CN1775949A - High-activity phytase composition - Google Patents
High-activity phytase composition Download PDFInfo
- Publication number
- CN1775949A CN1775949A CN 200410038539 CN200410038539A CN1775949A CN 1775949 A CN1775949 A CN 1775949A CN 200410038539 CN200410038539 CN 200410038539 CN 200410038539 A CN200410038539 A CN 200410038539A CN 1775949 A CN1775949 A CN 1775949A
- Authority
- CN
- China
- Prior art keywords
- phytase
- granulate
- feed
- animal
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 235000012204 lemonade/lime carbonate Nutrition 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N methionine Chemical compound CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 235000006109 methionine Nutrition 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229910000150 monocalcium phosphate Inorganic materials 0.000 description 1
- 235000019691 monocalcium phosphate Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000005789 organism growth Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000005453 pelletization Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- 229960002203 tilactase Drugs 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Landscapes
- Fodder In General (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
A process is disclosed for preparing aqueous phytase-containing liquids involving culturing microorganisms of the genus Aspergillus or Trichoderma in a medium containing assimilable carbon and nitrogen sources (e.g. glucose and ammonium ions), filtering the medium and subjecting the resulting filtrate to ultra-filtration to give an aqueous composition haying at least 14,000 FTU/g. This aqueous liquid can be used to prepare granulates that can be incorporated in animal feedstuffs.
Description
The application is to be that June 4, application number in 1998 are dividing an application of 98807104.5 patent application " high-activity phytase composition " applying date.
The present invention relates to the application aspect the granulate of the preparation of phytase and goods and their feed enzyme in the preparation animal-feed.
The application of various enzymes (for example phytase) in animal (for example livestock) feed is more common just day by day.Comprise these enzymes so that improve animal, but also may help to improve digestibility from the picked-up of beverage to nutrition and mineral substance.They are normally produced by culturing micro-organisms in by the large scale fermentation jar of industrial enzyme producer operation.When fermentation ends, " fermented liquid " of generation thus experience a series of filtration step separating biomass (microorganism) and required enzyme (in solution) usually.This enzyme solution or sell or be processed to dry products then with liquid form (usually after having added various stablizers).
Enzyme liquid and dry products are used to animal-feed industry on commercial size.Liquid preparation can be added to behind pelleting in the feed so that avoid the hot deactivation of the enzyme that meeting takes place in the pelleting process.
Dry products generally includes the steam pelleting, and wherein feed stands vapo(u)r blasting before pelleting.In follow-up pelleting step, the feed pressurized is by model or mould, and the strip of formation is cut into the suitable pill of all lengths.In this operation, temperature can be elevated to 60～95 ℃.
Phytase is such enzyme: (at least in part), hydrolysis of inositol six microcosmic salts become inositol and inorganic phosphate.These enzymes see in wheat bran, plant seed, the animal intestine, and can pass through microorganisms producing.Phytase is impregnated in animal-feed, is phytase because they can be degraded, and phytase can increase the utilization ratio of animal to phosphorus or other nutritive ingredient.Phytase also can increase the digestibility to calcium.
Phosphorus is the essential element of organism growth.With regard to livestock, feed has usually been augmented inorganic phosphorus so that monogastric animal is well grown.But usually do not need like this in the feed of ruminating animal, because the microorganisms that exists in the cud of ruminating animal energy catalysis phytase is to the enzyme of inositol and inorganic phosphate conversion.The degraded of phytate often needs, because phytic acid can be to be unfavorable for nutrition, because it is chelated with the mineral substance (for example calcium, zinc, magnesium produce iron) of usefulness, and can also be unfriendly and proteins react, so reduce the bioavailability of animal to them.The interpolation of phytase also can reduce the amount of the inorganic feed that needs interpolation, thereby reduces excretory phosphorus in the ight soil, and this more is of value to environment.
The gene of various phytases is cloned and has been expressed.EP-A-0,420,358 (Gist-Brocades) have described the expression of microbial phytase.
At recent application EP-A-0, among 684,313 (the Hoffmann-La Roche), the dna sequence dna of various many thuja acids that having described encodes has phytase activity.
EP-A-0,758,018 (Gist-Brocades) relate to the method for improving enzyme (especially being used as the enzyme of animal-feed) stability, and relate to phytase.
WO-A-94/03612 (Alko) has described with trichoderma (Trichoderma) and has produced the phytase degrading enzyme, and WO-A-97/16076 (Novo Nordisk) has then described the enzyme-containing formulation that is used to prepare animal-feed, and this feed comprises various lyophobic dusts.
Animal-feed has embodied one of costs on the higher scale of raise livestock and other animal.In addition, for example the additive of enzyme (such as phytase) can greatly increase the cost of animal-feed.The phytase composition that can provide production cost lower is provided.This can realize by producing highly active or highly spissated phytase composition.There are several factors to make the applicant can prepare these high-activity compositions, hereinafter these factors will be discussed.
Another advantage that can prepare high-activity phytase composition that the applicant has noticed is, these compositions can show enlarge markedly (especially in the pelleting process of preparation animal-feed (pill)) of stability, so more may keep higher phytase activity in time than prior art combinations.
The method that first aspect of the present invention provides a kind of preparation to comprise the aqueous liquid of phytase, this method comprises:
(a) under the condition that allows recombinant expressed phytase, in water-containing medium, cultivate Aspergillus or trichoderma microorganism with allos phytase gene, described allos phytase gene is under glucoamylase (for Aspergillus (Aspergillus)) promotor or the control of cellobiohydrolase (for trichoderma) promotor, and described substratum comprises as the assimilable carbon source of the food of described microorganism and assimilable nitrogen source;
(b) filter described water-containing medium and must contain filter liquor to remove microorganism; And
(c) filtrate that will derive from (b) is carried out uf processing and must phytase concentration be at least 14, the aqueous liquid of 000FTU/g.
