CN105517450A - Methods and compositions to improve the nutritional value of lignocellulosic biomass - Google Patents

Methods and compositions to improve the nutritional value of lignocellulosic biomass Download PDF

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Publication number
CN105517450A
CN105517450A CN201480045781.4A CN201480045781A CN105517450A CN 105517450 A CN105517450 A CN 105517450A CN 201480045781 A CN201480045781 A CN 201480045781A CN 105517450 A CN105517450 A CN 105517450A
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China
Prior art keywords
activity
feed
feed addictive
active
composition
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CN201480045781.4A
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Inventor
L·F·罗密欧米兰
S·于
M·沃尔什
S·兰茨
C·米奇森
B·鲍尔
S·阿伦特
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DuPont Nutrition Biosciences ApS
Danisco US Inc
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Danisco US Inc
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/32—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from hydrolysates of wood or straw
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The present invention relates to a method of preparing a feed additive composition comprising: (a) physically and/or chemically and/or biologically pre-treating lignocellulosic biomass, (b) admixing the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass with an enzyme composition, wherein the enzyme composition comprises at least the following activities: endoglucanase activity, beta-glucosidase activity and endoxylanase activity and wherein the enzyme composition comprises no, or substantially no, beta-xylosidase activity and/or no, or substantially no alpha-L- arabinofuranosidase activity, (c) incubating same for at least about 3-120 hours, (d) and optionally drying and/or optionally packaging.

Description

Improve the method and composition of lignocellulose biomass nutritive value
Technical field
The present invention relates to the method and composition improving lignocellulose biomass nutritive value.The method and composition that the present invention relates to can make lignocellulose biomass become the suitable substitutes of the usual cereal starch used in animal feed.
Background technology
The zootrophic cereal of annual supply about has 7.5 hundred million tons.The part using more cheap feed resource to substitute these cereal can make the whole mankind benefited a great deal.
Cereal (as corn) is replaced with more cheap cellulosic material (as lignocellulose biomass) meeting profound influence animal feed markets, and/or increase the stability of human food and energy market.
The nutritive value of lignocellulose biomass is also not enough to the viable substitute becoming high-energy feed raw material (such as grain).Nonruminant such as live pig or poultry, cannot digest the lignocellulosic in fibre composition (such as agricultural wastes) and absorb wherein glucose, such as, reach the degree allowing to substitute cereal a large amount of in animal diet followed.Animal, especially nonruminant, do not have the enzyme needed for digest cellulose and hemicellulose; Even if possess this fermentoid, the time of staying in intestines also can become the limiting factor that cellulose conversion is glucose.
Cellulose is chemical formula is (C 6h 10o 5) norganic compound, it is by the polysaccharide that forms of D-Glucose unit connected by β (1 → 4) glycosidic bond of straight chain.
Hemicellulose is any one in several heteropolymer (matrix polysaccharide), coexists in nearly all plant cell wall with cellulose.Hemicellulose comprises xylan, glucuronoxylan, araboxylan, glucomannans, and xyloglucan.Hemicellulose comprises many different sugar monomers.Such as, in addition to glucose, the sugar monomer in hemicellulose also can comprise wood sugar, mannose, galactolipin, rhamnose, and arabinose.Hemicellulose comprises most of D-pentose (C-5 sugar), sometimes also comprises a small amount of L-sugar.In most cases, wood sugar is the maximum sugar monomer of content, and mannose may be the maximum sugar of abundance in cork.Not only there is conventional sugar in hemicellulose, also there is the acidic form (such as glucuronic acid and galacturonic acid) of conventional sugar.
The people such as Schutte (BritishJ.ofNutrition1991,66,83-93) have studied the nutritional significance of D-wood sugar in pig body.The people such as Schutte propose the ileal digestibility of D-wood sugar and D-Glucose all close to 100%.In diet, the existence of D-wood sugar reduces ileum Chyme pH and enhances the flowing of volatile fatty acid in ileum, and this shows the microbial degradation that there occurs D-wood sugar in chitterlings.Schutte proposes in the pig body of high D-Xylose Content diet of feeding (in such as every kg diet, D-wood sugar is 200g), ileal digestibility, the ight soil digestibility of dry (DM), organic substance (OM), gross energy (GE) and nitrogen (N), and nitrogen retention rate significantly reduces.Schutte (1991) also finds the wood sugar adding 10% in the diet of pig, and wherein the wood sugar energy of 50% can be discharged with urine.
The people such as Verstegen (J.Animal.Physiol.a.Anim.Nutr.77 (1997) 180-188) test pig, have rated the feasibility of wood sugar as pig energy source.According to the computational methods that the document uses, when adding 10% wood sugar in diet, in diet, the wood sugar energy of 38% to 64% exists with the form of metabolizable energy (ME), the pig fed containing 10% wood sugar in diet of contrast with contain 5% glucose in diet of feeding contrast pig, it is found that: the ME in two kinds of diet is similar with the weightening finish of pig under two kinds of feed mode.
The people such as Savory (BritishJournalofNutrition (1992), 67,103-114) have studied in bird body by U- 14the metabolic fate of monose of C mark, and propose bird absorb wood sugar than absorption glucose and galactolipin much slow, but than absorption mannose and arabinose fast.Savory (1992) also observes in the excreta of bird by U- 14the wood sugar of C mark and arabinose larger than the rate of recovery of hexose, thus wood sugar and arabinose lower than the extent of metabolism of hexose (C6).
Accompanying drawing explanation
Fig. 1 illustrates the glucose and xylose output obtained by cellulase SC process DaCS (through the pretreated maize straw of weak aqua ammonia).
Fig. 2 illustrates by using trio tM, cellulase SC or cellulase SC+ endo-xylanase and glucose and xylose from DaCS release amount.
Fig. 3 illustrates by using trio tM, cellulase SC118, cellulase 151 and SC118+ zytase and sugar from DaCS release amount.
Fig. 4 illustrates by using trio tM, the cellulase SC endo-xylanase different with cellulase SC+ tetra-kinds and the DaCS that obtains is hydrolyzed situation.
Fig. 5 illustrates by using 30 μ l trio tMand the HPLC chromatogram of the DaCS hydrolysate obtained.
Fig. 6 illustrates by combinationally using cellulase SC, cellulase SC and endo-xylanase (SoloC118,50 μ L) and the HPLC chromatogram of the DaCS hydrolysate obtained.
Fig. 7 illustrates by using trio tM, cellulase SC and cellulase SC+ endo-xylanase and the hydrolysis situation of DaCS.
Fig. 8 illustrates and is adding or do not adding cellulase SC118 and endo-xylanase DaniscoXylanase tMwhen (see EP1222256, the document is incorporated herein by reference), under 50 DEG C and pH5.0, (weight of DaCS residue, in g) for the result of DaCS solubilising after 40 hours.
Fig. 9 illustrates and is adding or do not adding cellulase SC118 and endo-xylanase DaniscoXylanase tMwhen, under 50 DEG C and pH5.0, the result of DaCS solubilising after 40 hours (solubilising material %).
Figure 10 illustrates by using cellulase SC118 and zytase DaniscoXylanase tMdosage effect, under 50 DEG C and pH5.0, in 40 hours from DaCS release monose amount.
Figure 11 illustrates by using cellulase SC118 and zytase DaniscoXylanase tMdosage effect, under 50 DEG C and pH5.0, in 40 hours from DaCS release sugar (monomer+polymer form).
Figure 12 illustrates to take food wood sugar as the feed intake of the broiler chicken of energy source and weight gain.
Figure 13 illustrates to take food the broiler chicken of wood sugar as energy source in the body weight of feeding the 21st day.
Figure 14 illustrates to take food the broiler chicken of wood sugar as energy source at the feed conversion rate in the 0 to 21 day of feeding.
Figure 15 illustrates the nucleotide sequence (SEQIDNo.1) of coding endo-xylanase (FoxXyn6).Burst illustrates with black matrix (upper case).
Figure 16 illustrates the nucleotide sequence (SEQIDNo.2) of coding endo-xylanase (FoxXyn6).
Figure 17 illustrates the peptide sequence (SEQIDNo.3) of endo-xylanase (FoxXyn6).This sequence is the activity form (e.g., the mature form of enzyme) of enzyme.
Figure 18 illustrates the nucleotide sequence (SEQIDNo.4) of coding endo-xylanase (FoxXyn4).Burst illustrates with black matrix (upper case).
Figure 19 illustrates the nucleotide sequence (SEQIDNo.5) of coding endo-xylanase (FoxXyn4).
Figure 20 illustrates the peptide sequence (SEQIDNo.6) of endo-xylanase (FoxXyn4).This sequence is the activity form (e.g., the mature form of enzyme) of enzyme.
Figure 21 illustrates the nucleotide sequence (SEQIDNo.7) of coding from the endoglucanase (EG1) of aspergillus niger (Aspergillusniger) (CBS513.88).
Figure 22 illustrates the peptide sequence (SEQIDNo.8) of the endoglucanase (EG1) from aspergillus niger (CBS513.88).
Figure 23 illustrates the nucleotide sequence (SEQIDNo.9) of coding from the endoglucanase (EG2) of aspergillus niger CBS513.88.
Figure 24 illustrates the peptide sequence (SEQIDNo.10) of the endoglucanase (EG2) from aspergillus niger CBS513.88.
Figure 25 illustrates the nucleotide sequence (SEQIDNo.11) of coding endo-xylanase.
Figure 26 illustrates the peptide sequence (SEQIDNo.12) of endo-xylanase.
Figure 27 illustrates the nucleotide sequence (SEQIDNo.13) of coding from the beta-glucosidase of aspergillus niger CBS513.88.
Figure 28 illustrates the peptide sequence (SEQIDNo.14) of the beta-glucosidase from aspergillus niger CBS513.88.
Figure 29 illustrates the nucleotide sequence (SEQIDNo.15) of coding from the dissolubility polysaccharide monooxygenase of aspergillus niger CBS513.88.
Figure 30 illustrates the peptide sequence (SEQIDNo.16) of the dissolubility polysaccharide monooxygenase from aspergillus niger CBS513.88.
Figure 31 illustrates the nucleotide sequence (SEQIDNo.17) of coding from the CHB1A of aspergillus niger (CBS513.88).
Figure 32 illustrates the peptide sequence (SEQIDNo.18) from the CHB1A of aspergillus niger (CBS513.88).
Figure 33 illustrates the nucleotide sequence (SEQIDNo.19) of coding from the CHB1B of aspergillus niger (CBS513.88).
Figure 34 illustrates the peptide sequence (SEQIDNo.20) from the CHB1B of aspergillus niger (CBS513.88).
Figure 35 illustrates the nucleotide sequence (SEQIDNo.21) of coding from the CHB1 of trichoderma reesei (Trichodermareesei).
Figure 36 illustrates the peptide sequence (SEQIDNo.22) of the CHB1 from trichoderma reesei.
Figure 37 illustrates the nucleotide sequence (SEQIDNo.23) of coding from the CHB2 of trichoderma reesei.
Figure 38 illustrates the peptide sequence (SEQIDNo.24) of the CHB2 from trichoderma reesei.
Figure 39 illustrates the nucleotide sequence (SEQIDNo.25) of coding from the endoglucanase (EG1) of trichoderma reesei.
Figure 40 illustrates the peptide sequence (SEQIDNo.26) of the endoglucanase (EG1) from trichoderma reesei.
Figure 41 illustrates the nucleotide sequence (SEQIDNo.27) of coding from the endoglucanase (EG2) of trichoderma reesei.
Figure 42 illustrates the peptide sequence (SEQIDNo.28) of the endoglucanase (EG2) from trichoderma reesei.
Figure 43 illustrates the nucleotide sequence (SEQIDNo.29) of coding from the endoglucanase (EG3) of trichoderma reesei.
Figure 44 illustrates the peptide sequence (SEQIDNo.30) of the endoglucanase (EG3) from trichoderma reesei.
Figure 45 illustrates the nucleotide sequence (SEQIDNo.31) of coding from the dissolubility polysaccharide monooxygenase of trichoderma reesei.
Figure 46 illustrates the peptide sequence (SEQIDNo.32) of the dissolubility polysaccharide monooxygenase from trichoderma reesei.
Figure 47 illustrates the nucleotide sequence (SEQIDNo.33) of coding from the endo-xylanase of trichoderma reesei.
Figure 48 illustrates the peptide sequence (SEQIDNo.34) of the endo-xylanase from trichoderma reesei.
Figure 49 illustrates the nucleotide sequence (SEQIDNo.35) of coding from the beta-glucosidase of trichoderma reesei.
Figure 50 illustrates the peptide sequence (SEQIDNo.36) of the beta-glucosidase from trichoderma reesei.
Summary of the invention
In first aspect, the invention provides a kind of method preparing feed addictive composition, the method comprises:
A. physics and/or chemical and/or biological method preprocessing lignocellulose living beings are used,
B. will through physics and/or chemistry and/or the pretreated lignocellulose biomass of biological method mix with enzymatic compositions, wherein this enzymatic compositions at least has following activity: endoglucanase activity, beta-glucosidase activity and endo-xylanase are active, and wherein this enzymatic compositions does not have or does not substantially have xylobiase activity and/or α-L-arabinofuranosidase activity
C. by the mixture incubation of lignocellulose biomass and enzymatic compositions at least about 3 to 120 hours, preferably incubation 6 to 48 hours,
D. and optionally carry out drying and/or optionally pack.
In yet another aspect, the invention provides the purposes of enzymatic compositions, wherein manufacturing for improving lignocellulose biomass in the process of the feed addictive composition of the nutritive value of animal, enzymatic compositions at least has following activity: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, and wherein enzymatic compositions does not have or substantially do not have xylobiase activity and/or α-L-arabinofuranosidase activity.
Other one side of the present invention is that one can adopt method of the present invention to obtain the feed addictive composition of (such as, obtaining).
In yet another aspect, provide a kind of feed addictive composition or feed ingredient, its comprise by enzymatic compositions be hydrolyzed through physics and/or chemistry and/or the pretreated lignocellulose biomass of biological method, wherein enzymatic compositions at least has following activity: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, and does not have or substantially do not have xylobiase activity and/or α-L-arabinofuranosidase activity.
Another aspect provides a kind of feed or feedstuff, it comprises according to feed addictive composition of the present invention or feed ingredient, and method of the present invention or purposes maybe can be adopted to obtain the feed addictive composition of (preferably obtaining).
On the other hand, provide a kind of pre-composition, it comprises according to feed addictive composition of the present invention or feed ingredient, method of the present invention or purposes maybe can be adopted to obtain the feed addictive composition of (preferably obtaining), and at least one mineral matter and/or at least one vitamin.
Present invention also offers a kind of method preparing feedstuff, the method comprises and feed ingredient and feed addictive composition according to the present invention or feed ingredient, the feed addictive composition that maybe method of the present invention or purposes can be adopted to obtain (optionally obtaining) being contacted.
Present invention provides a kind of method improving animal organism physical characteristic, the method comprises to be used can obtain (such as by the inventive method to animal, obtain) feed addictive composition, or according to feed addictive composition of the present invention, or according to pre-composition of the present invention, or according to feedstuff of the present invention, or the feedstuff of (such as, obtaining) can be obtained by the inventive method.
In another, the invention provides and can be obtained (such as by method of the present invention or purposes, obtain) feed addictive composition or according to feed addictive composition of the present invention or according to pre-composition of the present invention or according to feedstuff of the present invention or can be obtained (such as, obtaining) by the inventive method feedstuff for improving the purposes of the biophysical properties of animal.
In a preferred embodiment, enzymatic compositions does not have or does not substantially have xylobiase activity, and it is active not have or substantially do not have α-L-arabinofuranosidase.
Aptly, enzymatic compositions can comprise one or both enzymes being selected from cellobiohydrolase I and cellobiohydrolase II further.
Aptly, enzymatic compositions can comprise dissolubility polysaccharide monooxygenase further.
Detailed description of the invention
The research (see example 5) of inventor has proved that the C-5 sugar monomer (such as wood sugar) of high-load in feed can cause adverse effect to the animal health of this feed of feed.
But, in feedstuff, use high-load lignocellulose biomass that the nutritive value of feedstuff can be caused not high enough, or for attempting making feedstuff have more high nutritive value and processing lignocellulose biomass, then can cause C-5 sugar monomer (such as wood sugar) too high levels in feedstuff and may work the mischief to animal health.
Therefore, inventor draws before feeding animals, should preferably be removed from any lignocellulose biomass feed by C-5 sugar monomer.
The present inventor has now found that a kind of unique method using enzymatic compositions treatment of lignocellulosic biomass, this enzymatic compositions can make C-5 sugar monomer (such as wood sugar) content in feedstuff or feed addictive composition reduce, and maintains (such as nutritious) the high-load C-6 oligomer to animal health and sugar monomer simultaneously.
Surprisingly, if remove the activity forming C-5 carbohydrate monomer, then can form a large amount of oxylooligomers under the cellulose conversion that do not detract is the efficiency of glucose or the prerequisite of time.Resulting composition to feed applications advantageously.
The present inventor surprisingly finds, eliminate that C5 sugar monomer generates and improve that C5 oligomer generates is favourable active complex effect with cellulase activity (such as, thus generation C6 oligomer and C6 sugar monomer) simultaneously.
