TVan Pareils, With Gum Arabic and Ti02 Coating Agents.
A Uni-Glatt laboratox~~ spray-coater is charged with 800 grams non pareil seeds. A 500 ml agueous p~atease concentrate weighing 520 grams and containing 8~ w/w prat~ase and ,24~ w/w total solids is madi~ied by dissolving 100 gams of stabiliser salts an the concentrate. These additions increase the solution volume 0 540 and and the solution weight ~0 620 grams. The modified conc~ntsate, with 6.7~ w/w en~ym~ solids and 36.3 w/w total eolids, is spra~red onto,t~ae fluidized cores at an inlet temperature of 50° C, an outlet temperatpre of 40°
C, and .~;n atomix~tion air px~ssure crf 3.5, bar. This increases the pacoduct weight to 1025 grams, a 28.1 w/w increase over the cox~ weaght: A coatang solution containing 120 grams gum ~t.l~t'i'1'U°i°~ '1°

Vlnp 93/0760 P~,"T/US91107~675 arabic and 60 grams Ti02 is then sprayed over these cores resulting in a final product weight of 1205 gramsr a net increase of 50.6 w/w.

Claims (6)

1. A process for providing a dry, dust-free particle from an enzyme containing fermentation broth, the process comprising:
a) introducing hydratable cores into a reaction chamber of a fluidized bed spray-coater or building up hydratable cores in the reaction chamber of a fluidized bed spray-coater;
b) providing a fermentation broth containing from about 5% to about 100% w/w of the total solids therein of a water soluble or water dispersible enzyme and a total solids content of about 4% to about 31% of the enzyme containing broth such that the broth has a viscosity of 10-5,000 cps at room temperature;
c) spraying the broth onto the core particle and evaporating liquid to leave a film of broth solids coated on the core particles, the broth solids added to the core particles providing a total dry weight gain of from about 1% to about 19%; and d) spraying a coating agent dissolved in liquid on the product of step c) and evaporating liquid to leave the coating agent over the solids such that the total broth solids and coating agent added to the core particles provide a total dry weight gain of from about 1% to about 24% w/w over the initial weight of the uncoated core particles.

2. A process for providing a dry, dust-free particle from an enzyme containing fermentation broth, the process comprising:
a) introducing hydratable cores into a reaction chamber of a fluidized bed spray-coater or building up hydratable cores in the reaction chamber of a fluidized bed spray-coater;
b) providing a fermentation broth containing from about 5% to about 100% w/w of the total solids therein of a water soluble or water dispersible enzyme and a total solids content of about 31% to about 55% of the enzyme containing broth such that the broth has a viscosity of 10-5,000 cps at room temperature;

c) spraying the broth onto the core particle and evaporating liquid to leave a film of broth solids coated on the core particles, the broth solids added to the core particles providing a total dry weight gain of from bout 1%
to about 100% w/w; and d) spraying a coating agent dissolved in liquid on the product of step c) and evaporating liquid to leave the coating agent over the solids such that the total broth solids and coating agent added to the core particles provide a total dry weight gain of from about 1% to about 100% w/w over the initial weight of the uncoated core particles.

3. A process according to claim 1 or 2 wherein the enzyme containing fermentation broth is filtered to remove microorganism cells or cellular debris.

4. A process according to claim 1 or 2 wherein the enzyme in the enzyme containing fermentation broth is processed by a protein purification or concentration technique.

5. A process according to claim 1 or 2 wherein the enzyme is selected from the group consisting of protease, lipase, cellulase and amylase.

6. An enzyme granule made by the process of claim 1 or 2.