Found that this method provides the phytase of special high density in the aqueous composition that generates.This can prepare also is other phytase composition of high reactivity level.It shows, this method cost lower (per unit enzymic activity) not only, and found that denseer to contain the phytase composition counterpart littler than their concentration much stable.
Described microorganism is these bacterial classifications preferably, i.e. aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae) or Trichoderma reesei.With regard to the Aspergillus organism, phytase gene is in glucoamylase suitably, and (or amyloglucosidase is AG) under the control of promotor.Belong to organism, advantageous applications cellobiohydrolase promotor with regard to Trichoderma.
Assimilable carbon source can comprise glucose and/or Star Dri 5, and/or assimilable nitrogenous source can comprise ammonium ion.Glucose and ammonium ion can be only assimilable carbon source or nitrogenous sources in the described water-containing medium.That is to say that compound carbon source or nitrogenous source are not used in expection.Ammonium ion can provide with ammonia or ammonium salts.Preferred ammonium salt comprises ammonium nitrate, ammonium sulfate and ammonium phosphate.
Preferably, described carbon source and/or nitrogenous source are to be supplied to substratum during the fermentation.The feeding speed of any nutrition source can to digest its speed roughly the same with microorganism.So carbon source and/or nitrogenous source can provide by lasting or continuous mode.Carbon source and nitrogenous source can provide independently, perhaps provide from same source.
The aqueous liquid that forms may have at least 16, and the phytase concentration of 000FTU/g also may even have 18,000FTU/g or higher.
By using these specific organisms under these conditions, described filtrate will be denseer.This just makes it can experience Ultrafiltration.In some art methods, the filtrate of formation contains too many residue makes it can not experience Ultrafiltration (filter obstruction) with other material.Yet in the method for the invention, filtrate is " limpid " comparatively, and it makes filtrate experience Ultrafiltration and does not need any further processing, and by Ultrafiltration, can therefore obtain special high-concentration aqueous composition.
Art methods has been discussed the possibility of described filtrate or aqueous composition experience crystallisation step and/or decolouring step (for example activated carbon filtration).But these other steps (they can increase the expense of producing phytase) all can be saved in the present invention.
Preferably, described microorganism does not have or does not express glucoamylase gene at least.This just shows that described microorganism can be the production contribution more energy of phytase.
Described microorganism may have the phytase gene of multiple copied.Found that this can increase the output of phytase, because there is more phytase gene to be expressed.
This aqueous composition may be substantially free of takadiastase.
In the method for the invention, preferably (basically) all before carbon sources and/or the filtration of nitrogenous source in carrying out (b) by microbial consumption.This can realize by continuation fermentation for some time after supplying carbon source and/or nitrogenous source the last time.Also can after having added whole carbon sources and/or nitrogenous source, continue fermentation.We can understand that the benefit of doing like this is that this aqueous composition just possible (basically) is carbonaceous sources and/or nitrogenous source (for example glucose and/or ammonium ion) not.Again, can facilitate more limpid aqueous liquid like this, it may contain by product still less.By reducing the amount of by product, we just can reduce to minimum with using this aqueous liquid or can obtaining the desired procedure of processing number of required high phytase activity.
Most preferred organism is an aspergillus niger.Further preferably, described phytase is expressed in the microorganism with glucoamylase signal sequence.
So the aqueous liquid that contains phytase of formation just can be used to various uses, but its being applied in animal-feed is special expectation here.Second aspect of the present invention relates to this aqueous liquid (for example can preparing by the method for first aspect), and it comprises that concentration is 14 at least, the phytase of 000FTU/g.
In this manual, " phytase " not only refers to natural phytase, also refer to have phytase activity any enzyme of [for example can catalysis relate to the reaction that removes or discharge inorganic inferior phosphorus (phosphoric acid salt) from inositolophosphate].Preferably, this phytase should belong to the EC3.1.3.8 class.This phytase itself is the fungi phytase preferably, for example derives from the phytase of Aspergillus or trichoderma bacterial classification.
The present invention can also provide the method for preparing the phytase preparation (its is used edible carbohydrate polymkeric substance and makes carrier) that is the granulate form.Described carrier can be granulate or powder type.The described aqueous liquid (for example solution or soup compound) that contains phytase can mix with solid carrier and can be attracted on the carrier.In mixing process or after mixing, described liquid and the carrier that contains phytase is processed to granulate, this granulate can be dried with that.The application of carbohydrate carrier makes can adsorb a large amount of composition (thereby adsorbing a large amount of phytases).Described mixture can be used to form the elasticity cream or the non-resilient paste that can easily be processed into particle (for example it can be extruded).Suitable carriers is a non-fibrous material, its feasible easier granulation: fibre object mass-energy stops passes through extruding pelletization.
Some prior art documents relate to the pill that contains various enzymes, but these pills are used as washing composition usually in cleaning composition.Otherwise the application is used for animal-feed, and for this reason, granulate of the present invention is that (animal) is edible and preferably or digestible.Therefore, unexpected, granulate of the present invention, particle and composition do not contain soap, stain remover and SYNTHETIC OPTICAL WHITNER or bleaching compounds, zeolite, binding agent, filler (TiO
2, kaolin, silicate, talcum powder etc.), only lift several examples that are.
Should select the edible carbohydrate polymkeric substance, it can be by animal edible (with regard to it, feed is desired) and preferably or digestible like this.This polymkeric substance preferably includes glucose (polymkeric substance that for example contains glucose), or (C
6H
10O
5)
nThe unit.Preferably, described carbohydrate polymer comprises α-D-glucopyranose units, amylose starch (line style (1 → 4) α-D-dextran polymer) and/or amylopectin (the branching D-dextran with α-D-(1 → 4) key and α-D-(→ 6) key).Starch is preferred carbohydrate polymer.Can be used to replace starch or other the suitable glucose polymer that contains except that starch to comprise: alpha-glucan, beta-glucan, pectin (for example protopectin-) and glycogen.The derivative of these carbohydrate polymers (for example their ether and/or ester) is also expected, but pasted starch is often avoided using.Suitable carbohydrate polymer is water-insoluble.