The method and the purposes that comprise this enzymatic compositions will allow to add the lignocellulose biomass of more high-load in animal feed and animal diet followed.
The present inventor surprisingly finds, significantly improves the nutritive value of lignocellulose biomass to animal (especially nonruminant): endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active by using the enzymatic compositions at least with following activity; Wherein in the process for the treatment of of lignocellulosic biomass, enzymatic compositions does not exist or substantially there is not the one or both in following activity: xylobiase is active and/or α-L-arabinofuranosidase is active.
Surprisingly find to reduce or remove xylobiase activity and α-L-arabinofuranosidase activity, the nutritive value of lignocellulose biomass to animal (especially nonruminant) can be significantly improved.
As can be seen from data, these benefits are that the while of coming from the reduction of sugar monomer in composition (especially C-5 sugar monomer) content, in composition, oligomer sugar (such as oxylooligomers) content significantly improves.
This discovery is astonishing, if because C6 monomer formation enzyme is shifted out system with regard to known by people before making the present invention, the feedback inhibition of reaction can make the accumulation of product (such as cellobiose), enzyme reaction slows down gradually.Therefore people are known before making the present invention, and when the sugar monomer content formed reduces, the feedback mechanism of reaction can cause enzyme reaction entirety to slow down.Surprisingly, obvious feedback mechanism is not observed for enzymatic compositions, the enzymatic compositions that xylobiase is active and/or α-L-arabinofuranosidase activity is very little that such as the present invention is used.
The present inventor surprisingly finds, use the method for enzymatic mixture can significantly improve the nutritive value of lignocellulose biomass to animal (especially nonruminant), this enzymatic mixture can decomposition of cellulose and hemicellulose, but does not have the activity that forms carbohydrate monomer or do not have the activity (especially not having the activity forming C-5 carbohydrate monomer) forming reduction monomer.
By using physics and/or chemical and/or biological method preprocessing lignocellulose living beings, this effect can be strengthened further.Therefore, first physics and/or chemical and/or biological method treatment of lignocellulosic biomass is used, then use enzymatic compositions process through physics and/or the chemical and/or pretreated lignocellulosic of biological method, wherein enzymatic compositions at least has following activity: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active; Wherein do not exist in enzymatic compositions or substantially there is not the one or both in following enzymatic activity: xylobiase is active and α-L-arabinofuranosidase is active.
The present invention means, available lignocellulose biomass substitutes the usual starch (e.g., from the starch of cereal) used in animal feed.This way has obvious benefit.Such as, the present invention is utilized to mean, without the need to using cereal to supply Animal nutrition with larger proportion in animal feed application as usual again.The present invention can make the cost preparing animal feed reduce.In addition or alternatively, present invention alleviates for a long time due to the resource pressure that population size increases and fossil fuel supply limits and causes supply chain.
The present invention is all relevant with ruminant with nonruminant, but the most relevant to nonruminant.
With regard to ruminant, can use and substitute cereal according to feed additive of the present invention or by feed additive prepared by the present invention, take meat and the dairy products of intensive manufacture ruminant, and/or increase system effectiveness.
The present invention relates to a kind of method preparing feed addictive composition, described method comprises:
A. physics and/or chemical and/or biological method preprocessing lignocellulose living beings are used,
B. will through physics and/or chemistry and/or the pretreated lignocellulose biomass of biological method mix with enzymatic compositions, wherein this enzymatic compositions at least has following activity: endoglucanase activity, beta-glucosidase activity and endo-xylanase are active, and wherein this enzymatic compositions does not have or does not substantially have xylobiase activity and/or α-L-arabinofuranosidase activity
C. by the mixture incubation of lignocellulose biomass and enzymatic compositions at least about 3 to 120 hours, preferably incubation 6 to 48 hours,
D. and optionally carry out drying and/or optionally pack.
Term used herein " does not exist " or " not having " refers to that enzymatic compositions does not have xylobiase activity and do not have α-arabinofuranosidase activity.
Herein the active term used of and/or α-L-arabinofuranosidase active in conjunction with xylobiase " substantially do not exist " and " substantially not having " refer to use " xylobiase activation measurement " in this paper to measure xylobiase activity lower than 3000 units/mg (aptly lower than 2000 units/mg, aptly lower than 1500 units/mg), and/or the α-L-arabinofuranosidase that uses " α-L-arabinofuranosidase activation measurement " in this paper to measure active lower than 1000 units/mg (aptly lower than 500 units/mg, aptly lower than 450 units/mg).
In one embodiment, the active term used of and/or α-L-arabinofuranosidase active in conjunction with xylobiase " does not exist " and " substantially not having " refers to that the xylobiase using " xylobiase activation measurement " in this paper to measure is active in 1500 units/mg substantially herein, and/or the α-L-arabinofuranosidase activity using " α-L-arabinofuranosidase activation measurement " in this paper to measure is lower than 450 units/mg.
In one embodiment, it is active that the present invention's enzymatic compositions used has the xylobiase lower than 3000 units/mg (aptly lower than 2000 units/mg, aptly lower than 1500 units/mg) using " xylobiase activation measurement " in this paper to measure, and the α-L-arabinofuranosidase lower than 1000 units/mg (aptly lower than 500 units/mg, aptly lower than 450 units/mg) using " α-L-arabinofuranosidase activation measurement " in this paper to measure is active.
In one embodiment, the active term used of and/or α-L-arabinofuranosidase active in conjunction with xylobiase " does not exist " and " substantially not having " refers to that the xylobiase using " xylobiase activation measurement " in this paper to measure is active in 1500 units/mg substantially herein, and the α-L-arabinofuranosidase using " α-L-arabinofuranosidase activation measurement " in this paper to measure activity is lower than 450 units/mg.
In a preferred embodiment, enzymatic compositions can not have or substantially not have xylobiase activity, and it is active not have or substantially do not have α-L-arabinofuranosidase.
In a preferred embodiment, enzymatic compositions can not have xylobiase activity, and it is active not have α-L-arabinofuranosidase.
In a preferred embodiment, the lignocellulose biomass that the present invention is used can be pretreated through physics, through Chemical Pretreatment, through Biological Pretreatment or through their the pretreated lignocellulose biomass of combination.
In another embodiment, through physics and/or chemistry and/or the pretreated lignocellulose biomass of biological method can together with enzymatic compositions incubation at least about 6 to 48 hours.
In another preferred embodiment, the enzymatic compositions that the present invention is used can be made up of (or being made up of following activity) following activity substantially: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active.
In one embodiment, the enzymatic compositions that the present invention is used also can have the one or both in following enzymatic activity: cellobiohydrolase I is active and cellobiohydrolase II is active.
In one embodiment, the enzymatic compositions that the present invention is used also can have dissolubility polysaccharide monooxygenase activity.
In certain embodiments, it is active that the present invention's enzymatic compositions used can have endoglucanase activity, activity of beta-glucosidase and endo-xylanase, and one or more in following enzymatic activity: cellobiohydrolase I is active, cellobiohydrolase II is active or dissolubility polysaccharide monooxygenase activity.
In certain embodiments, the present invention's enzymatic compositions used can be made up of (or being made up of following enzymatic activity) following enzymatic activity substantially: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, and one or more in following enzymatic activity: cellobiohydrolase I is active, cellobiohydrolase II is active or dissolubility polysaccharide monooxygenase activity.
In certain embodiments, it is active that the present invention's enzymatic compositions used can have endoglucanase activity, activity of beta-glucosidase and endo-xylanase, and in following enzymatic activity both or more person's (being suitably three): cellobiohydrolase I is active, cellobiohydrolase II is active or dissolubility polysaccharide monooxygenase activity.
In certain embodiments, the present invention's enzymatic compositions used can be made up of (or being made up of following enzymatic activity) following enzymatic activity substantially: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, and in following enzymatic activity both or more person's (being suitably three): cellobiohydrolase I is active, cellobiohydrolase II is active or dissolubility polysaccharide monooxygenase activity.
In one embodiment, the enzymatic compositions that the present invention is used characterizes by its enzymatic activity.
In one embodiment, the present invention's enzymatic compositions used has at least following enzymatic activity and (or to be substantially made up of at least following enzymatic activity, or be made up of at least following enzymatic activity): use the endoglucanase activity that " endoglucanase activity determination method " records, use the activity of beta-glucosidase that " activity of beta-glucosidase determination method " records, the endo-xylanase using " endo-xylanase activation measurement " to record is active.
Form below and paragraph thereafter list the scope of these activity that may be present in described enzymatic compositions:
Often kind of enzymatic activity in the field of activity of unit in composition
Endoglucanase activity 500-4000CMC U/g 1
Activity of beta-glucosidase 200-3500pNPG U/g 2
Endo-xylanase is active 1500-6000ABX U/g 3
Often kind of enzymatic activity in the field of activity of unit in composition
Endoglucanase activity 1000-3500CMC U/g 1(being preferably 1500-3000CMC U/g)
Activity of beta-glucosidase 300-3000pNPG U/g 2(being preferably 500-2500pNPG U/g)
Endo-xylanase is active 2000-5000ABX U/g 3(being preferably 3000-4000ABX U/g)
11 μm of ol reduced sugar (representing with glucose equivalent), under the condition of 50 DEG C and pH4.8, was disengaged in one minute in CMC active unit.
2pNPG unit representation, under the condition of 50 DEG C and pH4.8, per minutely discharges 1 μm of ol nitrophenol from p-nitrophenyl-β-D-glucopyranoside.
3aBX unit is defined as under the condition of 50 DEG C and pH5.3, the enzyme amount needed for generation per minute 1 μm of ol wood sugar reduced sugar equivalent.
In one embodiment, the enzymatic compositions that the present invention is used can have at least 500CMCU/g endoglucanase activity that use " endoglucanase activity determination method " measures (at least 1000CMCU/g is active aptly, at least 2000CMCU/g is active or at least 3000CMCU/g is active aptly aptly).
In another embodiment, the enzymatic compositions that the present invention is used can have at least 200pNPGU/g activity of beta-glucosidase that use " activity of beta-glucosidase determination method " measures (at least 1000pNPGU/g is active aptly, at least 1500pNPGU/g is active or at least 2500pNPGU/g is active aptly aptly).
In yet another embodiment, the enzymatic compositions that the present invention is used can have at least 1500ABXU/g endo-xylanase activity that use " endo-xylanase activation measurement " measures (aptly at least active, at least active, at least active or at least 5000ABXU/g aptly of 4500ABXU/g aptly of 4000ABXU/g aptly of 3000ABXU/g).
Aptly, the present invention's enzymatic compositions used can have following activity (or be substantially made up of following activity, or be made up of following activity): at least 500CMCU/g endoglucanase activity that uses " endoglucanase activity determination method " to measure (at least 1000CMCU/g is active aptly, at least 2000CMCU/g is active or at least 3000CMCU/g is active aptly aptly); At least 200pNPGU/g activity of beta-glucosidase that uses " activity of beta-glucosidase determination method " to measure (at least 1000pNPGU/g is active aptly, at least 1500pNPGU/g is active or at least 2500pNPGU/g is active aptly aptly); And at least 1500ABXU/g endo-xylanase activity using " endo-xylanase activation measurement " to measure (aptly at least active, at least active or at least active, at least 5000ABXU/g aptly of 4500ABXU/g aptly of 4000ABXU/g aptly of 3000ABXU/g).
In one embodiment, the enzymatic compositions that the present invention is used can have that use " endoglucanase activity determination method " measures at least about 500CMCU/g to about 4000CMCU/g endoglucanase activity (active at least about 1000CMCU/g to about 3500CMCU/g, active or active at least about 1500CMCU/g to about 2500CMCU/g aptly at least about 1500CMCU/g to about 3000CMCU/g aptly aptly).
In another embodiment, the enzymatic compositions that the present invention is used can have that use " activity of beta-glucosidase determination method " measures at least about 200pNPGU/g to about 3500pNPGU/g activity of beta-glucosidase (active at least about 300pNPGU/g to about 3000pNPGU/g, active or active at least about 1000pNPGU/g to about 2000pNPGU/g aptly at least about 500pNPGU/g to about 2500pNPGU/g aptly aptly).
In yet another embodiment, the enzymatic compositions that the present invention is used can have that use " endo-xylanase activation measurement " measures at least about 1500ABXU/g to about 6000ABXU/g endo-xylanase activity (active at least about 2000ABXU/g to about 5000ABXU/g, active or active at least about 3000ABXU/g to about 4000ABXU/g aptly at least about 2500ABXU/g to about 4500ABXU/g aptly aptly).
Aptly, the present invention's enzymatic compositions used can have following activity (or be substantially made up of following activity, or be made up of following activity): use " endoglucanase activity determination method " to measure at least about 500CMCU/g to about 4000CMCU/g endoglucanase activity (active at least about 1000CMCU/g to about 3500CMCU/g, active or active at least about 1500CMCU/g to about 2500CMCU/g aptly at least about 1500CMCU/g to about 3000CMCU/g aptly aptly); Use " activity of beta-glucosidase determination method " to measure at least about 200pNPGU/g to about 3500pNPGU/g activity of beta-glucosidase (active at least about 300pNPGU/g to about 3000pNPGU/g, active or active at least about 1000pNPGU/g to about 2000pNPGU/g aptly at least about 500pNPGU/g to about 2500pNPGU/g aptly aptly); And use " endo-xylanase activation measurement " to measure at least about 1500ABXU/g to about 6000ABXU/g endo-xylanase activity (active at least about 2000ABXU/g to about 5000ABXU/g aptly, active or active at least about 3000ABXU/g to about 4000ABXU/g aptly at least about 2500ABXU/g to about 4500ABXU/g aptly).
In a preferred embodiment, the present invention's enzymatic compositions used can have following activity and (or substantially be made up of following activity, or be made up of following activity): the endoglucanase activity of the about 1000CMCU/g to about 3500CMCU/g using " endoglucanase activity determination method " to measure, the activity of beta-glucosidase of the about 300pNPGU/g to about 3000pNPGU/g using " activity of beta-glucosidase determination method " to measure, and the endo-xylanase of the about 2000ABXU/g to about 5000ABXU/g using " endo-xylanase activation measurement " to measure is active.
In another embodiment, the enzymatic compositions that the present invention is used can also have in following enzymatic activity one or more: proteinase activity (such as serine protease (E.C.3.4.21) and/or alkaline subtilisins activity (E.C.3.4.21.62)), pectinase activity (E.C.3.2.1.15), α-glucosiduronic acid enzymatic activity (E.C.3.2.1.139), beta-glucuronidase activity (E.C.3.2.1.31) or esterase active (E.C.3.1.1.73).
Esterase active can be ferulaic acid esterase activity (E.C.3.1.1.73).
Endoglucanase activity as referred to herein can be inscribe-Isosorbide-5-Nitrae-callose enzymatic activity.Endoglucanase is the enzyme of (1 → 4)-β-D-glycosidic bond endo hydrolysis in catalyse cellulose, lichenin and cereal β-D glucan.In other words, endoglucanase activity defined herein refers to the activity of the enzyme of (1 → 4)-β-D-glycosidic bond endo hydrolysis in cellulose, lichenin and cereal β-D glucan.According to E.C. classification, endoglucanase activity can be classified as E.C.3.2.1.4.The another name of endoglucanase is 1,4 beta-glucanase.
The endoglucanase used in the enzymatic compositions that the present invention is used can be one or more endoglucanases comprising the encoded by nucleic acid being selected from following one or more nucleotide sequences (or form by being selected from one or more following nucleotide sequences, or substantially form by being selected from one or more following nucleotide sequences): SEQIDNo.29, SEQIDNo.27, SEQIDNo.25, SEQIDNo.9 and SEQIDNo.7.
The endoglucanase used in the enzymatic compositions that the present invention is used can be one or more endoglucanases comprising and be selected from following one or more peptide sequences (or form by being selected from one or more following peptide sequences, or substantially form by being selected from one or more following peptide sequences): SEQIDNo.30, SEQIDNo.28, SEQIDNo.26, SEQIDNo.10 and SEQIDNo.8.
" endoglucanase activity determination method " (CMCU/g)
Pipette 1mL1% sanlose (CMC) solution (using the preparation of 0.05M sodium-acetate buffer), add respectively in sample cell and blank tube.Each pipe is placed in 50 DEG C of water-bath incubations 10 minutes.Pipette 1mL enzyme dilution every 15 seconds, add various kinds QC.Shake up sample cell after adding enzyme dilution at every turn.After 10 minutes, add the identical order of enzyme and arrangement of time according to various kinds QC, add 3mL1%3,5-dinitrosalicylic acid sodium-salt (DNS).3mLDNS is added to sample blank pipe.After adding DNS, each test tube is transferred on another rack for test tube be not in 50 DEG C of water-baths.Diluted for 1mL enzyme is added in respective sample blank tube.For each pipe is added a cover, boil 5 minutes whole.From 100 DEG C of water-baths, take out each pipe, place 10 minutes in ice bath.Stay at room temperature 10 to 15 minutes.Transfer in 3mL cuvette.Using reagent blank to be returned to zero by spectrophotometer, take deionized water as reference, reads each sample at 540nm place.
The enzymatic activity that this process measures is the activity relatively with the enzyme reference material of specifying CMC unit.1 μm of ol reduced sugar (representing with glucose equivalent), under the condition of 50 DEG C and pH4.8, was disengaged in one minute in CMC active unit.