In embodiment described herein, W-Gum, yam starch and Starch rice have been used.But, can use the starch that derives from other (for example plant, such as vegetables or crop) source equally, other originate for example cassava, wheat, corn, sago, rye, oat, barley, sweet potato, Chinese sorghum or arrowroot.Similarly, can use native starch or treated starch (for example dextrin) in the present invention.Preferably, described carbohydrate (for example starch) contains on a small quantity or does not contain protein, for example is less than 5% (w/w), such as being less than 2% (w/w), preferably is less than 1% (w/w).
At least 15% (w/w) of described solid carrier may comprise carbohydrate polymer (for example starch).But, preferably, at least 30% (w/w) of described solid carrier comprises carbohydrate, the most suitable at least 40% (w/w).Advantageously, the main ingredient of described solid carrier is carbohydrate (a for example starch), for example more than 50% (w/w), and preferred at least 60% (w/w), at least 70% (w/w), and the most suitable at least 80% (w/w) suitably.These weight percentage are all based on the gross weight of non-enzyme component in the final dried granulate.
The water yield that the amount that contains phytase liquid on the carrier that can be attracted to is subjected to be adsorbed usually limits.With regard to natural granular starch, this amount can change between 25～30% (w/w), does not use high temperature (otherwise can cause the starch swelling).When implementing, the enzyme liquid percentage ratio that adds in the carbohydrate is more much bigger than this scope usually, contains relatively large solid usually because contain enzyme liquid.Phytase solution may contain the solid of have an appointment 25% (w/w), Just because of this, carbohydrate (for example starch) and phytase solution can be at carbohydrate: mix under the ratio of phytase solution=0.5: 1～2: 1 (for example 1.2: 1～1.6: 1), for example respectively with about 60% (w/w): the ratio of 40% (w/w).Preferably, add the carbohydrate absorption that the amount of liquid in the solid carrier should make (basically) the whole water in described (moisture) liquid be existed in the solid carrier to.
At high temperature, starch and other carbohydrate polymer can adsorb the water of much bigger amount when swelling.For this reason, wish that carbohydrate polymer can planar water the aqueous liquid of enzyme (or contain).For example, W-Gum is assigned ten times at 60 ℃ of triple water that can adsorb its weight down at 70 ℃.So the present invention expects to use higher temperature so that adsorb the more substantial enzyme liquid that contains, and especially is preferred really when relating to heat-staple phytase.Therefore concerning these enzymes, the mixing of solid carrier and liquid can be carried out under high temperature (for example being higher than room temperature), for example is higher than 30 ℃, preferably is higher than 40 ℃, should be higher than 50 ℃.Alternatively, perhaps in addition, described liquid can provide under this temperature.
But, in general, the preferred non-swelling condition under low temperature (for example room temperature) more, thus will reduce to minimum owing to the caused loss of activity of unstable of (heat sensitivity) phytase under higher temperature.Suitable temperature is 20～25 ℃ in the mixing process of described enzyme and described liquid.
Being used for mixture that the present invention will contain the liquid of phytase and solid carrier makes the mechanical workout of particle (in other words being granulation) and can adopt known technology commonly used in food, feed and the zymin processing.This can comprise expansion, extrude, spheroidizing, pelleting, high shear granulation, rotary drum granulation, fluidized-bed are reunited or its combination.The common input that is characterised in that mechanical energy of these methods, the for example driving of screw rod, the rotation of mixed organization, the pressure of pelleting device rolling mechanism, rotary bottom plate by fluidized-bed reunion machine makes the motion of particle or makes the motion of particle, perhaps its combination by air-flow.These methods can make solid carrier (for example being powder type) mix with the liquid that contains phytase (aqueous solution or soup compound), subsequently just by granulation.
Yet in yet another embodiment of the present invention, granulate (for example aggregate) is to be sprayed onto or to be coated onto that (for example in fluidized-bed reunion machine) forms on the carrier by the liquid that will contain phytase.Here, the particle of formation may comprise aggregate (for example can produce) in fluidized-bed reunion machine.
Preferably, the mixing that contains phytase liquid and solid carrier also comprises kneading of mixture in addition.Thereby this plasticity that can improve mixture is convenient to granulation (for example extruding).
If granulate is then extruded preferably and is under low pressure carried out by extruding formation.The advantage of doing like this is that the temperature of mixture will can not raise or just raise a little when being extruded.Low pressure is extruded and is comprised for example extruding in basket forcing machine of Fuji Paudal or domeshape forcing machine.Preferably, extrude and do not cause being extruded material temperature and be elevated to and be higher than 40 ℃.Extrude and to produce particle (this particle may come off) naturally or can adopt cutting machine after by mould.
Suitably, particle will have 30～40% water-content of (for example 33～37%).Enzyme content suitably is 3～10%, for example 5～9%.
The particle that obtains can experience finishing (for example spheroidizing), for example at balling machine (as MARUMERISEN
TMMachine) in and/or compacting.Particle can be by spheroidizing before dry because can reduce like this dust in the final granulate formation and/or can be so that any coating of granulate.
Then just can dry (for example in fluid bed dryer) this particle, perhaps under the situation that fluidized-bed is reunited, can be obtained (dried solid) granulate by convection drying (in the reunion machine).The technician can use other currently known methods of drying particulate in food, feed or the enzyme industry.Suitably, this granulate is flowable.
Drying is preferably carried out under the temperature of 25～60 ℃ (for example 30～50 ℃).The drying here may continue 10 minutes to a few hours (for example 15～30 minutes).The length of required time will depend on particle weight to be dried certainly, but the overall principle is every kg particle 1～2 second.
After the particle drying, form granulate and preferably have 3～10% the water-content of (for example 5～9%).
Be coated with the last layer coating and provide other (for example favourable) characteristic or performance for this granulate, for example low dust content, color, protective enzyme are in case are made up by enzymic activitys different in the influence of surrounding environment, a kind of granulate or its.This particle can be coated with fat, wax, polymkeric substance, salt, ointment and/or ointment or contain the coating of (second kind) enzyme (for example liquid) or its combination.Should understand, if necessary, can be coated with several layers of (different) coating.To can utilize some known methods in the granulate coated, comprise the application of fluidized-bed, high shear granulator, mixer-granulator or Nautamixer.
In other embodiments, other component can be mixed this granulate (for example as processing aid), in order that further improve the pelleting stability and/or the package stability of granulate.Some these class preferred additives are discussed below.
Salt can be included in this granulate, (for example being contained in described solid carrier or the liquid).Preferably can add inorganic salt (as EP-A-0, in 758,018 suggestion like that), inorganic salt can improve the processibility and the package stability of dried phytase preparation.Preferred inorganic salt comprise divalent cation, for example zinc, magnesium and calcium.Sulfate radical is most preferred negatively charged ion.
Preferably can add inorganic salt (as EP-A-0, in 758,018 suggestion like that), inorganic salt can improve processibility and the package stability of doing zymin.Preferred inorganic salt are water miscible.They can comprise divalent cation, for example zinc (especially preferred), magnesium and calcium.Sulfate radical is most preferred negatively charged ion, but causes water miscible other negatively charged ion also can use.This salt can be solid form and add (for example adding in the mixture).But, this salt can be dissolved in water before solid carrier mixes or contain in the liquid of enzyme.Suitably, the amount that this salt is provided is 15% (w/w is based on enzyme) at least, for example is 30% at least.But it may be up at least 60% or even 70% (also being w/w, based on enzyme).This tittle can be applicable to described particle or granulate.Thereby described granulate may comprise the salt that is less than 12% (w/w), for example 2.5～7.5%, and such as 4～6%.
If this salt provides in water, its amount may be 5～30% (w/w) so, for example, and 15～25%.
The further improvement of pelleting stability can reach by mixing hydrophobic gelationization compound or slowly dissolving (for example in water) compound.The amount that these compounds can be provided is 1～10wt%, for example 2～8wt%, preferably 4～6wt% (based on the weight of water and solid carrier component).Suitable material comprises the derivatize Mierocrystalline cellulose, for example HPMC (Vltra tears), CMC (carboxymethyl cellulose), HEC (Natvosol); Polyvinyl alcohol (PVA); And/or edible oil.Edible oil (for example soya-bean oil or Tower rape oil) can be used as processing aid and is added (for example adding in the mixture to be granulated), and but, in general, lyophobic dust (for example plam oil) does not preferably exist.
Preferably, this particle has narrower distribution of sizes (for example they are monodispersities).This can promote phytase in particle and/or the uniform distribution of enzyme granulates in animal-feed.Method inclination of the present invention is in the granulate of production narrow size distribution.But, if necessary, can comprise an other step in the method and the distribution of sizes of described particle is narrowed down, for example screening.The distribution of sizes of described granulate is suitably between 100 μ m～2000 μ m, and is preferably between 200 μ m～1800 μ m, the most suitable between 300 μ m～1600 μ m.That described particle can have is irregular (but preferably rule) shape, for example subglobular.
In animal-feed (it comprises pet food), can comprise the enzyme that other is suitable.The function of these enzymes is normally improved feed conversion rate, for example the viscosity by reducing some feed compound or by reducing its anti-nutritive validity.Feed enzyme for example can also be used for reducing the amount that muck is harmful to the compound of environment.For the preferred enzyme of these purposes is: carbohydrase, for example amylolytic enzyme and plant cell-wall degrading enzymes, they comprise cellulase, beta-glucanase for example, hemicellulose, for example zytase, or Galactanase; Peptase, tilactase, polygalacturonase, esterase; Proteolytic enzyme preferably has neutrality and/or acid optimal pH; And lipase, preferably Phospholipid hydrolase, for example Mammals pancreatic phospholipase a2.Preferably, this enzyme does not comprise starch degrading enzyme (for example amylase).May get rid of proteolytic enzyme in some embodiments, because if these enzymes of words of digestion may cause detrimentally affect.If this enzyme is a plant cell-wall degrading enzymes; (for example cellulase), and hemicellulose (for example zytase) especially, the enzymic activity scope that so final granulate may have is 3; 000～10; 000EXU/g, preferably 5,000～80; 000EXU/g; should be 8,000～70,000EXU/g.If this enzyme is a cellulase, beta-glucanase for example, the enzymic activity that so final granulate may have is 500～15,000BGU/g, preferably 1,000～10,000BGU/g should be 1,500～7,000BGU/g.
Described particle may comprise 5～20% enzyme of (for example 7～15%).This enzyme can be natural alcohol or recombinant chou.
Therefore, preferable methods of the present invention comprises:
A. will contain the aqueous liquid of phytase and comprise that the solid carrier of at least 15% (w/w) edible carbohydrate polymkeric substance mixes, and for example mixes this solid carrier with the aqueous liquid that contains enzyme;
B. the optional mixture of kneading and forming;
C. granulation (for example passing through mechanical workout) this mixture is so that obtain to contain enzyme particle, for example by the application nodulizer or by extruding;
D. optional with this particle cluster glomeration;
E. the dry particle that forms and must contain enzyme granulates.
In whole process, should note the top temperature of enzyme contact is remained on below 80 ℃.
Granulate of the present invention is applicable to the preparation animal-feed.At its wide region, the granulate that this aspect of the present invention covers comprises phytase and edible carbohydrate polymkeric substance, and the activity of this granulate is 6 at least, 000FTU/g.In these methods, granulate is mixed with feed substances, perhaps mix, perhaps as the part of premixture like this.The feature of granulate of the present invention makes them be used as the component of mixture, and this mixture is well suited for as animal-feed, if especially this mixture by steam treatment after then by pelleting.In such pill, can see or can recognize described dried particle.