In one embodiment, the dissolubility polysaccharide monooxygenase that the present invention is used can be the dissolubility polysaccharide monooxygenase to propose in Publication about Document: the people such as Levasseur, BiotechnologyforBiofuels2013,6:41; The people such as Kittle, BiotechnologyforBiofuels2012,5:79; These documents are incorporated herein by reference.
The dissolubility polysaccharide monooxygenase used in the enzymatic compositions that the present invention is used can be one or more dissolubility polysaccharide monooxygenase: SEQIDNo.31 and SEQIDNo.15 comprising the encoded by nucleic acid being selected from following one or more nucleotide sequences (or form by being selected from one or more following nucleotide sequences, or substantially form by being selected from one or more following nucleotide sequences).
The dissolubility polysaccharide monooxygenase used in the enzymatic compositions that the present invention is used can be one or more dissolubility polysaccharide monooxygenase: SEQIDNo.32 and SEQIDNo.16 comprising and be selected from following one or more peptide sequences (or form by being selected from one or more following peptide sequences, or substantially form by being selected from one or more following peptide sequences).
Cellobiohydrolase (CBH) activity can be CBHI class (CBHI) activity or CBHII class (CBHII) activity, or the combination of CBHI and CBHII.Desirably, (1 → 4)-β-D-glycosidic bond in cellobiohydrolase hydrolyzable cellulose and cellotetrose, from the non-reducing end release cellobiose of chain.Another term of cellobiohydrolase activity can be exocellobiohydrolase activity or cellulose Isosorbide-5-Nitrae beta fibers bioside enzymatic activity.According to E.C. classification, cellobiohydrolase II class activity can be classified as E.C.3.2.1.91.According to E.C. classification, cellobiohydrolase I class activity can be classified as E.C.3.2.1.176.
The cellobiohydrolase (CBH) used in the enzymatic compositions that the present invention is used can be one or more cellobiohydrolases comprising the encoded by nucleic acid being selected from following one or more nucleotide sequences (or form by being selected from one or more following nucleotide sequences, or substantially form by being selected from one or more following nucleotide sequences): SEQIDNo.23, SEQIDNo.21, SEQIDNo.19 and SEQIDNo.17.
The cellobiohydrolase (CBH) used in the enzymatic compositions that the present invention is used can be one or more cellobiohydrolases comprising and be selected from following one or more peptide sequences (or form by being selected from one or more following peptide sequences, or substantially form by being selected from one or more following peptide sequences): SEQIDNo.24, SEQIDNo.22, SEQIDNo.20 and SEQIDNo.18.
It is active that endo-xylanase activity can be inscribe-Isosorbide-5-Nitrae-beta-xylanase.Preferably, (1 → 4)-β-D-wood sugar glycosidic bond in endo-xylanase endo hydrolysis xylan.Preferably, endo-xylanase is classified as E.C.3.2.1.8.
The endo-xylanase used in the enzymatic compositions that the present invention is used can be one or more endo-xylanases comprising the encoded by nucleic acid being selected from following one or more nucleotide sequences (or form by being selected from one or more following nucleotide sequences, or substantially form by being selected from one or more following nucleotide sequences): SEQIDNo.33, SEQIDNo.11, SEQIDNo.5, SEQIDNo.4, SEQIDNo.2 and SEQIDNo.1.
The endo-xylanase used in the enzymatic compositions that the present invention is used can be one or more endo-xylanases comprising and be selected from following one or more peptide sequences (or form by being selected from one or more following peptide sequences, or substantially form by being selected from one or more following peptide sequences): SEQIDNo.34, SEQIDNo.12, SEQIDNo.6 and SEQIDNo.3.
" endo-xylanase activation measurement " (ABXU/g)
Pipette 1.8mL1% birch 4-O-methylglucuronic acid xylan substrate solution, add in every root test tube.50 DEG C of incubations 10 to 15 minutes, make substrate solution balance.Positive displacement pipette or equivalent of the apparatus is used to pipette 0.2mL enzyme dilution.Vortex mixes.50 DEG C of incubation every increment product 5 minutes are whole.Add 3mL1%3,5-dinitrosalicylic acid sodium-salt (DNS) solution and mix.With the top of each test tube of cap covers, avoid evaporating.Test tube is put in boiling water bath, stop 5 minutes whole.Each test tube is placed in ice/water bath cooling 10 minutes.Incubation at room temperature test tube 10 minutes.Being transferred to by tube contents in cuvette, take deionized water as reference, measures at 540nm place.Deduct the absorbance of corresponding enzyme blank, carry out the absorbance of correcting background look.This determination method measure endo-xylanase act on birchwood xylan substrate discharge the speed of reduced sugar.The reduced sugar rate of release using DNS to measure is directly proportional to enzymatic activity.
ABX unit is defined as under the condition of 50 DEG C and pH5.3, the enzyme amount needed for generation per minute 1 μm of ol wood sugar reduced sugar equivalent.
Activity of beta-glucosidase defined herein, disengages the activity of β-D-Glucose for hydrolysis end irreducibility β-D-glucosyl residue.According to E.C. classification, activity of beta-glucosidase can be classified as E.C.3.2.1.21.
The beta-glucosidase used in the enzymatic compositions that the present invention is used can be one or more beta-glucosidases comprising the encoded by nucleic acid being selected from following one or more nucleotide sequences (or form by being selected from one or more following nucleotide sequences, or substantially form by being selected from one or more following nucleotide sequences): SEQIDNo.35 and SEQIDNo.13.
The beta-glucosidase used in the enzymatic compositions that the present invention is used can be one or more beta-glucosidases comprising and be selected from following one or more peptide sequences (or form by being selected from one or more following peptide sequences, or substantially form by being selected from one or more following peptide sequences): SEQIDNo.36 and SEQIDNo.14.
" activity of beta-glucosidase determination method " (pNPGU/g)
Pipette 1mL3% nitrobenzophenone-β-D-glucopyranoside (pNPG) solution (using the preparation of 0.05M sodium-acetate buffer), add every increment product respectively with in the parallel test tube of contrast.Each pipe is placed in 50 DEG C of water-baths, stops 5 minutes.Every 15 to 30 seconds, 200 μ L contrasts or sample are added in corresponding parallel pipe.200 μ L sodium-acetate buffers are added in reagent blank pipe.After adding sample, vortex is respectively managed.The each pipe of incubation 10 minutes is whole.Incubation, after 10 minutes, adds 500 μ L1M sodium carbonate liquors, reaction is stopped.After adding sodium carbonate liquor, vortex is respectively managed, and is placed in by test tube on the rack for test tube outside water-bath.Add 10mLmilli-Q water to each pipe, vortex mixes.Use reagent blank to be returned to zero by spectrophotometer, read each sample at 400nm place, measure the concentration of 4-nitrophenols thus.
PNPG unit representation, under the condition of 50 DEG C and pH4.8, per minutely discharges 1 μm of ol nitrophenol from p-nitrophenyl-β-D-glucopyranoside.
Xylobiase activity can from the non reducing end hydrolysis continuous print xylose residues of (1 → 3)-β-D-xylan, and such as xylobiase can be 1,3 β-D-xylosidases.1,3 β-D-xylosidases can be classified as E.C.3.2.1.72 according to E.C. classification, or can catalysis (1 → 4)-β-D-xylan hydrolysis, remove continuous print D-xylose residues from irreducibility or reducing end under neutral, such as xylobiase can be Isosorbide-5-Nitrae xylobiase.Isosorbide-5-Nitrae xylobiase can be classified as E.C.3.2.1.37 according to E.C. classification.
" xylobiase activation measurement "
Xylobiase enzyme analytical plan is the front analytical plan (Ruttersmith proposed therewith, L.D., DanielR.M.1993.Thermostable β-glucosidaseand β-xylosidasefromThermotogasp.strainFjSS3-B.1.Biochim.Bioph ys.Acta.1156:167-172, the instruction content of the document is incorporated herein by reference) similar, but have some to revise.
Xylobiase activation measurement according to the present invention is implemented according to the following step: the substrate of xylobiase is p-nitrophenyl-β-D-xylopyranose glucosides (pN β xp) (Sigma – N2132).For the enzyme of each test sets up dose response curve.Under 7 kinds of dosage, (100ppm, 50ppm, 25ppm, 12.5ppm, 6.25ppm, 3.13ppm, 1.56ppm) tests each enzyme, wherein only comprises substrate and analysis buffer without enzyme control sample.All solution all uses 50mM sodium-acetate buffer to prepare.Standard reaction mixture comprises 280 μ L0.1M sodium-acetate buffer (pH5.0, ultimate density is 50mM) and the 80 μ L substrate solutions (ultimate density is 1mM) prepared by 50mM sodium-acetate buffer, then add the enzyme of the various dose concentration prepared by 50mM sodium-acetate buffer, the final volume of reactant mixture is settled to 400 μ l.Reactant mixture (buffer solution and substrate) is placed in 70 DEG C of water-bath preheatings, then adds enzyme.Add enzyme and start reaction.By sample mixture incubation 10 minutes, then add the Na of 0.8ml0.1M 2cO 3solution makes reaction stop.Use molar extinction coefficient ε 400=18,300M -1cm -1calculate the paranitrophenol concentration of release.Absorbance containing enzyme hole is deducted the absorbance of control group, then convert gained absorbance (A400) to concentration value according to analytical plan.Active unit is defined as per minutely under analysis condition generating 1 μm of ol paranitrophenol by pN β xp.
Araban can be hydrolyzed to Arabinose by α-L-arabinofuranosidase (E.C.3.2.1.55).
" α-L-arabinofuranosidase activation measurement "
This analytical plan is the front α-L-arabinofuranosidase activity analysis scheme (Miyazaki proposed therewith, K. (2005) Hyperthermophilic α-L-arabinofuranosidasefromThermotogamaritimaMSB8:molecula rcloning, geneexpression, andcharacterizationoftherecombinantprotein.Extremophiles 9 (5): 399-406, the instruction content of the document is incorporated herein by reference) similar, but have some to revise.
α-L-arabinofuranosidase activation measurement according to the present invention is implemented according to the following step: the substrate of α-L-arabinofuranosidase is p-nitrophenyl-α-L-arabinofuranosidase (pN α Laf) (Sigma-N3641).For the enzyme of each test sets up dose response curve.Under 7 kinds of dosage, (100ppm, 50ppm, 25ppm, 12.5ppm, 6.25ppm, 3.13ppm, 1.56ppm) tests each enzyme, wherein only comprises substrate and analysis buffer without enzyme control sample.All solution all uses 50mM sodium-acetate buffer to prepare.Standard reaction mixture comprises 280 μ L0.1M sodium-acetate buffer (pH5.0, ultimate density is 50mM) and the 80 μ L substrate solutions (ultimate density is 1mM) prepared by 50mM sodium-acetate buffer, then add the enzyme of the various dose concentration prepared by 50mM sodium-acetate buffer, the final volume of reactant mixture is settled to 400 μ l.Reactant mixture (buffer solution and substrate) is placed in 70 DEG C of water-bath preheatings, then adds enzyme.Add enzyme and start reaction.By reactant mixture incubation 10min, then add the Na of 0.8ml0.1M 2cO 3solution makes reaction stop.At 400nm place, use molar extinction coefficient ε=10,500M -1cm -1calculate the paranitrophenol concentration (Miyazaki, 2005) of release.Absorbance containing enzyme hole is deducted the absorbance of control group, then convert gained absorbance (A400) to concentration value according to analytical plan.Unit of enzyme activity is defined as per minutely under analysis condition generating 1 μm of ol paranitrophenol by substrate.
It is active that term used in the linguistic context of the present invention's enzymatic compositions activity used " substantially by ... composition " refers to that enzymatic compositions has one or more through characterizing, and do not have other enzymatic activity and/or do not have the enzymatic activity that other can digest lignocellulose biomass.
Only for example, following enzyme may be applicable to used according to the invention.
In one embodiment, the enzymatic compositions that the present invention is used can be the combination (such as enzymatic mixture) of single enzyme or multiple enzyme.
In a preferred embodiment, enzymatic compositions according to the present invention is enzymatic mixture.
In one embodiment, each different enzymatic activity defined herein is preferably provided by independent protein.In other words, often kind of enzymatic activity belongs to different zymoproteins.The enzymatic activity of definition is preferably main (or unique) activity of protein.In other words, the enzymatic activity of definition is not preferably the pair activity of protein.
living beings
Term " lignocellulosic " refers to and comprises lignin and cellulosic composition.Ligno-cellulosic materials also can comprise hemicellulose.
Term " cellulose " refers to the composition comprising cellulose and annexing ingredient (comprising hemicellulose).
In one embodiment, lignocellulose biomass is any one cellulose or ligno-cellulosic materials.Term " lignocellulose biomass " refers to any one ligno-cellulosic materials, and comprises containing cellulosic material.Lignocellulose biomass optionally also can comprise hemicellulose, lignin, starch, compound sugar and/or monose.
Living beings also can comprise annexing ingredient, such as protein, lipid, ash content and/or extract.Living beings can be derived from single source, or living beings can comprise the mixture being derived from more than one sources; Such as, living beings can comprise the mixture of corn ear and maize straw, or the mixture of grass and blade.
Preferably, lignocellulose biomass comprises at least 15% cellulose.
In one embodiment, lignocellulose biomass comprises at least 20% cellulose.
Lignocellulose biomass can be any one cellulose or ligno-cellulosic materials, such as the combination of agricultural residue, bioenergy crops, industrial solid wastes, municipal solid waste, sludge, yard waste, timber refuse, forestry waste and these materials from papermaking.
In one embodiment, lignocellulose biomass can be selected from corn ear, crop residue is as maize peel, maize straw, grass, beet pulp, wheat stalk, wheat shell, oat straw, wheat time powder, wheat prostitute, rice bran, rice husk, wheat bran, oat shell, palm kernel, citrus pulp, cotton, lignin, Barley straw, hay, rice straw, rice husk, switchgrass, Chinese silvergrass, Value of Spartina Anglica, reed canary grass, paper discarded object, bagasse, Chinese sorghum slag, forage sorghum, broomcorn straw, soybean stalk, soybean, pulverize the component that trees obtain, branch, root, leaf, wood chip, sawdust, shrub and bushes, vegetables, fruit, flower.
In one embodiment, lignocellulose biomass can be selected from moist forage cake, zein fiber, corn germ cake, corn bran, hominy grits slag, maize gluten feed, cereal wheat gluten, wheat prostitute, wheat time powder, based on distiller's dried grain (DDG) and the distiller's dried grain DDGS (DDGS) of (such as) corn, wheat, Chinese sorghum, or the combination of these materials.
In a preferred embodiment, lignocellulose biomass can be selected from maize straw, wheat stalk, corn ear, bagasse, switchgrass, forage sorghum and rice straw.
In another embodiment, lignocellulose biomass can be selected from maize straw, wheat stalk, corn ear, bagasse, switchgrass, forage sorghum and rice straw.
In one embodiment, living beings used in the present invention comprise the living beings with relative Hi CHO value, and this living beings are relatively fine and close, and/or are relatively easy to collect, transport, store and/or process.
In one embodiment, lignocellulose biomass can comprise and be less than 50% starch, is preferably less than 40% starch.
In one embodiment, lignocellulose biomass can comprise and be less than 30% starch, is preferably less than 10% starch.
In one embodiment, lignocellulose biomass can comprise and be less than 3% starch.
In one embodiment, lignocellulose biomass can comprise and be less than 1% starch.
Desirably, lignocellulose biomass does not comprise starch.
In one embodiment, lignocellulose biomass does not comprise cereal.
pretreatment
In one embodiment, adopt any preprocess method preprocessing lignocellulose living beings known in the art, this can destroy the compact texture of lignocellulose biomass, exposed fibers cellulose fiber, and/or cause living beings degree of crystallinity (such as cellulose crystallity) to decline, and/or the accessible surface increasing enzymatic activity amasss.
In the past in decades, develop multiple preprocess method.In general, these pretreatment can be divided into machinery/physics pretreatment, physical chemistry pretreatment, Chemical Pretreatment and Biological Pretreatment, or these pretreated combinations.Various pretreated general introduction is found in (such as) XZhao, LZhang, DLiuReview:Fundamentalsofdifferentpretreatmentstoincreas etheenzymaticdigestibilityoflignocelluloses, Biofuels, Bioprod.Bioref.6:561 – 579 (2012), the instruction content of the document is incorporated herein by reference.
Pretreatment can be any process known in the art, described process or cause cellulose crystallity to reduce, as adopted the method (people such as Segal of Segal, " AnEmpricalMethodforEstimatingtheDegreeofCrystallinityofN ativeCelluloseUsingtheX-RayDiffractometer ", TextileResearchJournal, Oct.1959, Vol.29, No.10,786-794, the instruction content of the document is incorporated herein by reference) record; The accessible surface increasing enzymatic activity amasss; Or realize this two kinds of effects simultaneously.
In one embodiment, described pretreatment is the pretreatment reducing cellulosic degree of crystallinity in lignocellulose biomass.
In one embodiment, described pretreatment is, when using Segal method (aforementioned) to measure, degree of crystallinity cellulosic in lignocellulose biomass is reduced the pretreatment at least about 20%.
In one embodiment, described pretreatment is the pretreatment that the accessible surface increasing enzymatic activity in lignocellulose biomass amasss.
In one embodiment, described pretreatment be the accessible surface in lignocellulose biomass is amassed increase at least about 20%, aptly at least about 30%, aptly at least about 40% pretreatment.Accessible surface is long-pending can adopt BET (Brunauer, Emmett and Teller) to measure, the people such as Guo, BioresourceTechnology99 (2008) 6046-6053 (this bibliography is incorporated herein by reference) teaches this determination method.
Brunauer, Emmett and Teller (BET) analytic approach can be adopted to measure the useable surface area of lignocellulose biomass, and wherein said method comprises (i) and weighs 0.5g dry-eye disease; (ii) use room temperature (such as 25 DEG C) high pure nitrogen (99.999%) to sample degassing overnight; (iii) use Surface Area Analyzer (QuantachromeNOVA2000) to perform BET to analyze.
In one embodiment, lignocellulose biomass can stand (or subjected to) physics and/or chemistry and/or carry out a biological disposal upon.This process can be described as physics and/or chemical and/or biological method pretreatment in this article.
In one embodiment, can be physical treatment or chemical treatment or physical chemistry process according to physics of the present invention and/or Chemical Pretreatment.
Term " physics and chemistry process " and " physical chemistry process " are used interchangeably in this article.
In one embodiment, lignocellulose biomass can stand (or subjected to) physics and/or Chemical Pretreatment.
In one embodiment, lignocellulose biomass can stand the pretreatment of (or subjected to) physics.
In one embodiment, lignocellulose biomass can stand (or subjected to) Chemical Pretreatment.
In one embodiment, lignocellulose biomass can stand the pretreatment of (or subjected to) physical chemistry.
Physics and/or chemistry and/or biological treatment can be any physics known in the art and/or chemistry and/or biological treatment (such as, pretreatment).
machinery/physics pretreatment
Machinery/physics pretreatment refers to mechanical milling processes chip, grinding, dry grinding, wet-milling, vibratory milling, rotation ball milling combined.The size of raw material after chip is generally 10mm to 30mm, and the size after grinding or grinding is generally 0.2mm to 2mm, and cellulose crystallity declines and/or the long-pending increase of accessible surface.With hot water, acid or niter cake, pretreatment in early stage is carried out with softening raw material to biomass solid, grinding can be made to consume energy less.In one embodiment of the invention, the method preprocessing biomass adopting machinery to decrystallize.
Term " physics pretreatment " and " mechanical pretreatment " are used interchangeably in this article.
physical chemistry pretreatment
The chemical modification of biomass compositions and the physical rupture of cell wall structure combine by physical chemistry pretreatment.A kind of physical chemistry pretreatment of practicality is steam blasting.In this pretreatment, process living beings with high-pressure saturated steam (high pressure and high temperature), optionally comprise the interpolation of acid, alkali or other chemicals; Then rapid depressurization, makes material stand explosive decompression.
In one embodiment, physics and/or chemical treatment (such as, pretreatment) can be aquathermolysis or wet oxidation, and comprise: the high pressure provided with aqueous water and/or steam and/or high temperature, optionally comprise the interpolation of acid, alkali or other chemicals.
In one embodiment, pressure in 300psi to 600psi scope, preferably in 350psi to 550psi scope, preferably in 400psi to 500psi scope.
In one embodiment, high temperature means temperature at about 100 DEG C within the scope of 300 DEG C, preferably at about 140 DEG C within the scope of 240 DEG C, such as at about 170 DEG C within the scope of 200 DEG C.
Only for example, pretreatment can be the physical treatment comprising steam blasting.
With regard to cellulose crystallity, after steam blasting, the crystallinity index (CrI) of substrate increases.Crystallinity index (CrI) is taught in the document (aforementioned) of Segal.
Physical chemistry another example pretreated is ammonia fiber explosion (AFEX), and wherein ligno-cellulosic materials soaks into through liquefied ammonia, is then warming up to about 90 DEG C.Do not wish to be bound by theory, the ammonia formed and living beings under stress (such as 17 bar to 20 cling to and continue 5 minutes to 10 minutes) interact, then rapid release pressure, this may cause, and cellulose decrystallizes, hemicellulose prehydrolysis and lignin structure changes and/or accessible surface is long-pending increases.
AFEX pretreatment condition slightly change can cause forming different cellulose crystals structures, so crystallinity index (CrI) may affect by AFEX pretreatment condition.
Another kind of practical physical chemistry preprocess method is liquid heat water pretreatment, and this pretreatment uses water as medium preprocessing biomass under stress, and the object of adherence pressure makes water at high temperature still keep liquid.Usually also this pretreatment is called aquathermolysis or hydrothermal pretreatment.About 40% to 60% of the total living beings of this pretreatment solubilized, form liquid soluble oligosaccharide, and these total living beings of dissolving comprise 4% to 22% cellulose and almost whole hemicellulose.Therefore, can be observed the long-pending increase of accessible surface.In addition or alternatively, can be observed that cellulose crystallity declines, cellulose reduces and cellulose depolymerization with the associating intensity of lignin, these factors all help lifting cellulose accessibility.
In one embodiment, pretreatment can be the physical chemistry pretreatment comprising aquathermolysis or hydrothermal treatment consists.
In one embodiment, lignocellulose biomass is wheat stalk, and pretreatment comprises use hydrothermal treatment consists.Desirably, can comprise the hydrothermal treatment consists proposed in WO2011/125056 (IBICON) according to pretreatment of the present invention, the instruction content of this patent is incorporated herein by reference.
Radiation pretreatment relates to the multiple method using γ radiation, ultrasonic, electron beam or Microwave Pretreatment lignocellulose biomass.Radiation pretreatment also can be arranged in pairs or groups with other pretreatment and be used, to improve cellulosic accessibility further.Do not wish to be bound by theory, because cellulose, hemicellulose and lignin, under high dose of radiation, radiation degradation occurs, so substrate becomes frangible.Cellulose crystallity may by γ radiation destruction to a certain extent.Microwave Pretreatment is carried out usually in the environment having water, organic solvent, alkali or dilute acid soln.
chemical Pretreatment
Chemical Pretreatment relates to the multiple method using different chemical product preprocessing biomass under various conditions.Different with pretreatment condition according to the chemicals used, these pretreated mechanism are different.The example of suitable process for chemically pretreating include, but not limited to dilute acid pretreatment, oxygenation pretreatment, sulphite pretreatment, oxidation pre-treatment (such as wet oxidation), cellulose solvent pretreatment, ammonia diafiltration (APR) and organic solvent pretreatment.
In one embodiment, pretreatment can be the chemical treatment comprising and use acid or alkali treatment.
Only for example, physics and/or chemical treatment can be interpolation base catalyst, or other method known in the art.
In one embodiment, lignocellulose biomass is maize straw, and physics and/or chemical treatment comprise use acid treatment.
In one embodiment, pretreatment is dilute acid pretreatment.Such as, lignocellulose biomass material and diluted acid (can be generally H 2sO 4) and water mixing to form slurries, with being steam heated to temperature required (usually between 160 DEG C and 220 DEG C), and after one period of time of staying, be flashed to atmospheric pressure.
In a specific embodiment, pretreatment can comprise following step: a) use 0.2%H 2sO 4dipping lignocellulose biomass (such as wheat stalk), such as, with immersion way process; B) pressed material, makes dry matter content between 40% to 50%; And c) with 190 DEG C of steam pre-treatment 10min.Optionally obsession obtains slurries, filtering liquid; And optionally clean solid residue two to three times, suppress afterwards, make final solids content become about 40% to 50%.
In another specific embodiment, pretreatment can comprise following step: a) flood lignocellulose biomass (such as wheat stalk) with 1% acetic acid with immersion way; B) pressed material, makes dry matter content between 40% to 50%; And c) with 200 DEG C of steam pre-treatment 10min.Optionally suppress follow-up slurries, filtering liquid; And optionally clean solid residue two to three times, suppress afterwards, make final solids content become about 40% to 50%.
Oxygenation pretreatment may relate to use various alkali, such as NaOH, KOH, CaOH 2, ammoniacal liquor, peroxide and lime (utilizes CaOH 2), carry out treatment of lignocellulosic biomass, to carry out pretreatment to lignocellulose biomass.Do not wish to be bound by theory, it is believed that during oxygenation pretreatment, the intermolecular ester bond generation saponification of crosslinked xylan hemicellulose and lignin, thus cause living beings delignification.It is documented, alkali is used to swollen cellulose and changes cellulose crystallite polymorph, thus affects the suitable agent of cellulose digestibility, and wherein the change of cellulose crystallite polymorph shows that crystallization hydrogen bond network changes.
In one embodiment, physics and/or chemical treatment can be the chemical treatment comprising and use alkali (such as ammonia) to process.
In one embodiment, pretreatment is anhydrous ammonia pretreatment.
In a specific embodiment, pretreatment can be anhydrous ammonia pretreatment, comprise the following steps: a) lignocellulose biomass material (such as maize straw) is put into container, for making anhydrous ammonia can permeate biological material more swimmingly, this container emptying of having exerted pressure; B) at 140 DEG C, biological material and the contact of the aqueous solution comprising 12% ammonia is implemented; C) the sustainable maximum 25h of ammonia pretreatment, but the best release also just may observing glucose and xylose in the shorter time (as 15 minutes) afterwards; D) apply vacuum, remove extra ammonia spirit, obtain the dry that final solids content is 50% to 60%.
Or, physical chemistry process can be the use ammonia described in WO2006/110891, WO2006/11899, WO2006/11900 and WO2006/110901 (DuPont) (such as, rare ammonia) preprocess method, the instruction content of these documents is incorporated herein by reference.
In one embodiment, pretreated living beings are the maize straw (DaCS) crossed with rare ammonia pretreatment.
In one embodiment, pretreatment is causticity delignification.
In one embodiment, pretreatment can be the pretreatment of causticity delignification, comprise the following steps: 1) between 25 to 200 DEG C and under the condition of pH in 9 to 11 scopes, process living beings 0.25h to 20h with the nucleophilic alkali (such as NaOH (NaOH), lithium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide or their combination) between 0.5% to 3%; 2) reclaim pretreated living beings filtrate, and use washed with de-ionized water, remove the accessory substance of any excessive alkali and dissolving.
In a specific embodiment, pretreatment can be the pretreatment of causticity delignification, comprise the following steps: 1) under the condition of pH in 9 to 11 scopes, process living beings 0.25h to 1.5h with the NaOH (NaOH) between 0.5% to 3% at about 121 DEG C; 2) reclaim pretreated living beings filtrate, and use washed with de-ionized water, remove the accessory substance of any excessive alkali and dissolving.
The people such as Xu (2011) Bioresources6 (1) 707-720 (this bibliography is incorporated herein by reference) teaches a kind of method performing causticity delignification.In one embodiment, pretreated living beings can be the living beings with the causticity delignification method process proposed in the people such as aforementioned Xu (2011).
In one embodiment, pretreated living beings are causticity delignification maize straw (DLcs) and/or causticity delignification switchgrass (DLswg).
In one embodiment, pretreated living beings are causticity delignification maize straw (DLcs) and/or causticity delignification switchgrass (DLswg).
In one embodiment, sulphite pretreatment or sulfite pulping process comprise and carry out sulphite process to biological material in acid condition, carry out mechanicalness subsequently reduce size with disk refining (diskrefining).Sulphite pretreatment makes a large portion hemicellulose be removed, and cellulosic degree of polymerization reduces, and makes Sulphonation of Lignin, increases lignin hydrophily.
Another kind of Chemical Pretreatment can be oxidation pre-treatment, refers to the method using oxidant to remove delignification and reducing substances.The oxidant being usually used in being oxidized delignification comprises ozone, hydrogen peroxide, oxygen and peracetic acid.This process optionally can be combined with other chemistry or hydrothermal treatment consists.Different with reaction condition (such as pH) according to the oxidant used, the mechanism of lignin oxidation's degraded is different.With regard to ozone decomposed, wet oxidation and peracid delignification, parent's electricity that aromatic series and alkene race structural degradation relate to oxidant initial is attacked, but at alkaline H 2o 2between pre-treatment period, these structures are destroyed by the nucleophillic attack of hydroperoxides anion.
In one embodiment, physics and/or chemical treatment are wet oxidation.
In an available embodiment, wet oxidation relates to the high pressure and/or high temperature that provide with aqueous water and/or steam, optionally comprises the interpolation of acid, alkali or other chemicals.
High pressure may mean that pressure is in 300psi to 600psi scope, preferably in 400psi to 500psi scope, and such as about 450psi.
High temperature may mean temperature about 100 DEG C within the scope of 300 DEG C, preferably about 180 DEG C to 200 DEG C within the scope of keep 5 minutes to 15 minutes (SchmidtandThomsen, 1998, BioresourceTechnol.64:139-151, the instruction content of the document is incorporated herein by reference).In one embodiment, high temperature may be about 140 DEG C to 235 DEG C.
Desirably, wet method explosion is the modification of wet oxidation preprocess method, is wherein combined with wet oxidation as above and steam blasting.In wet method explosion, after the specific time of staying, introduce oxidant during pre-processing.Then by being flashed to atmospheric pressure, termination (WO2006/032282, this patent is incorporated herein by reference) is carried out.
Only by way of example, in one embodiment, physics and/or Chemical Pretreatment can relate to the high pressure and/or high temperature that provide with aqueous water, and wherein water is with liquid and vapour mixture existence, optionally comprise the interpolation of acid, alkali or other chemicals.High pressure in this embodiment can be in the scope of 50psi to 300psi, be suitably 100psi to 200psi scope in, be such as about 150psi.High temperature in this embodiment can be in about 100 DEG C in the scope of 300 DEG C, is about 170 DEG C to 220 DEG C suitably, such as, from about 170 DEG C to 200 DEG C.
Another available Chemical Pretreatment is organic solvent pretreatment, and it extracts 30-60 minute and by cellulose biomass material delignification by using hydrous ethanol (such as 40% to 60% ethanol) at 160-200 DEG C.Sulfuric acid can be added as catalyst.In organic solvent pretreatment, most hemicellulose can be removed.
The pretreatment of cellulose solvent type is also the Chemical Pretreatment processed lignocellulose biomass based on cellulose solvent (such as phosphoric acid (CPA) and ionic liquid (IL)).
biological Pretreatment
In one embodiment, (such as Biological Pretreatment) can be carried out a biological disposal upon to lignocellulose biomass.This type of biological treatment can be any biological treatment known in the art.
In Biological Pretreatment, microorganism can be utilized to carry out pretreatment to improve the enzymic digestion rate of remaining solid to living beings.The microorganism used usually can lignin degrading and carbohydrate polymer.Such as, the fungi of some types can produce lignocellulolyticenzymes (these enzymes can Synergistic degradation plant cell wall), and the fungi of other type can produce hydrogen peroxide.
After Biological Pretreatment, accessible surface is long-pending can be increased, thus improves cellulosic digestibility.
Only by way of example, Biological Pretreatment can comprise use white-rot fungi process.More known white-rot fungis are Phanerochaete chrysosporium (Phanerochaetechrysosporium) such as, worm intends wax bacterium (Ceriporiopsissubvermispora), Phlebiasubserialis and Produced from Pleurotus ostreatus (Pleurotousostreatus) can (see by the lignin effectively in the multiple ligno-cellulosic materials of metabolism, such as SinghD and ChenS. " Thewhite-rotfungusPhanerochaetechrysosporium:conditionsf ortheproductionoflignin-degradingenzymes. ", " AppliedMicrobiologyBiotechnology ", 2008, 81st volume, 399-417 page, the instruction content of the document is incorporated to herein by reference).
Before pre-processing (such as before physics and/or chemistry and/or biological method pretreatment), methods known in the art also can be used to carry out the process such as granularity reduction, pre-soaking, wetting, washing or conditioning to cellulosic material.
Method of the present invention also can comprise the step contacted with feed addictive composition according to the present invention by feed ingredient.
The present invention also relates to method and the purposes of improving animal organism physical characteristic.
Preferably, can obtain (such as by the inventive method to detoxification, obtain) feed addictive composition, or according to feed addictive composition of the present invention, or according to pre-composition of the present invention, or according to feedstuff of the present invention, or the feedstuff of (such as, obtaining) can be obtained by the inventive method.
Biophysical properties can be selected from following one or more: animal performance, growth of animal performance, food conversion ratio (FCR), the raw material digestion power (digestibility of such as nutriment, comprise starch digestibility, fat digestibility, protein digestibility, digestibility of fiber), nitrogen retention rate, carcass yield, growth rate, weightening finish, body weight, quality, feed efficiency, body fat percentage, the distribution of body fat, growth, egg size, egg size amount, egg quality, laying rate and ambient influnence, such as muck output and/or nitrogen excretion.
In the present invention, will through physics and/or the chemical and/or pretreated lignocellulose biomass of biological method incubation together with enzymatic compositions.
Aptly, the duration of this type of incubation is 6 little of 120 hours, be preferably 10 little up to 60 hours, be preferably 20 little up to 50 hours, be preferably 35 little up to 50 hours, be preferably 40 little of 48 hours.
In certain embodiments, incubation period can be 4 little of 72 hours.In one embodiment, incubation period is 6 little of 48 hours.In one embodiment, incubation period is at least about 6 hours.In another embodiment, incubation period is less than 72 hours.It will be understood by those of skill in the art that incubation period is by the impact by dry addition, when dry addition increases, incubative time must increase to reach identical effect pari passu.