So the 3rd aspect of the present invention relates to preparation animal-feed or the premixture of animal-feed or the method for precursor, this method comprises that the composition with second aspect mixes with one or more animal-feed materials (for example particle) or component.Then just can be to this mixture sterilization (for example heat-treating).Again the composition that forms suitably is processed into pill.
The 4th aspect of the present invention relates to the composition of the granulate that comprises second aspect, its preferably edible feed composition (for example animal-feed).Said composition preferably is the form (can be that every ball has 1～5 (for example 2～4) dried particle) of pill.
Suitably, said composition comprises 0.05～2.0FTU/g, for example 0.3～1.0FTU/g, the phytase of the most suitable 0.4～0.6FTU/g.The zytase of (for example 1～40) EXU/g that may have 0.5～50.Alternatively, the cellulase of (for example 0.2～0.4) BGU/g that perhaps also may have 09.1～1.0 in addition.
Said composition may have 10～20% water-content of (for example 12～15%).The amount of enzyme suitably 0.0005～0.0012%, for example 5ppm at least.
The 5th aspect of the present invention relates to the method that promotes growth of animal, and this method comprises: with the food nutrition purposes, especially for feeding animals of the composition that comprises the composition of second aspect or the 4th aspect.Here, animal foodstuff may comprise described granulate itself, or is present in the granulate in the feed.
The 6th aspect of the present invention relate to composition in animal-feed application or as the application of the component of animal-feed, the perhaps application in animal foodstuff.
The 7th aspect of the present invention also relates to a kind of application of composition, and said composition comprises at least 15% (w/w) thereby improves the edible carbohydrate polymkeric substance of the pelleting stability of phytase as the carrier of phytase.
Suitable animal comprises farming animals (pig, poultry, livestock), non-ruminant animal or monogastric animal (pig, wild fowl, poultry, marine animal (for example fish)), ruminating animal (ox or sheep, for example milk cow, sheep, goat, deer, calf, lamb).Poultry comprises chick, hen and turkey.
Preferable feature of one aspect of the invention and characteristic are fit to do necessary correction on the other hand equally.
Provide following embodiment and only be in order to set forth the present invention, be not intended as or can not be considered to restrictive.
Embodiment
Embodiment 1
The fermentation of aspergillus niger CBS513.88
Aspergillus niger fungal spore preparation prepares by standard method.
Spore and cell subsequently forwarded to by a series of batch fermentations in Erlenmeyer flask in one 10 liters the fermentor tank.In batch culture, after the growth, the inclusion in this fermentor tank is used as the inoculum of 500 liters of final batch fermentations.
The substratum of using contains: 91g/l W-Gum (BDH Chemicals), ammonium 38g/l one DEXTROSE MONOHYDRATE; 0.6g/l MgSO
47H
2O; 0.6g/l KCl; 0.2g/l FeSO
47H
2O and 12g/l KNO
3By using 4N NaOH or 4N H
2SO
4Automatically titration remains on 4.6 ± 0.3 with pH.
Under being 25% air saturation, the dissolved oxygen concentration of automatically control makes the cell growth under 28 ℃.Ferment after 10 days, phytase output reaches the maximum value of 5～10U/ml.
Using sulfated ammonium instead of corn starch (providing suitable assimilable nitrogen content) repeats fermentation.
Embodiment 2
The purifying of phytase and sign: phytase activity analysis
100 μ l ferment filtrates (if necessary just dilution) or supernatant liquor or 100 μ l softening waters (demiwater is as reference) are added in the culturing mixt with following composition:
-0.25M sodium acetate buffer (pH5.5), or
-glycine HCl damping fluid, pH2.5;
-1mM phytic acid, sodium salt
-softening water is added 900 μ l
The mixture that forms was cultivated 30 minutes down at 37 ℃.By adding 1ml 10%TCA (trichoroacetic acid(TCA)) stopped reaction.Behind reaction terminating, add 2ml reagent [3.66g FeSO
47H
2O is in 50ml ammonium molybdate solution (2.5g (NH
4)
6Mo
7O
244H
2O and 8ml H
2SO
4, be diluted to 250ml with softening water) in].
Measure blue light intensity at the 750nm place with spectrophotometry.The amount of phosphate that measuring result indication (with respect to phosphoric acid salt calibration curve in the scope of 0～1mMol/l) discharges.
Embodiment 3
A. phytase (is containing and is merging aspergillus niger amyloglucosidase (AG) gene with expression vector
Fructus Fici aspergillus (A.Ficuum) phytase gene of promotor and/or signal sequence) what transformed is black
Expression among the aspergillus CBS513.88
In order to reach overexpression phytase in aspergillus niger, a kind of expression cassette of having derived, wherein the Fructus Fici aspergillus niger phytase gene is under the jointly controlling of aspergillus niger amyloglucosidase (AG) promotor and signal sequence.For longer leader sequence, described AG promoter sequence merged into the phytase encoding sequence that comprises phytase leader sequence (this phytase leader sequence is merged into the proteic phytase gene fragment of encoding mature) (see EP-A-0,420,358 reference example 10).
B. phytase gene expression under the control of AG promotor in aspergillus niger
Transform aspergillus niger strain CBS513.88 (being preserved in Dutch fungi strain preservation center on August 10th, 1988, classification called after aspergillus niger DS2975) by currently known methods (for example seeing EP-A-0,420,358 embodiment 9) with 10 μ gDNA fragments.Separate the single aspergillus niger transformant that derives from each expression cassette, streak culture spore on selected ethanamide-agar plate.From under 37 ℃ in 0.4% potato-glucose (Oxoid, England) spore of each transformant of cell collection of having grown on the agar plate 3 days.In shaking bottle, under following condition, survey sufficient phytase output:
With about 1 * 10
8Individual spore inoculating is gone into the pre-culture medium that 100ml contains following material (every liter): 1g KH
2PO
430g maltose; The 5g yeast extract; The 10g casein hydrolysate; 0.5gMgSO
47H
2O and 3g Tween 80.With pH regulator to 5.5.
In 34 ℃ rotary shaking table, grow after the night, the 1ml grown culture is inoculated the main medium that contains following material (every liter) into 100ml: 2g KH
2PO
470g maltodextrin (maldex MDO
3Amylum); 12.5 yeast extract; The 25g casein hydrolysate; 2g K
2SO
45gMgSO
47H
2O; 0.03g ZnCl
20.02g CaCl
20.05MnSO
44H
2O and FeSO
4With pH regulator to 5.6.
Mycelial growth at least 140 hours.Phytase output is to measure by the method for describing among the embodiment 2.Glucose and the ammonium sulfate of using equivalent repeat fermentation as carbon source and nitrogenous source.Filtering fermentation liquor is provided filtrate, it is separated with biomass.Use the maximum phytase activity that expression cassette PFYT3 (AG-promotor/phytase leader sequence) gets 280U/ml.
Embodiment 4
From filtrate purifying phytase
Obtain highly purified phytase by following method purifying:
1. cation-exchange chromatography when pH4.9
2. cation-exchange chromatography when pH3.8
3. anion-exchange chromatography when pH6.3
4. ultrafiltration
1. water is with 20 times of described phytase filtrate dilutions and with pH regulator to 4.9.Allow this material by the post that flows fast with 20mM citric acid/NaOH (pH4.9) damping fluid equilibrated S Sepharose.Collection contains the unconjugated material of phytase and is used for next step.
2. the material with pH4.9 is adjusted to pH3.8, and phytase is bonded to on the mobile fast post of 2mM citric acid/NaOH (pH3.8) damping fluid equilibrated S Sepharose.Use 20mMNaPO
4, 50mM NaCl (pH7.6) damping fluid wash-out phytase from the post.
3. the phytase fraction pH regulator to 6.3 that will compile from second cation-exchange step is combined in phytase and uses 10mM KPO
4(pH6.3) on the mobile fast post of damping fluid equilibrated Q Sepharose.Be applied in the identical damping fluid gradient elution phytase to 1M NaCl.
| Purification result is summed up | |
| Sample | Purification of factor |
| Initial filtrate | 1 |
| After the cationic exchange of pH4.9 | 1.07 |
| After the cationic exchange of pH3.8 | 1.2 |
| After the anionresin | 1.46 |
The Amicon Stirred Cell (2L assembly) that application has Kalle E35 film concentrates ten times by ultrafiltration under 3 crust with the product that last (anionresin) contains 10mg protein/ml.
The ultimate density of the phytase behind the purifying reaches 280～300g/l (28～30%).Specific activity is a 100FTU/mg protein, and this causes phytase activity is 28,000～30,000FTU/g.
Embodiment 5
The high-activity phytase stability test
Provide higher pelleting stability, the pelleting stability that has prepared granulate and tested these samples in order to illustrate higher enzyme concn (in the particle of using the preparation of high-activity phytase liquid) with high enzyme concn.
Comparative sample A: by mix, knead, extrude, spheroidizing and dry preparation be based on the low activity enzyme granulates of W-Gum.
By being mixed and knead, 73% (w/w) W-Gum and lower concentration 4% (w/w) phytase ultrafiltrated and 23% (w/w) water prepared a kind of mixture.Use the basket forcing machine of Nica E-220 and extrude this mixture and the extrudate that must wet, with it at Fuji Paudal Marumerise
TMMiddle spheroidizing obtained round shaped grain that mean diameter is 600 μ m in 2 minutes.Subsequently that these particles are dry in Glatt GPCG 1.1 fluid bed dryers.The final activity of this granulate is 610FTU/g.
Comparative sample B: by mix, knead, extrude, spheroidizing and dry preparation be based on the medium activity enzyme granulates of W-Gum.
By being mixed and knead, 70% (w/w) W-Gum and 17% (w/w) phytase ultrafiltrated and 13% (w/w) water prepared a kind of mixture.Use that the basket forcing machine of Nica E-220 is extruded this mixture and the extrudate that must wet obtained round shaped grain that mean diameter is 600 μ m in 2 minutes with its spheroidizing in Fuji Paudal Marumeriser.Subsequently that these particles are dry in GlattGPCG 1.1 fluid bed dryers.The final activity of this granulate is 4170FTU/g.
Sample C
By mix, knead, extrude, spheroidizing and dry preparation be based on the high active enzyme granulate of W-Gum.
By will be among 67% (w/w) W-Gum and 30% (w/w) embodiment 4 preparation the phytase ultrafiltrated (but be diluted to 18,400FTU/g) and 3% (w/w) water mix and knead and prepared a kind of mixture.Use the basket forcing machine of Nica E-220 and extrude this mixture and the extrudate that must wet, it is round shaped grain of 600 μ m that its spheroidizing in Fuji Paudal Marumeriser was got mean diameter in 2 minutes.Subsequently that these particles are dry in Glatt GPCG 1.1 fluid bed dryers.The final activity of this granulate is 6830FTU/g.
The comparison of pelleting stability
Then described different enzyme granulates is placed relatively their pelleting stability of pelleting test.Pelleting test comprise with described enzyme granulates and feed premixure respectively 1500,320 and 200ppm under mix.Make temperature be raised to 75 ℃ by these mixtures of vapo(u)r blasting pre-treatment, then in pellet press, these mixture pelletings are obtained temperature and be 82 ℃ feed pill, subsequently with they dryings.This method is the common method that fodder industry obtains the feed pill.
Table 1 has been concluded the result of pelleting test.Obviously, having, two kinds of particles of high enzyme concn have much higher pelleting stability.