Aptly, described incubation can make at the end of incubation period, and in living beings, the pentose (wood sugar and arabinose) of about 5 % by weight to 20 % by weight (being preferably about 10 % by weight to 13 % by weight) and the sugar of about 1 % by weight to 10 % by weight (being preferably about 1 % by weight to 4 % by weight) are oligomer and polymer form.
Aptly, described incubation can make at the end of incubation period, and the wood sugar of the glucose (wood sugar and arabinose) of about 10 % by weight to 29 % by weight (being preferably about 16 % by weight to 21 % by weight) and about 1 % by weight to 10 % by weight (being preferably about 4 % by weight to 7 % by weight) is monomeric unit.
The duration of incubation period can be depending on enzyme used and/or enzyme concentration used.The object of incubation period makes enzyme degradation biological matter fully.For example, by those skilled in the art such as by adopting the reduction of the insoluble living beings of weight analysis determining after the enzymatic treatment, can determine whether living beings fully degrade.
In one embodiment, dry according to product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff.
Term " drying " refers to be reduced to according to the water content (% by weight) of product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff and is less than 15 % by weight, is preferably less than 10 % by weight.
Therefore, present invention also offers according to the product of drying of the present invention or substantially dry product and/or feed addictive composition and/or pre-composition and/or feedstuff, its water content, lower than 30 % by weight, is preferably lower than 15 % by weight, is preferably lower than 10 % by weight, is preferably lower than 5 % by weight.
In certain embodiments, the present invention can provide semi liquid state product or pasty products.According to the water content of semi liquid state product of the present invention or pasty products lower than 90 % by weight, be preferably lower than 80 % by weight, be preferably lower than 70 % by weight or more preferably lower than 60 % by weight.
In one embodiment, product according to the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff are packed, and/or under being stored in dry or substantially dry state.
Term used herein " dry " or " drying regime " refers to according to product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff is not moisture or only contain the water seldom measured.In other words, term used herein " dry " or " drying regime " can to refer to according to the water content (% by weight) of product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff lower than 5 % by weight, be preferably lower than 1 % by weight.
Term used herein " substantially drying regime " refers to according to product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff only containing the water of little amount.In other words, term used herein " substantially drying regime " can to refer to according to the water content (% by weight) of product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff lower than 30 % by weight, is preferably lower than 15 % by weight, is preferably lower than 10 % by weight.
In one embodiment, according to the water content of product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff lower than 20 % by weight.
In another embodiment, according to the water content of product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff lower than 15 % by weight.
In another embodiment, according to the water content of product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff lower than 10 % by weight.
In another embodiment, according to the water content of product of the present invention and/or feed addictive composition and/or pre-composition and/or feedstuff lower than 5 % by weight.
Preferably, the xylose units be present in oligomer or polymer form in material of at least 70%, preferably at least 60% is comprised according to product of the present invention and/or feed addictive composition.
The present invention's enzymatic compositions used preferably generates to have and comprises wood sugar, rhamnose, glucose, mannose, galactolipin, glucuronic acid, the soluble oligomer of sugar unit of galacturonic acid and soluble polymer (such as xylo-oligosaccharide, araboxylan oligomer) in product according to the present invention and/or feed addictive composition.
In a preferred embodiment, enzymatic compositions can not discharge, maybe can not generate or discharge the C-5 sugar (such as wood sugar) of a large amount of monomeric form.
Preferably, the total C-5 sugar (such as wood sugar) being less than 50% in feed addictive composition and/or feed ingredient exists with monomeric form.
Inventor has found, by using the enzymatic compositions that there is not or substantially do not exist xylobiase activity and/or α-arabinofuranosidase activity, C-5 sugar (such as wood sugar and/or arabinose and/or arabinofuranose) content in feed addictive composition according to the present invention to be maintained reduced levels.
Also can comprise to feed addictive composition described in detoxification according to method of the present invention.
In one embodiment, imagine method of the present invention and also comprise feed ingredient is mixed with feed addictive composition, thus such as obtain a kind of feed or feedstuff.
The present invention relates to by effective dose is applied to animal according to feed addictive composition of the present invention or the feed addictive composition prepared by method of the present invention of effective dose or the feedstuff comprising this type of feed addictive composition of effective dose, thus improve purposes and the method for animal organism physical characteristic.
Term used herein " biophysical properties " refer to be selected from following group one or more: animal performance, growth of animal performance, feed conversion rate (FCR), raw material digestion power (such as nutritive digestibility, comprise starch digestibility, fat digestibility, protein digestibility, digestibility of fiber), nitrogen retention rate, carcass yield, growth rate, weightening finish, body weight, quality, feed efficiency, body fat percentage, body fat distributes, growth, egg size, egg size amount, egg quality, laying rate and ambient influnence, such as muck output and/or nitrogen excretion.
Term used herein " improvement " refers to and not to make moderate progress according to the lignocellulose biomass of process of the present invention compared to detoxification.
For guaranteeing that lignocellulose biomass is degraded effectively, in living beings, enzyme can be added with suitable concentration.In certain embodiments, the consumption of enzyme can add in the scope of about 2.5g to 20kg activated protein in pretreated living beings per ton, adds about 5g to 14kg activated protein suitably in pretreated living beings per ton.
Only by way of example, suppose to add 5% to 60% living beings in per metric ton (MT) feed, then in per metric ton feed, activated protein is per metric ton feed 0.125g to 12kg.
In one embodiment, in feed enzyme amount can in per metric ton feed about 0.125g to about between 12kg (be that in per metric ton feed, about 500g, to about between 10kg, is about 1kg extremely about between 8kg in per metric ton feed suitably suitably).
In imagination feedstuff, treated biomass content can be change.In one embodiment, treated in feed biomass content be lower than total feedstuff 90 % by weight, be preferably lower than 80 % by weight, suitably for lower than 70 % by weight, suitably for lower than 60 % by weight, suitably for lower than 50 % by weight, suitably for lower than 40 % by weight.Preferably, treated in feedstuff biomass content is lower than 60 % by weight.
In one embodiment, biomass content treated in feed higher than 30 % by weight of total feedstuff, preferably higher than 5 % by weight, preferably higher than 10 % by weight, preferably higher than 20 % by weight, preferably higher than 30 % by weight, preferably higher than 40 % by weight, suitably higher than 50 % by weight, suitably higher than 60 % by weight.Preferably, treated in feedstuff biomass content is higher than 5 % by weight or 10 % by weight.
In another embodiment, biomass content treated in feed is in the scope of about 5 % by weight to 70 % by weight of total feedstuff, suitably in the scope of 5 % by weight to 60 % by weight, suitably in the scope of 5 % by weight to 50 % by weight, suitably in the scope of 10 % by weight to 40 % by weight.
In another embodiment, treated in feed biomass content is in the scope of 10 % by weight to 70 % by weight.
In another embodiment, treated in feed biomass content is in the scope of 10 % by weight to 40 % by weight.
In one embodiment, " mixing " used herein comprises any mixed method, such as, stir, merge, sprinkling etc.
In one embodiment, enzymatic compositions, before mixing with lignocellulose biomass (such as through physics and/or chemistry and/or the pretreated lignocellulose biomass of biological method), can be dryness enzyme preparation (such as granular form or be positioned at (such as wheat carrier) on carrier).In another embodiment, enzymatic compositions, before mixing with lignocellulose biomass (such as through physics and/or the chemical and/or pretreated lignocellulose biomass of biological method), can be liquid preparation.
When enzyme was liquid preparation before mixing with lignocellulose biomass (such as through physics and/or the chemical and/or pretreated lignocellulose biomass of biological method), mix this enzyme by such as spraying enzyme preparation or being immersed in enzyme preparation by lignocellulose biomass (such as through physics and/or the chemical and/or pretreated lignocellulose biomass of biological method).
(before or after ferment treatment) through grinding and/or powder process and/or can form meal through the living beings of physics and/or chemistry and/or Biological Pretreatment and/or drying solid part.
animal
Term used herein " animal " refers to will be used or use according to feed addictive composition of the present invention or the animal of feedstuff comprising feed addictive composition according to the present invention.
Preferably, animal is mammal, birds, fish or shellfish, comprises such as livestock or performing animal (such as pet).
In one embodiment, " animal " is livestock.
Term used herein " livestock " refers to any cultivated animals.Preferably; livestock is one or more in following animal: milk cow or bull (comprising calf), pig (comprising piglet, live pig), poultry (comprising broiler chicken, laying hen, chicken and turkey), bird, fish (comprise fresh-water fishes; such as salmon, cod, trout and carp (such as bright and beautiful carp) and ocean fish, such as sea bass), shellfish (such as shrimp, mussel and scallop), horse (comprising horse racing), sheep (comprising lamb).
In another embodiment, " animal " is domesticated animal in performing animal or pet or zoo environment.
Term used herein " performing animal or pet or zoo environment in domesticated animal " refers to any relevant animal, comprise canid (as dog), cats (as cat), rodent (as mouse, rat, mouse), bird, fish (comprising fresh-water fishes and ocean fish), and horse.
In one embodiment, animal is nonruminant.In a preferred embodiment, nonruminant can be poultry or pig (or their combination).
In another embodiment, animal is ruminant.
packaging
In one embodiment, enzymatic compositions according to the present invention and/or feed addictive composition and/or feed ingredient and/or pre-composition and/or feed or feedstuff are packed.
In a preferred embodiment, enzymatic compositions and/or feed addictive composition and/or feed ingredient and/or pre-composition and/or feed or feedstuff are packaged in bag (such as paper bag).
In an alternative embodiment, enzymatic compositions and/or feed addictive composition and/or feed ingredient and/or pre-composition and/or feed or feedstuff can be sealed in container.Any suitable container can be used.
feed
Feed addictive composition of the present invention can be used as feed, or can be used for preparing feed.
Term used herein " feed " and " feedstuff " are synonyms.
Feed can be the form of solution or solid, and this depends on its purposes and/or application model and/or mode of administration.
When composition of the present invention be used as feed or for the preparation of feed (such as functionalization feed) time, said composition can be combined with following one or more: acceptable adjuvant, neutraceutical active ingredients in acceptable auxiliary material, nutrition in acceptable diluent, nutrition in acceptable carrier, nutrition in nutrition.
In a preferred embodiment, feed addictive composition of the present invention and feed ingredient are mixed to form feedstuff.
Term used herein " feed ingredient " refers to all or part of feedstuff.What feed a part raw material can refer to a kind of part of feedstuff or feedstuff exceedes a kind of part, such as 2 kinds or 3 kinds or 4 kinds of parts.In one embodiment, pre-composition or pre-composition part contained in term " feed ingredient ".
Preferably, feed is fodder material or its pre-composition, mixed feed or its pre-composition.In one embodiment, feed addictive composition according to the present invention can be mixed with mixed feed, mixed feed component, or feed addictive composition according to the present invention be mixed into the pre-composition of mixed feed or be mixed into fodder material, fodder material component or fodder material pre-composition.
Term used herein " fodder material " refers to any food (instead of animal must oneself be looked for food) being supplied to animal.Fodder material contains plant chopped.
Term " fodder material " comprises hay, stalk, ensilage, compressed feed and pellet, oil and mixing provisions, also comprises germinated ceral and beans.
Fodder material can derive from and be selected from one or more following plants: clover (alfalfa), barley, crowtoe, Btassica, Chaumoellier, collard, rapeseed (Kano is drawn), turnip (Sweden's wild cabbage), radish, clover, Alsike, red clover, subterranean clover, butch clover, grass, tall oat grass, fescue, bermuda grass, bromegrass, heath grass, English grass (grassland from mixing naturally), orchard grass, rye grass, timothy grass, corn (maize), millet, oat, Chinese sorghum, soybean, tree (the tree spray as under the pruning of trunk grass), wheat, and legume.
Term " mixed feed " refers to the commercial feed into meal, pellet, nut, cattle cake or debris form.Mixed feed can be mixed by multiple raw material and additive.These blends can be prepared according to the particular demands of target animal.
Mixed feed can be the complete feeding-stuffs providing all daily required nutriments, and providing unit divides the concentrate of provisions (albumen, energy), or only provides additional micronutrient as the replenishers of minerals and vitamins.
Main component for mixed feed is feed grain, and they comprise corn, soybean, Chinese sorghum, oat and barley.
Aptly, pre-composition as referred to herein can for the composition that must be become to be grouped into other by micro constitutent such as vitamin, mineral matter, chemical preservative, antibiotic, fermented product.Pre-composition is generally the composition being applicable to being mixed into business provisions.
Any feedstuff of the present invention can comprise the Feed Material that one or more are selected from the group comprising the following: a) cereal, such as minimand (as wheat, barley, naked barley, oat and their combination) and/or large grain cereal, as maize or Chinese sorghum; B) from the accessory substance of plant (such as cereal), such as moist forage cake, distiller's dried grain (DDG) and distiller's dried grain DDGS (DDGS), zein fiber, maize germ meal, corn bran, corn granulated slag, maize gluten feed, wheat prostitute, wheat time powder or their combination (being preferably the accessory substance according to method of the present invention); C) draw from such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, Kano, the albumen obtained originated by fish powder, dried plasma albumen, meat meal tankage, potato protein, whey powder, copra, sesame; D) to originate the grease and fat that obtain from plant and animal; E) minerals and vitamins.
Feedstuff of the present invention can comprise corn and dregs of beans or corn and full-fat bean or sizing or the sunflower meal of at least 30 % by weight, at least 40 % by weight, at least 50 % by weight or at least 60 % by weight.
Or alternatively, feedstuff of the present invention can comprise at least one accessory substance of at least one high-fiber feeding stuff material and/or at least one high-fiber feeding stuff material, to provide high-fiber feeding stuff raw material in addition.The example of high-fiber feeding stuff material comprises: wheat, barley, naked barley, oat, accessory substance from plant (such as cereal), such as moist forage cake, distiller's dried grain (DDG) and distiller's dried grain DDGS (DDGS), zein fiber, maize germ meal, corn bran, corn granulated slag, maize gluten feed, wheat prostitute, wheat time powder or their combination.Also can think that some protein sources are rich in fiber: such as from the protein that the source of such as sunflower, lupin, broad bean and cotton obtains.
In the present invention feed can be following one or more: mixed feed and pre-composition, comprise pellet, nut or (domestic animal) cattle cake; Following crop or crop residue: corn, soybean, Chinese sorghum, oat, barley, maize straw, copra, stalk, husk, sugar beet refuse; Fish meal; The grass newly cut and other forage plant; Meat meal tankage; Molasses; Oil cake and filter cake; Compound sugar; The forage plant of preservation: hay and ensilage; Marine alga; Seed that is complete or that processed through crushing, grinding etc. and cereal; The cereal germinateed and beans; Yeast extract.
Term " feed " in the present invention also contains pet food in certain embodiments.Pet food is intended to by the plant of pets or animal material, such as dog grain or cat grain.Pet food (such as dog grain or cat grain) can be dried forms (such as roughly grinding dog grain), or wet can form.Cat grain can comprise amino acid taurine.
Fish food also contained in certain embodiments in term " feed " in the present invention.Fish food comprises usually makes cultivation fish keep the necessary Macronutrient of good health state, trace element and vitamin.Fish food can be the form of flake, particle or tablet.Particle form (some of them can be sunk rapidly) is generally used for feed larger fish or demersal fishes.Some fishes food also comprises additive, such as beta carotene or sex hormone, manually to improve the color and luster of pet fish.
Term " feed " in the present invention also contains birdseed in certain embodiments.Birdseed comprises the food for bird feeder and feeding of pets bird.Birdseed generally includes multiple seed, also can contain leaf fat (tallow or suet).
Term used herein " contact " refers to indirectly or is directly applied in product (as feed) by composition of the present invention.The example of spendable application process comprises, but be not limited to, in the material comprising feed addictive composition treatment product, feed addictive composition and Product mix are directly applied, feed addictive composition is sprayed onto product surface or product are immersed in feed addictive composition preparation.
In one embodiment, preferably feed addictive composition of the present invention is mixed with product (as feedstuff).Alternatively, feed addictive composition can be comprised in the emulsion of feedstuff or raw material.
For some application, importantly make composition waiting to affect/pending product surface on or part available.This makes composition give below animal one or more favourable characteristics: biophysical properties, such as, wherein biophysical properties be selected from following one or more: animal performance, growth of animal performance, feed conversion rate (FCR), raw material digestion power (such as nutritive digestibility, comprise starch digestibility, fat digestibility, protein digestibility, digestibility of fiber), nitrogen retention rate, carcass yield, growth rate, weightening finish, body weight, quality, feed efficiency, body fat percentage, body fat distributes, growth, egg size, egg size amount, egg quality, laying rate and ambient influnence, such as muck output and/or nitrogen excretion.
(one or more) enzyme of feed addictive composition of the present invention and controlled quatity can be scattered, is coated with and/or infusion product (raw material of such as feedstuff or feedstuff).
Preferably, enzymatic compositions of the present invention and/or feed addictive composition are to being heat-staple up to about 70 DEG C, up to about 85 DEG C or up to the heat treatment of about 95 DEG C.Can carry out reaching about 1 minute, reach about 5 minutes, reach about 10 minutes, reach about 30 minutes, reach the heat treatment of about 60 minutes.Exist in additive before term " thermally-stabilised " refers to and is heated to specified temp/effectively enzyme component still exists after cooling to room-temperature at least about 75%/effectively.Preferably, exist in additive before being heated to specified temp and effective enzyme component still exist after cooling to room-temperature at least about 80% and effectively.
In a particularly preferred embodiment, by enzymatic compositions and/or feed addictive composition homogenizing, to make powder.
In an alternative preferred embodiment, enzymatic compositions and/or feed addictive composition are mixed with the granule (being called TPT granule) described in WO2007/044968, the document is incorporated to herein by reference.
In another preferred embodiment, when enzymatic compositions and/or feed addictive composition are mixed with granule, granule comprises the hydrated barrier salt be coated in above albumen core.The advantage of this kind of salt coating is improved heat resistance, improved storage stability and avoiding is subject to the adverse effect that other feed addictive causes enzyme.
Preferably, the salt for salt coating has the water activity being greater than 0.25 at 20 DEG C, or is greater than the constant humidity of 60%.
Preferably, salt coating comprises Na 2sO 4.
The method preparing feed addictive composition also can comprise further step powder being made pellet.Powder can be mixed with other component known in the art.The mixture making powder or comprise powder that can exert a force is forced through mould, and gained line material is cut into the pellet of suitable different length.
Optionally, before forming pellet, granulation step can comprise steam treatment or conditioning stage.The mixture comprising powder can be placed in conditioner, such as, with the agitator of vapor injection.In conditioner, mixture is heated to specified temp, such as, from 60 DEG C to 100 DEG C, typical temperature is 70 DEG C, 80 DEG C, 85 DEG C, 90 DEG C or 95 DEG C.The time of staying can be changed to several minutes even a few hours from the several seconds.Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.
Should be appreciated that feed addictive composition of the present invention is suitable for being added in any suitable Feed Material.
Term used herein " Feed Material " refers to the basic Feed Material by animal edible.It is also understood that Feed Material may comprise, such as at least one or multiple unprocessed cereal and/or finished plant and/or animal material (such as dregs of beans or bone meal).
Term used herein " feedstuff " refers to the Feed Material adding one or more feed addictive compositions.
Technical staff should be appreciated that different animals needs different feedstuffs, and different according to the object of letting animals feed, even if same animal also may need different feedstuffs.
Preferably, feedstuff can comprise Feed Material, this Feed Material comprises any accessory substance of maize or corn, wheat, barley, triticale, naked barley, rice, cassava, Chinese sorghum and/or aforementioned each, and high protein component, as dregs of beans, canola meal, canola meal, cotton seed meal, sunflower seeds meal, animal byproducts meal and their mixture.More preferably, feedstuff can comprise animal tallow and/or vegetable oil.
Optionally, feedstuff also can comprise additional mineral matter, such as, and calcium and/or additional vitamin.
Preferably, feedstuff is corn-soybean meal mixture.
In yet another aspect, the method preparing feedstuff is provided.Feedstuff is prepared usually in feed-processing plant, in feed-processing plant first by grinding raw material to appropriate particle size size, then by grinding after raw material mix with suitable additive.Next, feedstuff can be made pasty state or granular, the latter is usually directed to such method, by the method, temperature is increased to target level, then makes feed by mould thus prepares the pellet of specific size.Pellet is cooled.Liquid additive (such as fat and enzyme) can be added subsequently.Prepare feedstuff and also can relate to extra step, this step comprise granulation before extrude or expanded, especially by least comprising extruding of using the appropriate technology of steam to carry out or expanded.
Feedstuff can be the feedstuff of nonruminant of feeding, such as poultry (such as broiler chicken, laying hen, Breeder hens, turkey, duck, goose, aquatic bird), live pig (all age brackets), pet (such as dog, cat) or fish, preferably, feedstuff is poultry feed raw material.
Only for example, one or more compositions (%) that the feedstuff of chicken (as broiler chicken) can be listed by following table in the stage that such as following table provides form:
Composition Brood (%) Be bred as (%)
Maize 46.2 46.7
Wheat time powder 6.7 10.0
Cornstalk vinasse DDGS 7.0 7.0
Containing the dregs of beans of 48% crude protein 32.8 26.2
Animal/vegetable fat blend 3.0 5.8
L lysine HCL 0.3 0.3
DL-methionine 0.3 0.3
L-threonine 0.1 0.1
Salt 0.3 0.4
Lime stone 1.1 1.1
Dicalcium Phosphate 1.2 1.2
Poultry vitamin and trace mineral 0.3 0.3
Only for example, the dietary standards of chicken (as broiler chicken) can be as shown in the table:
Dietary standards
Crude protein (%) 23.00 20.40 26 -->
Poultry metabolizable energy (kcal/kg) 2950 3100
Calcium (%) 0.85 0.85
Phosphorus (%) can be utilized 0.38 0.38
Sodium (%) 0.18 0.19
Apparent digestible lysine (%) 1.21 1.07
Methionine (%) can be digested 0.62 0.57
Methionine+cysteine (%) can be digested 0.86 0.78
Threonine (%) can be digested 0.76 0.68
Only for example, one or more compositions (%) that the feedstuff of laying hen can be listed by following table in the stage that such as following table provides form:
Only for example, the dietary standards of laying hen can be as shown in the table:
Dietary standards
Crude protein (%) 16.10
Poultry metabolizable energy (kcal/kg) 2700
Lysine (%) 0.85
Methionine (%) 0.42
Methionine+cysteine (%) 0.71
Threonine (%) 0.60
Calcium (%) 3.85
Phosphorus (%) can be utilized 0.42
Sodium (%) 0.16
Only for example, one or more compositions (%) that the feedstuff of turkey can be listed by following table in the stage that such as following table provides form:
Only for example, the dietary standards of turkey can be as shown in the table:
Dietary standards
Crude protein (%) 29.35 26.37 22.93 20.00
Poultry metabolizable energy (kcal/kg) 2.850 2.900 2.950 3.001
Calcium (%) 1.43 1.33 1.22 1.02
Phosphorus (%) can be utilized 0.80 0.71 0.65 0.53
Sodium (%) 0.16 0.17 0.17 0.17
Apparent digestible lysine (%) 1.77 1.53 1.27 1.04
Methionine (%) can be digested 0.79 0.71 0.62 0.48
Methionine+cysteine (%) can be digested 1.12 1.02 0.90 0.74
Threonine (%) can be digested 1.03 0.89 0.73 0.59
Only for example, one or more compositions (%) that the feedstuff of piglet can be listed by following table in the stage that such as following table provides form:
Composition Stage 1 (%) Stage 2 (%)
Maize 20.0 7.0
Wheat 25.9 46.6
Naked barley 4.0 10.0
Wheat time powder 4.0 4.0
Cornstalk vinasse DDGS 6.0 8.0
Containing the dregs of beans of 48% crude protein 25.7 19.9
Whey powder 10.0 0.0
Soybean oil 1.0 0.7
L lysine HCL 0.4 0.5
DL-methionine 0.2 0.2 28 -->
L-threonine 0.1 0.2
L-Trp 0.03 0.04
Lime stone 0.6 0.7
Dicalcium Phosphate 1.6 1.6
Live pig vitamin and trace mineral 0.2 0.2
Salt 0.2 0.4
Only for example, the dietary standards of piglet can be as shown in the table:
Dietary standards
Crude protein (%) 21.50 20.00
Live pig digestible energy (kcal/kg) 3380 3320
Live pig net energy (kcal/kg) 2270 2230
Calcium (%) 0.80 0.75
Phosphorus (%) can be digested 0.40 0.35
Sodium (%) 0.20 0.20
Apparent digestible lysine (%) 1.23 1.14
Methionine (%) can be digested 0.49 0.44
Methionine+cysteine (%) can be digested 0.74 0.68
Threonine (%) can be digested 0.80 0.74
Only for example, one or more compositions (%) that the feedstuff of grower pigs/growing and fattening pigs can be listed by following table in the stage that such as following table provides form:
Composition Grow/fatten (%)
Maize 27.5
Containing the dregs of beans of 48% crude protein 15.4
Cornstalk vinasse DDGS 20.0
Wheat bran 11.1
Rice bran 12.0
Mustard seed meal 10.0
Lime stone 1.6
Dicalcium Phosphate 0.01
Salt 0.4
Live pig vitamin and trace mineral 0.3
FE-5 0.2
Vegetable oil 0.5
Only for example, the dietary standards of grower pigs/growing and fattening pigs can be as shown in the table:
form
Enzymatic compositions of the present invention and/or feed addictive composition of the present invention and other component can be used in any suitable form and/or comprise the feedstuff of this enzymatic compositions, feed addictive composition and other component.
The form of solid or liquid preparation or their alternative form can use enzymatic compositions of the present invention and/or feed addictive composition of the present invention.The example of solid pharmaceutical preparation comprises wettable, spraying dry or cryodesiccated powder, paste, bolus, capsule, granula, tablet, pulvis and granule.The example of liquid preparation includes, but are not limited to water-based, organic or water-based organic solution, suspension and emulsion.
In some applications, feed addictive composition of the present invention can mix with feed, or is applied in drinking water.
For quick-releasing type, slowbreak type, adjust the Suitable examples of form releasing the application of type, spacetabs type, pulse release type or control release type to comprise in powder, paste, bolus, granula, tablet, pill, capsule, pearl agent, solution or suspension one or more, and flavor enhancement or coloring agent can be comprised.
For example, if composition of the present invention in solid form (such as pellet form) uses, said composition also can comprise following one or more: excipient, such as microcrystalline cellulose, lactose, natrium citricum, calcium carbonate, calcium dihydrogen phosphate and glycine; Disintegrant, the such as silicate of starch (being preferably corn, potato or tapioca), Sodium Starch Glycolate, Ac-Di-Sol and some complexity; Adhesive for granulating, such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin and Arabic gum; Lubricant, such as dolomol, stearic acid, glyceryl behenate and talcum powder.
The example preparing acceptable carrier in the nutrition used of above-mentioned form comprises, such as water, salting liquid, alcohol, silicones, wax, vaseline, vegetable oil, polyethylene glycol, propane diols, liposome, sugar, gelatin, lactose, amylose, dolomol, talcum powder, surfactant, silicic acid, viscous paraffin, aromatic oil, mono fatty acid glyceride and fatty acid diglyceride, petroethral fatty acid esters, CMC, polyvinylpyrrolidone etc.
The preferred excipient of above-mentioned form comprises lactose (lactose), starch, cellulose, lactose (milksugar) or high molecular weight polyethylene glycol.
For water slurry and/or elixir, composition of the present invention can be combined from different sweeteners and flavor enhancement, pigment or dyestuff, combine with emulsifying agent and/or suspending agent, and combine with diluent (such as water, propane diols and glycerine and their combination).
with other combination of components
Feed addictive composition of the present invention or feed ingredient or feed or feedstuff or pre-composition can use with other combination of components.
Medical treatment or physiological function benefit can be provided for consumer by feed addictive composition of the present invention or feed ingredient or feed or feedstuff or pre-composition and the another kind of combination of components that is suitable for animal edible.
In one embodiment, " another kind of component " can be one or more enzymes.
The present invention's suitable additional enzymes used can be one or more enzymes being selected from following enzyme: endoglucanase (E.C.3.2.1.4), cellobiohydrolase (E.C.3.2.1.91), beta-glucosidase (E.C.3.2.1.21), cellulase (E.C.3.2.1.74), lichenase (E.C.3.1.1.73), lipase (E.C.3.1.1.3), acyltransferase (being usually classified as E.C.2.3.1.x), phosphatidase (E.C.3.1.1.4, E.C.3.1.1.32 or E.C.3.1.1.5), phytase (such as 6-phytase (E.C.3.1.3.26) or 3-phytase (E.C.3.1.3.8)), AMS (E.C.3.2.1.1), zytase (E.C.3.2.1.8, E.C.3.2.1.32, E.C.3.2.1.37, E.C.3.1.1.72, E.C.3.1.1.73), glucoamylase (E.C.3.2.1.3), protease (such as subtilopeptidase A (E.C.3.4.21.62) or Bacillus subtilis neutral protease (E.C.3.4.24.28) or alkaline serine protease (E.C.3.4.21.x) or keratinase (E.C.3.4.x.x)) and/or mannase (such as 'beta '-mannase (E.C.3.2.1.78)).
In one embodiment (especially the embodiment of feed applications), other component can be one or more enzymes being selected from following enzyme: zytase (E.C.3.2.1.8, E.C.3.2.1.32, E.C.3.2.1.37, E.C.3.1.1.72, E.C.3.1.1.73), amylase (comprises AMS (E.C.3.2.1.1), G4-forms amylase (E.C.3.2.1.60), beta amylase (E.C.3.2.1.2) and gamma amylase (E.C.3.2.1.3)), and/or protease (such as subtilopeptidase A (E.C.3.4.21.62) or Bacillus subtilis neutral protease (E.C.3.4.24.28) or alkaline serine protease (E.C.3.4.21.x) or keratinase (E.C.3.4.x.x)).
In one embodiment (especially the embodiment of feed applications), other component can be the combination of amylase (such as AMS (E.C.3.2.1.1)) and protease (such as subtilopeptidase A (E.C.3.4.21.62)).
In one embodiment (especially the embodiment of feed applications), other component can be 1,4 beta-glucanase, such as inscribe-1,3 (4)-1,4 beta-glucanase (E.C.3.2.1.6).
In one embodiment (especially the embodiment of feed applications), other component can be mannase (such as 'beta '-mannase (E.C.3.2.1.78)).
In one embodiment (especially the embodiment of feed applications), other component can be lipase (E.C.3.1.1.3), acyltransferase (usually classifying as E.C.2.3.1.x) or phosphatidase (E.C.3.1.1.4, E.C.3.1.1.32 or E.C.3.1.1.5), suitably for lipase (E.C.3.1.1.3).
In one embodiment (especially the embodiment of feed applications), other component can be protease (such as subtilopeptidase A (E.C.3.4.21.62) or Bacillus subtilis neutral protease (E.C.3.4.24.28) or alkaline serine protease (E.C.3.4.21.x) or keratinase (E.C.3.4.x.x)).
In one embodiment, annexing ingredient can be stabilizing agent or emulsifying agent or binding agent or carrier or excipient or diluent or disintegrant.
The composition that term used herein " stabilizing agent " is defined as preventing product (as feed product) from passing in time and changes or composition combination.
Term used herein " emulsifying agent " refers to and prevents emulsion from the composition (as feed ingredient) be separated occurring.Emulsion is two kinds of immiscible materials, and wherein a kind of material existed with drops is contained among another kind of material.Emulsion can comprise emulsion oil-in-water or water-in-oil emulsion, and in emulsion oil-in-water, drop or decentralized photo are oil, and continuous phase is water; And in water-in-oil emulsion, water becomes decentralized photo, continuous phase is oil.Also make foam (namely gas dispersion is in liquid) and suspension (namely solid dispersal is in liquid) stablize by using emulsifying agent.
Term used herein " binding agent " refers to the composition (as feed ingredient) be bonded together by product by physics or chemical reaction.Such as in " gelation " process, water is absorbed, thus produces bond effect.But, other liquid of binding agent Absorbable rod (such as oil), and hold it within product.In linguistic context of the present invention, binding agent will be used for the product of solid product or low moisture content usually, such as, bake and bank up with earth product: short crust pastry, baked donut, bread and other products.The example of adhesive for granulating comprise in following material one or more: polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, maltose, gelatin and Arabic gum.
" carrier " refers to the material being applicable to use enzyme, and comprise this type of material any known in the art, such as any liquid, gel, solvent, liquid diluent, solubilizer etc., this type of material non-toxic and not interacting in harmful mode with any component of composition.
The invention provides the method preparing composition (such as feed addictive composition), the method comprises and being mixed with the physiologically acceptable carrier of at least one by feed addictive of the present invention (and preferably corn or corn's by-products), and this carrier is selected from least one in following material: maltodextrin, lime stone (calcium carbonate), cyclodextrin, wheat or wheat component, sucrose, starch, Na 2sO 4, talcum powder, PVA, sorbierite, benzoate, sorbate, glycerine, sucrose, propane diols, 1,3-PD, glucose, p-hydroxybenzoate, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formates and their mixture.
The example of " excipient " comprise in following material one or more: microcrystalline cellulose and other cellulose, lactose (lactose), natrium citricum, calcium carbonate, calcium dihydrogen phosphate, glycine, starch, lactose (milksugar) and high molecular weight polyethylene glycol.
The example of " disintegrant " comprise in following material one or more: the silicate of starch (being preferably corn, potato or tapioca), sodium starch glycollate, Ac-Di-Sol and some complexity.
The example of " diluent " comprise in following material one or more: water, ethanol, propane diols and glycerine and their combination.
Other component can simultaneously (as being present in mixture or even when being provided by different approaches when them when other component simultaneously) or successively (as other component provides by different approaches) in feed addictive of the present invention.
In one embodiment, preferably, chromium or Organic Chromium is not comprised according to feed addictive composition of the present invention or feed ingredient or feed or feedstuff or pre-composition.