Table 1
The result of pelleting test
| Sample number | Granulate activity (is unit with FTU/g) | The temperature of fodder meal (℃) | The temperature of pill (℃) | Enzyme yield (%) behind the pelleting |
| Contrast (A) | 610 | 75 | 82 | ＜17 |
| Contrast (B) | 4,170 | 75 | 82 | 37 |
| (C) | 6,830 | 75 | 82 | 48 |
Embodiment 6
Usually mix, knead, pelleting and dry preparation contain soya-bean oil andMgSO
4 Additive based on potato The enzyme granulates of starch
In a mixing tank/kneading machine, add the 30kg yam starch and sneak into 2.5kg soya-bean oil.Then add and contain MgSO
47H
2O (3.5kg MgSO
47H
2O is dissolved in the 14kg ultrafiltrated) the phytase ultrafiltrated that derives from Aspergillus (16,840FTU/g).This product of thorough mixing in kneading machine is extruded and drying in fluid bed dryer then as in Example 1.This produces the product of 5870FTU/g.
Embodiment 7
By mix, knead, extrude, spheroidizing and dry preparation be based on the enzyme granulates of Starch rice
Mix and knead by the identical phytase ultrafiltrated that will use among 62% (w/w) Starch rice and 38% (w/w) embodiment 6 and prepared a kind of mixture.Use the basket forcing machine of Fuji Paudal and extrude this mixture and the extrudate that must wet, again with it at MARUMERISER
TMIt is round shaped grain of 785 μ m that middle spheroidizing got mean diameter in one minute.Dry these particles in fluid bed dryer as in Example 1 subsequently.The final activity of this granulate is 7280FTU/g.
Embodiment 8
By mix, mediate, extrude, spheroidizing and dry preparation be based on the HPMC that contains of W-Gum
The enzyme granulates of additive
Knead by the mixture of the phytase ultrafiltrated will be among 54% (w/w) W-Gum, 5%HPMC (Vltra tears) and 41% (w/w) embodiment 6 used and to have obtained a kind of enzyme preparation.Use the basket forcing machine of Fuji Paudal and extrude this mixture and the extrudate that must wet, with it at MARUMERISER
TMIt is round shaped grain of 780 μ m that middle spheroidizing got mean diameter in one minute.Subsequently in fluid bed dryer under 40 ℃ of bed temperatures and 75 ℃ of temperature ins with dry 20 minutes of these particles.The dried enzyme granulates activity of Huo Deing is 8470FTU/g like this.
Embodiment 9
By mix, knead, extrude, spheroidizing and dry preparation contains the HEC additive, based on corn
The enzyme granulates of starch
Mix and knead by the identical phytase ultrafiltrated that will use among 54% (w/w) W-Gum, 5% (w/w) HEC (Natvosol) and 41% (w/w) embodiment 6 and obtained a kind of enzyme preparation.Use the basket forcing machine of Fuji Paudal and extrude this mixture and the extrudate that must wet, with it at MARUMERISER
TMIt is round shaped grain of 780 μ m that middle spheroidizing got mean diameter in one minute.Subsequently in fluid bed dryer under 40 ℃ of bed temperatures and 75 ℃ of temperature ins with dry 20 minutes of these particles.The dried enzyme granulates activity of Huo Deing is 8410FTU/g like this.
Embodiment 10
Used 18 of the ultrafiltrated that derives from embodiment 4, the ultrafiltrated of 000FTU/g, and diluted.
Sample
The activity of three samples of preparation is 610FTU/g (A, a contrast sample); 4170FTU/g (B, contrast sample) and 6830 (C) FTU/g.This has provided and actively has been respectively 1.153,1.685 and three kinds of feeds of 1.745FTU/g feed.
With first sample (150g) and 20kg feed by following such mixing.After this this premixture is mixed and be divided into two parts with the 80kg feed, become the feed of two following two tests of differing temps.Second sample is that 153.6g is in the 20kg feed.Two parts of equivalent that this 20,153.6 sample are divided into 10.076kg.Then every part is mixed with the 230kg feed and must test the fodder meal of usefulness.
As for the 3rd test, the 96g granulate is mixed two parts that are divided into 10.048g again with the 20kg feed.Then every part is mixed with the 230kg feed and must test the fodder meal of usefulness.Pelleting speed is 600kg/h.
Described fodder mixtures comprises:
Corn 20.00%
Wheat 30.00%
Soybean (heating) 10.00%
Soybean (meal 46.7/3.7) 18.20%
Cassava (65% starch) 6.97%
Bone powder fodder (56.5/10.9) 4.00%
Fish meal (70.6%re) 1.00%
Feather meal, moisture 1.00%
Soya-bean oil/Semen Maydis oil 1.30%
Animal tallow 4.00%
Vitamin-mineral premix (corn) 1.00%
Lime carbonate 0.85%
Mono-calcium phosphate 1.05%
Salt 0.26%
L-Methionin HCl 0.16%
DL-methionine 0.21%
Then with these three kinds of mixture pelletings.Feed is fed into even wet device, wherein steam directly is added on the fodder meal.Temperature is elevated to 75 ℃.Discharge fodder meal from tablets press subsequently, fodder meal in tablets press, be pushed through have the 5mm hole, template that 65mm is thick.The temperature of feed raises again 4 ℃ and reaches 79 ℃ at this moment.
The activity of these three kinds of feeds is 10.11 (A); 10.04 (B) with 9.81 (C).
The residual activity result of this test is: 63% (A); 66% (B) and 72% (C) are respectively corresponding to original 610FTU/g sample; 4170FTU/g sample and 6830FTU/g sample.This shows, even similar activity (B and C), the most highly active prescription (C are arranged; 6830FTU/g, prescription of the present invention) provides much higher pelleting stability.This is higher by 6% than (correlated) sample B, and this value is quite remarkable, because only observe 3% increase from the sample (from A to B) of activity increase very big (610 to 4170FTU/g).