In one embodiment, preferably, sorbic acid is not comprised according to feed addictive composition of the present invention or feed ingredient or feed or feedstuff or pre-composition.
biophysical properties
" biophysical properties " used herein refers to any animal organism physical property improving animal health and/or performance and/or output.For example, the biophysical properties paid close attention to can be one or more characteristics being selected from following characteristic: animal performance, growth of animal performance, feed conversion rate (FCR), raw material digestion power (such as nutritive digestibility, comprise starch digestibility, fat digestibility, protein digestibility, digestibility of fiber), nitrogen retention rate, carcass yield, growth rate, weightening finish, body weight, quality, feed efficiency, body fat percentage, body fat distributes, growth, egg size, egg size amount, egg quality, laying rate and ambient influnence, such as muck output and/or nitrogen excretion.
In a preferred embodiment, derive from and can be according to the biophysical properties of method of the present invention or purposes one or more characteristics being selected from following characteristic: animal performance, growth of animal performance, feed conversion rate (FCR), raw material digestion power (the digestibility of such as nutriment, comprise starch digestibility, fat digestibility, protein digestibility, digestibility of fiber), nitrogen retention rate, carcass yield, growth rate, weightening finish, body weight, quality, feed efficiency, body fat percentage, body fat distributes, growth, egg size, egg size amount, egg quality, laying rate and ambient influnence, such as fertilizer output and/or nitrogen excretion.
In one embodiment, the biophysical properties of animal refers to animal performance.
performance
" animal performance " used herein can be increased weight by feed efficiency and/or animal and/or digestible energy or metabolizable energy and/or nitrogen retention rate and determine in feed conversion rate and/or feed Middle nutrition substance digestion rate (such as amino acid whose digestibility) and/or feed.
Preferably, " animal performance " is increased weight by feed efficiency and/or animal and/or feed conversion rate and determining.
" animal performance improvement " refer to compared to detoxification not according to the lignocellulose biomass of process of the present invention, use feed addictive composition of the present invention that the feed efficiency of animal is improved and/or weightening finish becomes large and/or feed conversion rate reduces and/or feed Middle nutrition material or energy digestibility increases and/or nitrogen retention rate increases.
Preferably, " animal performance improvement " refers to that feed efficiency improves and/or weightening finish becomes large and/or feed conversion rate reduces.
Term used herein " feed efficiency " refers within a period of time to the recruitment of the weight of animals occurred during the food of animal unrestrictedly feeding or ormal weight of feeding.
" feed efficiency raising " refers to compared to feeding not according to the animal of the lignocellulose biomass of process of the present invention, uses and the weightening finish caused by per unit Feed consumption can be made to become large according to feed addictive composition of the present invention in feed.
feed conversion rate (FCR)
Term used herein " feed conversion rate " refers to as making the specific amount of the weight of animals increase feed to the amount of the feed of animal.
Feed conversion rate improvement refers to that feed conversion rate reduces.
" feed conversion rate reduction " or " feed conversion rate improvement " refer to compared to when not according to the lignocellulose biomass of process of the present invention for feed in or be used as feed time for making animal increase forage volume needed for the body weight of specified quantitative, the forage volume of the required feeding animals that weighs sb. such as use feed addictive composition that animal can be made to increase in feed is less.
nutritive digestibility
Nutritive digestibility used herein refers to the ratio of the nutriment disappeared from intestines and stomach or intestines and stomach particular section (such as small intestine).Nutritive digestibility by be administered to this nutriment in the nutriment amount of application of animal and animal wastes discharge rate between difference measure, or to be measured by the difference between the reserved of this nutriment in the nutriment amount of application that is administered to animal and intestines and stomach particular section (such as ileum) inner digest.
" nutritive digestibility " used herein can be measured by the difference between the intake of nutriment within a period of time and the nutriment discharge rate of being collected completely by excreta and drawing; Or do not measured by inert labels's thing that animal absorbs by using, this label enables researcher calculate the nutrition content disappeared at whole intestines and stomach or intestines and stomach section.This type of inert labels's thing can be titanium dioxide, chromium oxide or acid-insoluble ash.The percentage that digestibility can be expressed as feed Middle nutrition material or be expressed as every mass unit in feed nutriment in the mass unit number of contained digestible nutriment.
" nutritive digestibility " used herein contains starch digestibility, fat digestibility, protein digestibility and amino acid digestibility.
" energy digestibility " used herein refers to that the gross energy of eaten feed deducts the gross energy of ight soil, or the gross energy of edible feed deduct the gross energy of residue digest in animal gastrointestinal tract particular section (such as ileum)." metabolizable energy " used herein refers to Apparent metabolizable energy, and the gross energy meaning eaten feed deducts the gross energy in ight soil, urine and the gaseous product being included in digestion and producing.The method identical with measuring nutritive digestibility can be used, difference between the gross energy of being discharged by intake and the ight soil of gross energy, or and intestines and stomach particular section (such as ileum) in difference between the digest gross energy that exists measure energy digestibility and metabolizable energy, suitably correction is carried out to elimination of nitrogen simultaneously and calculates the metabolizable energy of feed.
nitrogen retention rate
Nitrogen retention rate used herein refers to that the nitrogen in diet is possessed the ability for body weight by animal.When elimination of nitrogen amount exceedes daily intake, there will be negative nitrogen balance, when muscle reduces, usually can observe this phenomenon.Positive nitrogen balance is usually relevant to muscle growth, especially for animal in growth period.
Nitrogen retention rate can be measured by the difference between the intake of nitrogen within a period of time and the nitrogen output collected completely and drawn by excreta and urine.Should be appreciated that the nitrogen of discharge comprises the secretion of indigested protein, endogenous protein in feed, microprotein and urinary nitrogen.
carcass yield and meat yield
Term used herein " carcass yield " refers to after business or experiment slaughter process, as the amount of the carcass of a live weight part.Term " carcass " refers to as edible slaughtered, and removes the animal body of head, internal organ, extremity portion and feather or skin.Term used herein " meat yield " refers to the edible meat amount as a live weight part, or as the specific cube meat amount of a live weight part.
weightening finish
Present invention also offers and increase the method for animal (as poultry or live pig) body weight, the method comprises and comprises feedstuff according to feed addictive composition of the present invention to described detoxification.
" weightening finish become large " refers to and to comprise compared to feeding not according to the feed of the lignocellulose biomass of process of the present invention or feed by the animal of the feed form according to the lignocellulose biomass of process of the present invention, the weight of animals increase of the feed comprising feed addictive composition of feeding.
Unless otherwise defined, what implication and the disclosure those of ordinary skill in the field of all scientific and technical terminologies used herein understood usually is the same.The people such as Singleton, DICTIONARYOFMICROBIOLOGYANDMOLECULARBIOLOGY, 20ED., JohnWileyandSons, NewYork (1994) and Hale & Marham, THEHARPERCOLLINSDICTIONARYOFBIOLOGY, HarperPerennial, NY (1991) provide the universaling dictionary of many terms used in the disclosure for technical staff.
The disclosure by the restriction of illustrative methods disclosed herein and material, or any method that be equal to similar with material with method as herein described and material all can be used for embodiment of the present disclosure practice or in testing.Number range comprises the end value of limited range numerical value.Except as otherwise noted, any nucleotide sequence is all from left to right write with 5' to 3' orientation, and amino acid sequence is from left to right write to carboxyl orientation with amino.
During description integrally, title used herein is not the restriction to disclosure different aspect or embodiment.Therefore, during description integrally, the term being about to definition below just defines more comprehensively.
The abbreviation of amino acid name, trigram or single letter abbreviation is used to represent amino acid herein.
Term used herein " protein " comprises protein, polypeptide and peptide.
Term used herein " amino acid sequence " is synonym with term " polypeptide " and/or term " protein ".In some cases, term " amino acid sequence " and term " peptide " are synonyms.In some cases, term " amino acid sequence " and term " enzyme " are synonyms.
Term " albumen " and " polypeptide " exchange use in this article.In the disclosure and claims, traditional single-letter and three-letter codes can be used for amino acid residue.This three-letter codes is the amino acid three-letter codes defined according to IUPACIUB Biochemical Nomenclature joint committee (JCBN).It is also understood that the degeneracy due to genetic code, polypeptide can by more than one nucleotide sequence coded.
Must be noted that, as used herein, and in the appended claims, singulative " ", " one " and " described " comprise plural references, unless clearly separately indicated in context.Therefore, such as, the connotation of " a kind of enzyme " comprises this type of candidate agent multiple, and the connotation of " described feed " comprises the connotation of one or more feeds and their equivalent known to those skilled in the art, etc.
Publication as herein described only submits to the disclosure before day to provide for reference to it at this patent.Content herein all should not be construed as admits that such publication can be used as the prior art in appended claims.
Now only with reference to the following drawings and example, the present invention is described by way of example.
example
summary
Due to gold brick four countries (Brazil, Russia, India and China), population increases and living standard improves constantly, and food and feed price are rising violently always.The present invention aims to provide the alternative feed ingredient for animal productiong.
The present inventor surprisingly finds, eliminate C5 sugar monomer generates and improve C5 oligomer generate simultaneously with cellulase activity (such as, thus generate C6 oligomer and C-6 monomer sugar) to for or production as the vegetable material (such as lignocellulose biomass) of feedstuff be favourable.
materials and methods
The enzyme of test is:
Cellulase SC is a kind of enzymatic compositions, there is following enzymatic activity: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, and not there is or substantially do not have xylobiase activity and α-L-arabinofuranosidase activity.Cellulase SC thaws before using at being stored in-20 DEG C.
Endo-xylanase (inscribe-Isosorbide-5-Nitrae-beta-xylanase)-DaniscoXylanase tM40000L (synonym: zytase Y5 or Y5) can derive from DaniscoAnimalNutrition, DuPontA/S-be diluted to 9293U/g.
Endo-xylanase (inscribe-Isosorbide-5-Nitrae-beta-xylanase)-EconaseXT tMaBVista can be derived from.This enzyme is the purified form of 0.51mg albumen/ml.EconaseXT tMbe the variant of zytase, come from actinomyces Nonomuraeaflexuosa (people such as Leskinen, ApplMicrobiolBiotechnol (2005) 67:495 – 505).
Endo-xylanase (inscribe-1,4-beta-xylanase)-(FoxXyn6) as in PCT/CN2012/079655 (document be incorporated to by reference herein) of instructing, encoded by nucleotide sequence SEQIDNo.1 or SEQIDNo.2 and there is peptide sequence SEQIDNo.3.
Endo-xylanase (inscribe-1,4-beta-xylanase)-(synonym: FveABC5, FoxXyn4, FveXyn4) as in PCT/CN2012/079650 (document be incorporated to by reference herein) of instructing, encoded by nucleotide sequence SEQIDNo.4 or SEQIDNo.5 and there is peptide sequence SEQIDNo.6.
. trio tMbe that one compares type enzymatic compositions, have endoglucanase, beta-glucosidase and endo-xylanase activity and C-5 monomer formation enzymatic activity, such as xylobiase is active and α-L-arabinofuranosidase is active. trio tMrecommended doses be 0.05 to 0.3mL every gram cellulose or roughly 0.03 to 0.16mL every gram living beings (depending on that living beings form).Use front general trio tMbe kept at-20 DEG C.
pretreated living beings: through the pretreated maize straw of weak aqua ammonia (DaCS) from DuPont (product batch number HP90-HP93).This maize straw has about 65% solids content, and containing a small amount of acetamide.This maize straw is according to DuPont patent application (WO2006/110901A2-this patent application is incorporated to herein by reference) preparation.
The AminexHPX-87P chromatographic column (300mm × 7.8mm) from Biorad (Hercules, CAUSA) is used to analyze sugar.The free list open-birth substance undergoes hydrolysis of sugar and oligomer.According to manufacturer, following sugar can realize good separation: 1. cellobiose, 0.1%; 2. glucose, 10%; 3. wood sugar, 0.1%; 4. galactolipin, 0.1%; 5. arabinose, 0.1%; 6. mannose, 0.5%.The analysis condition used is: column oven temperature is set to 80 DEG C, and flow velocity is set to 0.5mlmilliQ water/min.HPLC is DionexSummit, is equipped with P580 pump and ShodexRI detector.Use glucose, wood sugar and arabinose reference material (1% (w/v)).Sample size is 10 or 20 μ l.
DionexHPLC use the CarboPac chromatographic column from Dionex analyze glucose, wood sugar, arabinose and compound sugar.
Equally also in the DionexCarboPac chromatographic column of DionexHPLC, the sugar of release, oligomer and polymer are analyzed.
The mensuration of free monosaccharide: Centrifuge A sample 5min under 16000 × g, retains supernatant.2ml methyl alcohol and 2ml water is used strong anion exchange solid-phase extracting column (SAXSPE) (can SigmaFineChemicals be derived from) (500mg adsorbent) to be nursed one's health.0.1ml sample solution is added solid-phase extraction column, and then sample is inhaled into test tube under vacuum.Use 2ml water cleaning solid-phase extraction column, and cleaning fluid is collected in same test tube.According to different monosaccharide concentration, with water by each Sample Dilution to 10ml (or other appropriate volume).The mensuration of total reducing sugar after acid hydrolysis: add 0.100ml sample, 0.100ml water and 0.200ml2MH in 35ml test tube 2sO 4.Clog this test tube with stopper and leave standstill 3 hours at 100 DEG C.29.6ml water is added after test tube cooling.Then by sample centrifugal 5min under 13000 × g, and 0.45 μm of Filter paper filtering is used.According to different sugar concentration, further dilute with water sample.Polymerization sugar content is according to following calculating:
DionexHPLC uses CarboPac tMpA1 (4mm × 250mm) analyzes process sample, uses CarboPac tMpA1 (4mm × 40mm) is as pretreatment column (can derive from Dionex).Column oven temperature: 30 DEG C.Mobile phase: A:HPLC level water, B:0.2MNaOH, sample size: 25l, flow velocity: 1ml/min.The Gradient program used is: 0min, 10%B; 1.1min, 10%B; 5.0min, 0%B; 33min, 0%B; 34min, 100%B; 41min, 100%B; 42min, 10%B; 56min, 10%B.Use PED detector (pulsed electrochemical detector).The Standard for Sugars thing used is: L (+) arabinose (Merck1488), D-galactolipin (Merck4058), D (+)-wood sugar (Merck8689), D (+) glucose (Merck8337), D (+) mannose (Merck5984).Total reducing sugar (mg/l) after polymerization sugar (mg/l)=acid hydrolysis-free monosaccharide (mg/l).
example 1 uses cellulase SC to be hydrolyzed through the pretreated maize straw of weak aqua ammonia (DaCS).
Through the pretreated maize straw of weak aqua ammonia (DaCS): add 15gDaCS and running water in 50mlFalcon centrifuge tube, and with 2NHCl, pH is adjusted to 5.0.Add 1.33ml cellulase SC (production lot number 118 (protein concentration is 76.6g/L), is called cellulase SC118 herein), start reaction.The ultimate density of living beings DaCS is 18.4% (w/v).Cellulase SC118: biomass (w/w) is 1.08:100.
This reacts on 50 DEG C, vibrate under 200rpm rotating speed 70h.Respectively when reacting 21.5h, 45.5h, 62.5h and 70.5h (namely at the end of reaction), the living beings in brief centrifugation Falcon centrifuge tube, and take out fluid sample.Sample size in each time interval is 60 μ l, is then mixed with the water that 60 μ l are used for the pipette tip rinsing pipette samples by the sample of taking-up.Before carrying out HPLC analysis by collect Sample storage at 5 DEG C.Then each sample is mixed with 120 μ lmilliQ water again, at 100 DEG C, heat 5min and filter.By 10 μ l filtrate sample introductions to HPLC, analyze the sugar of release.Add 14mlmilliQ water respectively in three Falcon centrifuge tubes, wash centrifugal rear remaining living beings.By the residual biomass dry 20h at 80 DEG C after washing, weigh.The alleged weight obtained is compared with the biomass weight without enzymatic compositions process.
the HPLC analysis result of example 1
Fig. 1 is the glucose and xylose output that cellulase SC process DaCS obtains.
Fig. 1 is based on the data listed in detail in following table (table 1).It can be seen from the table, under 50 DEG C and pH=5.0, the interior Xylose Content discharged by DaCS of 70.5h can be reached lower than 1/3 of the glucose content discharged.
table 1
the gravimetric analysis result of example 1
As can be seen from Table 2, under in vitro conditions, the DaCS of 56% transforms.The conversion ratio of DaCS is improved by optimizing mixing further.
table 2: at 50 DEG C, during 70.5h, cellulase SC is to the biomass by hydrolyzation situation of maize straw
Result shows trio tM.Can find out that from HPLC chromatogram hydrolysate is glucose, wood sugar and a small amount of arabinose.
Cellulase SC mixture has been hydrolyzed DaCS, but with compare type enzymatic compositions trio tM(Fig. 2) unlike, cellulase SC generally hydrolysis efficiency is lower, and produces less C5 sugar (wood sugar and arabinose).Because nonruminant utilizes the efficiency of C5 sugar lower, so it is favourable for forming less C5 sugar.This result shows that cellulase SC has low (or not having) xylobiase activity and α-L-arabinofuranosidase activity.
Found that cellulase SC adds endo-xylanase (DaniscoXylanase tM) release of glucose and xylo-oligosaccharide can be promoted.This is very favorable for applying it in feedstuff.
example 2 uses cellulase SC be hydrolyzed the maize straw (DaCS) of weak aqua ammonia process and add endo-xylanase effect.