Claims (20)
1. granulate, it comprises the dried particle that is formed by phytase and solid carrier, wherein said solid carrier comprises at least 15% (w/w) edible carbohydrate polymkeric substance.
2. the granulate of claim 1, wherein said particle comprises at least a divalent cation.
3. claim 1 or 2 granulate, wherein said particle comprises one or more hydrophobicity gelationization compound or water-insoluble compounds.
4. the granulate of claim 3, wherein said hydrophobicity gelationization compound or water-insoluble compound comprise derivatize Mierocrystalline cellulose, polyvinyl alcohol (PVA) or edible oil.
5. the granulate of claim 4, wherein said derivatize Mierocrystalline cellulose is that Vltra tears, carboxymethyl cellulose or Natvosol and/or described edible oil are soya-bean oil or Tower rape oil.
6. each granulate of claim 1-5, zytase and/or beta-glucanase in it also comprises in addition.
7. each granulate of claim 1-6, wherein said carrier comprises starch.
8. each granulate of claim 1-7, wherein said phytase is not a kind of heat-stable (heat-staple) phytase.
9. each granulate of claim 1-8, wherein said phytase is the fungi phytase.
10. each composition of claim 1-9, wherein said fungi phytase derives from Aspergillus or trichoderma bacterial classification.
11. a method for preparing the premixture or the precursor of animal-feed, animal-feed, this method comprise that the phytase granulate that contains with each definition of claim 1-10 mixes with one or more animal-feed materials or component.
12. the method for claim 11, the mixture experience steam treatment of wherein said feed substances and composition or granulate is by pelleting and optional being dried.
13. a composition, it comprises that each granulate and/or activity of claim 1-10 is at least 6, and 000FTU/g contains phytase granulate.
14. the composition of claim 13, it is a kind of edible feed composition.
15. the composition of claim 14, it is a kind of animal-feed.
16. the composition of claim 14 or 15, it comprise with claim 1-9 each one or more feed substances of granulate blended or the pill of component.
17. each composition of claim 13-16, it is the premixture or the precursor of a kind of animal-feed or animal-feed, can be by the method preparation of claim 11 or 12.
18. a method that promotes that animal increases, this method comprises that to a kind of food of feeding animal this food comprises the granulate of each definition of claim 1-9 or the composition of each definition of claim 13-17.
19. the granulate of each definition of claim 1-9 in animal-feed application or as the application of the component of animal-feed, perhaps be used for animal foodstuff.
20. the application of a composition, composition comprise 15% (w/w) edible carbohydrate polymkeric substance, this polymkeric substance as the carrier of phytase to improve the pelleting stability of phytase.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US4861197P | 1997-06-04 | 1997-06-04 | |
| US60/048,611 | 1997-06-04 | ||
| EP97201641.4 | 1997-06-04 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB988071045A Division CN1315397C (en) | 1997-06-04 | 1998-06-04 | High-activity phytase granulate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1775949A true CN1775949A (en) | 2006-05-24 |
| CN100526459C CN100526459C (en) | 2009-08-12 |
Family
ID=21955490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100385392A Expired - Fee Related CN100526459C (en) | 1997-06-04 | 1998-06-04 | High-activity phytase composition |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN100526459C (en) |
| HU (1) | HUP0002767A3 (en) |
| TW (1) | TWI223002B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168734B (en) * | 2007-09-29 | 2010-06-02 | 北京昕地美饲料科技有限公司 | Coating micro-pill phytase and preparation method thereof |
| CN102651973A (en) * | 2009-12-18 | 2012-08-29 | 希尔氏宠物营养品公司 | Pet food compositions including probiotics and methods of manufacture and use thereof |
| CN102885208A (en) * | 2011-07-22 | 2013-01-23 | 北京奕农顺丰生物技术有限公司 | Duplex enzyme preparation containing phytase |
| CN111073880A (en) * | 2020-01-07 | 2020-04-28 | 南京工业大学 | Pichia pastoris immobilization method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI77359C (en) * | 1986-08-22 | 1989-03-10 | Suomen Sokeri Oy | Feed fix and process for making the same. |
| CZ289014B6 (en) * | 1989-09-27 | 2001-10-17 | Dsm N. V. | Purified and isolated DNA sequence encoding fungal phytase, construct for expression, vectors and transformed host cells as well as phytase preparation process |
-
1998
- 1998-06-04 HU HU0002767A patent/HUP0002767A3/en unknown
- 1998-06-04 CN CNB2004100385392A patent/CN100526459C/en not_active Expired - Fee Related
- 1998-06-30 TW TW87110748A patent/TWI223002B/en not_active IP Right Cessation
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168734B (en) * | 2007-09-29 | 2010-06-02 | 北京昕地美饲料科技有限公司 | Coating micro-pill phytase and preparation method thereof |
| CN102651973A (en) * | 2009-12-18 | 2012-08-29 | 希尔氏宠物营养品公司 | Pet food compositions including probiotics and methods of manufacture and use thereof |
| US10212954B2 (en) | 2009-12-18 | 2019-02-26 | Colgate-Palmolive Company | Pet food compositions including probiotics and methods of manufacture and use thereof |
| US11510424B2 (en) | 2009-12-18 | 2022-11-29 | Hill's Pet Nutrition, Inc. | Pet food compositions including probiotics and methods of manufacture and use thereof |
| CN102885208A (en) * | 2011-07-22 | 2013-01-23 | 北京奕农顺丰生物技术有限公司 | Duplex enzyme preparation containing phytase |
| CN111073880A (en) * | 2020-01-07 | 2020-04-28 | 南京工业大学 | Pichia pastoris immobilization method and application thereof |
| CN111073880B (en) * | 2020-01-07 | 2021-08-27 | 南京工业大学 | Pichia pastoris immobilization method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100526459C (en) | 2009-08-12 |
| HUP0002767A1 (en) | 2000-12-28 |
| TWI223002B (en) | 2004-11-01 |
| HUP0002767A3 (en) | 2001-01-29 |
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