Be add 1gDaCS, 5mlMilliQ water in the plastic tube of 12ml and mix respectively to 9 volumes, by pipe at 5 DEG C hold over night to guarantee DaCS well-hydrated.Within second day, add 150 μ l1NHCl, pH is adjusted to 4.6 to 5.1.Add enzyme (listed in following table 3), start reaction.This reacts on 50 DEG C, vibrate under 180rpm rotating speed 40h.Be collected in the fluid sample (120 μ l) when reaction time 6h, 19h and 40h respectively, use 120 μ lMilliQ water dilute samples, in order to analyzing at being stored in 5 DEG C.At the end of enzyme reaction, in each sample of collecting, add 240 μ lmilliQ water again, then in 100 DEG C of water-baths, heat 5min, afterwards centrifugal 10min under 10000g.Filter 250 μ l supernatants subsequently, and analyze in 10 μ l filtrate sample introductions to HPLC.Filter the glucose and xylose reference material of 1% (w/v), use the condition identical with the sample water hydrolysis products collected and sample size to carry out HPLC analysis.
Living beings loss of weight is analyzed: by the residual biomass obtained after centrifugal in 9 pipes, after removing supernatant, adds 4ml water, and vibration is to wash any resolvability material in precipitation off.The biomass residuals after washing is obtained, then dry 48h at 60 DEG C by again centrifugal for suspension.
table 3
the HPLC analysis result of example 2
The wood sugar that cellulase SC (cellulase SC118 and cellulase SC151) produces far less than trio tM(see Fig. 2 and Fig. 3).The glucose that cellulase preparation cellulase SC produces also is less than trio tM.Infer from the analysis result of reaction time 6,19 and 40h, this result is equally applicable to whole course of reaction (lower Fig. 2).
Cellulase SC facilitates the release of glucose and xylose in whole reaction time process together with endo-xylanase (FoxXyn4).Wood sugar that cellulase SC discharges together with zytase (such as C5 sugar) far less than trio tMthe wood sugar of release.
In DionexHPLC, use CarboPac chromatographic column to analyze the hydrolysate generated in table 2 further.Result provides in figure 3.As can be seen from Figure 3, the monose total amount of generation is similar to Fig. 2 result.In addition, Fig. 3 show total solubility polymerization sugar that cellulase SC process or cellulase SC add that endo-xylanase process obtains more than trio tMprocess the total solubility polymerization sugar obtained.
As can be seen from Figure 3, in interpolation or when not adding zytase, the total soluble polymer type sugar discharged by cellulase SC is by having xylobiase activity and α-L-arabinofuranosidase activity trio tM4 times of total soluble polymer type sugar of release.Use the CarboPac chromatogram column analysis sugar of Dionex.
example 3 is poly-through the pretreated maize straw of weak aqua ammonia (DaCS) wood different from interpolation to use cellulase SC hydrolysis the further research of the effect of carbohydrase
Taking 1gDaCS to each volume that table 3 is listed is in the pipe of 12ml, adds 4.6ml water and 0.15ml1NHCl, and mixes.To survey pH be 5.03 to 5.17.Then add at 50 DEG C trio tM, cellulase SC118 or cellulase SC118 adds the zytase that 10 to 120 μ l5 kinds are different, starts reaction, to vibrate 45h with 200rpm.Pipe 22 to pipe 24 is the control samples of not adding enzyme.Pipe 3k, pipe 6k, pipe 12k, pipe 15k, pipe 18k and pipe 21k are blank samples.
related component is not added in table 3 "-" expression
* pure EconaseXT is a kind of form purer than EconaseXT, and SDS-PAGE gel only shows the band that corresponds to zytase.
At the end of reaction, all pipes are collected supernatant with the centrifugal 10min of 3500rpm.Use 4ml and 3.5mlmilliQ water washing precipitation twice respectively, then repeated centrifugation operation.Merge all supernatants and be adjusted to final volume 12ml.Get wherein 0.3ml and heat 7min at 100 DEG C, filter, and 10 μ l filtrate sample introductions are carried out glycan analysis to HPLC.Equally also by arabinose, xylose and glucose (quality percent by volume) reference material filter and by 10 μ l filtrate sample introductions to HPLC.Each sample carries out twice HPLC operation.
Biomass weight is analyzed: dry 48h at the living beings of final residual precipitation is placed in 80 DEG C also weighs, and obtains net weight.
the HPLC analysis result of example 3
As can be seen from Figure 4, combined from all different zytases by cellulase SC, glucose yield all improves about 30% to 40%, this with by trio tMthe glucose yield of gained is consistent.Wood sugar output also improves, but its output is starkly lower than trio tMthe output of gained.
By trio tMthe typical HPLC chromatogram of the DaCS hydrolysate of gained illustrates the monose peak (see Fig. 5) of glucose, wood sugar and arabinose, and this monose peak has very smooth peak (retention time is the peak 2 at 9.22min place) in polymer areas.
With in Fig. 5 by trio tMthe oligomer of the more Gao Gengkuan of gained is compared with polymer peak, by cellulase SC (lower trace) and cellulase SC and DaniscoXylanase tMthe typical HPLC chromatogram of the DaCS hydrolysate of coupling (upper trace) gained shows glucose peaks, wood sugar peak and arabinose peak (see Fig. 6).
Fig. 5 and Fig. 6 illustrates that the wood sugar peak value that cellulase SC and cellulase SC and zytase coupling generate is lower, and the glucose peak of generation is also lower.On the other hand, in retention time 6.5 to 11.5min region, the content of oligomer and polymer is higher.
The present inventor surprisingly finds, when hydrolysate being used as feed ingredient (during in particular as the feed ingredient of nonruminant), reducing wood sugar (and even glucose) content and increases oligomer and polymer content is favourable.
The present inventor finds, wood sugar is not effective source of nonruminant energy, because have about 50% to discharge with urine form in edible wood sugar.On the other hand, the micropopulation that oligomer and polymer can be occupied large intestine is fermented into SCFA, and SCFA can be rapidly absorbed into blood flow and is used as energy source by animal.
the gravimetric analysis result of example 3
Reduce according to the weight of gained residual biomass after enzyme reaction as can be seen from lower Fig. 7, deposit in case at cellulase SC, endo-xylanase (such as DaniscoXylanase tM, EconaseXT tM, FoxXyn4 or FoxXyn6, or DaniscoXylanase tM, EconaseXT tMmore high purity form) interpolation all enhance hydrolysis.Hydrolysis shown in Fig. 7 uses non-industrial processes condition and shorter time to carry out, to adapt to the restriction of bench scale test.Therefore, overall hydrolysis degree is suboptimum, but still reflects to be hydrolyzed as cellulase SC and endo-xylanase coupling and improve.
example 4 is hydrolyzed the maize straw (DaCS) of weak aqua ammonia process and cellulase SC and interior to using cellulase SC cut zytase (DaniscoXylanase tM ) the further research of dosage effect
In each pipe that table 4 is listed, add 1gDaCS, add water and 0.15ml1NHCl makes volume reach 12ml, mixing.Then at 50 DEG C, add 50 to 200 μ l cellulase SC118 or 50 to 200 μ l cellulase SC118 add 10 μ lDaniscoXylanase tM, start reaction, and with 250rpm vibration 40h.After reaction terminates, will manage centrifugal, and collect supernatant.4ml and 3.5ml water is used to be precipitated twice by resuspended and centrifuge washing respectively.Merge supernatant, and by adding extra water by volume-adjustment to 12ml.The supernatant (2ml) merged is placed 10min and made enzyme deactivation at 100 DEG C, then centrifugal and filter.Use HPLC to analyze filtrate, sample size is 10 μ l.After washing be deposited in 80 DEG C at dry 48h.
table 4
the gravimetric analysis result of example 4
As can be seen from Figure 8, the dry matter content of DaCS is 63.3% (left side first row).At 50 DEG C, pH to be adjusted to incubation 40h in the water of 5 using HCl by DaCS, and 16.0% dry becomes solvable (in Fig. 9 left side secondary series).Add 10 μ l (93 unit) endo-xylanase, make the solubility of DaCS be increased to 26.9% further.Independent interpolation cellulase SC118 makes the solubility of DaCS be increased to 41.7%.10 μ l endo-xylanases and 50 μ l cellulase SC118 combine, and make the solubility of DaCS reach about 51.7%, this shows that zytase and cellulase SC have additive effect.For reaching the solubility level obtained by 10 μ l zytases and 50 μ l cellulase SC couplings, need the cellulase SC (150 μ l and 200 μ l) of interpolation 3 times or more separately.Conclusion is, for the solubilising of DaCS, 50 μ l cellulase SC and 10 μ l endo-xylanase couplings are better than adding 50,100 and 150 μ l cellulase SC separately.For the DaCS that dry load is 10% (w/v), 50 μ l cellulase SC add that 10 μ l endo-xylanases are best, because 100,150 and 200 μ l cellulase SC add that 10 μ l endo-xylanases can not increase solubility further.It is generally acknowledged that solubility is higher, the digested rate of body is also higher.
the HPLC analysis result of example 4
As can be seen from Figure 10, do not adding enzyme or adding 10 μ l (93 unit) DaniscoXylanase tMwhen, the glucose that the soluble fraction of DaCS is not dissociated.This shows by adding DaniscoXylanase separately tMthe solable matter of discharge 26.9% is substantially oligomer and polymer form (Fig. 9).In reaction, additive capacity is 50,100,150 to 200 μ l cellulase SC, can increase the release of glucose and xylose, but the non-linear relation of the dosage of increment and cellulase SC (Figure 10).
As can be seen from Figure 10 also, 10 μ lDaniscoXylanase are added by 50 μ l cellulase SC tMthe glucose content of release is higher than the glucose content discharged separately by 100 μ l cellulase SC; 100 μ l cellulase SC add 10 μ lDaniscoXylanase tMeffect equal independent 150 μ l cellulase SC haply, and 150 μ l cellulase SC add 10 μ lDaniscoXylanase tMeffect equal independent 200 μ l cellulase SC haply.
Conclusion is, clearly for the release of glucose and xylose, and DaniscoXylanase tMwith cellulase SC, there is cooperative effect, because adding 10 μ lDaniscoXylanase by 50 μ l cellulase SC tMthe glucose content of release is higher than only by 10 μ lDaniscoXylanase tMthe glucose content (there is no that glucose discharge) of release and only by the summation (Figure 10) of glucose content of 50 μ l cellulase SC releases.
Figure 11 DaCS enzymolysis, then centrifugal and washing are shown after gained 12ml supernatant in the concentration of total soluble sugar.Can find out that the rear main sugar of hydrolysis is glucose, wood sugar, arabinose, accessory constituent is galactolipin and mannose.
conclusion
Have studied the sugar release of different cellulase preparation.For Animal nutrition, commercial enzyme combines, particularly those enzymes for decomposing lignocellulose living beings combination in bio-ethanol industry, for being undesirable in Animal nutrition, main because this based composition can generate a large amount of wood sugar.We have found that, nonruminant can not effectively digest and utilize wood sugar.
Enzymatic compositions at least has following activity: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, wherein do not exist in enzymatic compositions or substantially there is not the one or both in following enzymatic activity: xylobiase is active and α-L-arabinofuranosidase active, while this enzymatic compositions is considered to lignocellulose degradation living beings, producing the most compositions useful being most suitable for being used as animal feed composition or the product as feedstuff.
example 5: wood sugar is as the energy source of broiler chicken
This research employs the male broiler chicken (1 age in days) of 240 Ross308 strains altogether.Weighed one by one by chicken when hatching, and be divided into 12 groups, often group comprises the chicken of 5 body weight equilibriums.Then weigh often organizing chicken, and will often organize on electrical heating Petersime tier rack type brooder that chicken is housed in cage.First week, respectively brooder and room temperature are set to 32 DEG C and 29 DEG C.After this, close the heat supply of brooder, but room temperature maintains 29 DEG C all the time in whole experimentation.Research continues 21 days, feeds to the one in four kinds of inorganic agents below chicken during this period: 1: basal diet (diet based on corn-soybean meal)+cornstarch 25%, D-wood sugar 0%; 2: basal diet+cornstarch 20%, D-wood sugar 5%; 3: basal diet+cornstarch 10%, D-wood sugar 15%; And 4: basal diet+cornstarch 0%, D-wood sugar 25%.In whole research, for all chickens provide fresh water and feed, make its free choice feeding.During research, monitoring feed amount and chicken body weight are for calculating body weight increase, arbitrarily feed intake and feed conversion rate weekly.
Research continues 21 days, but after 14 days of process, because the chicken death rate of inorganic agent 4 of feeding is higher and have disease symptoms and they removed from research.Basal diet is corn-soybean meal diet.
At first 14 days of research, compared with the chicken of other inorganic agents all of feeding, the feed food-intake and the weightening finish that comprise the chicken of 25% wood sugar in diet of feeding significantly reduce (P<0.05) (see Figure 12).The 14th day that studies, all chickens of inorganic agent 4 of feeding are removed from research.Now, the death rate of processed group (25% wood sugar) reaches 10%, and more chicken demonstrates the symptoms such as disorientation, blind and body shakes.
Feed after inorganic agent diet 21 days, with comprise 25% starch in diet of feeding and compare with the chicken of 0% wood sugar, the weightening finish comprising the chicken of 10% cornstarch and 15% wood sugar in diet of feeding significantly reduce and feed efficiency obviously poorer (P<0.05) (see Figure 13 and Figure 14).Feed comprise the chicken of 5% wood sugar and 20% cornstarch in diet weightening finish and FCR between other two kinds of inorganic agent gained weightening finishes and FCR, but with both all without significant difference.
Conclusion is, it is harmful that 25% energy source in diet is replaced with wood sugar to broiler health.Before producing remarkable adverse effect to growth performance, broiler chicken seems to tolerate in diet the wood sugar added up to 5%, but compared with control diet, even if add 5% wood sugar in the diet, growth performance numerically also can reduce.
example 6 uses 1500 tM with xT tM the corn of hydrolysis weak aqua ammonia processstraw stalk and causticity delignification switchgrass (DLswg) (DaCS)
Be add 1gDaCS and 5mlMilliQ water in the plastic tube of 12ml respectively to 8 volumes, mixing.Equally, be add 1gDLswg and 5mlMilliQ water in the plastic tube of 12ml to other 8 volumes, mixing.By all pipes at 5 DEG C hold over night to guarantee DaCS and DLswg well-hydrated.Within second day, add 150 μ l1NHCl, pH is adjusted to 4.6 to 5.1.Add enzyme (listed in following table 5), start reaction.This reacts on 50 DEG C, vibrate under 180rpm rotating speed 40h.Collect the fluid sample (120 μ l) during reaction time 6h, use 120 μ lMilliQ water dilute samples, in order to analyzing at being stored in 5 DEG C.At the end of enzyme reaction, in each sample of collecting, add 240 μ lmilliQ water again, then in 100 DEG C of water-baths, heat 5min, afterwards centrifugal 10min under 10000 × g.Filter 250 μ l supernatants subsequently, and analyze in 10 μ l filtrate sample introductions to HPLC.Filter the glucose and xylose reference material of 1% (w/v), use and carry out HPLC analysis with the described identical condition of " materials and methods " part and sample size.
table 5
1500 is the commercial enzymes that can derive from Danisco (now belonging to DuPont), and has some cellulase activity such as endoglucanase activity, activity of beta-glucosidase, and certain hemicellulase activity such as endo-xylanase is active.
xT5P is the commercial enzyme that can derive from ABVista, has inscribe 1-4 β xylanase activity.
result
Result shows to add in the pretreated maize straw of weak aqua ammonia and causticity delignification switchgrass 1500 tMwith the release of XT5P to glucose has cooperative effect, because serving as reasons 1500 tM(0.3ml/g) and the glucose of XT5P (1.4mg/gDM) coupling release is more than the glucose (table 6) discharged separately by often kind of enzyme.In this example, by adding 1500 tMwith xT5P and the endoglucanase activity, the endo-xylanase combination that is active and activity of beta-glucosidase that provide make glucose discharge in a large number, this with add trio tMresult similar. 1500 tMwith the combination of XT5P makes wood sugar discharge significantly minimizing, because with trio tMcomparing, there is not a large amount of α-L-arabinofuranosidase activity and xylobiase activity in the combination of this enzyme.
table 6. uses trio tM , 1500 tM with xT5P hydrolysis is through rare the release of xylose and glucose when the maize straw (DAcs) of pretreatment with agueous Ammonia and causticity delignification switchgrass (DLswg).
1dAcs-through the pretreated maize straw of weak aqua ammonia
2dLswg-causticity delignification switchgrass
N.d.-do not detect
Example 7 use daniscoXylanase tM with bG tM the process of hydrolysis weak aqua ammonia maize straw (DaCS) and causticity delignification switchgrass (DLswg)
Be add 1gDaCS and 5mlMilliQ water in the plastic tube of 12ml respectively to 12 volumes, mixing.Equally, be add 1gDLswg and 5mlMilliQ water in the plastic tube of 12ml to other 12 volumes, mixing.By all pipes at 5 DEG C hold over night to guarantee DaCS and DLswg well-hydrated.Within second day, add 150 μ l1NHCl, pH is adjusted to 4.6 to 5.1.Add enzyme (listed in following table 7), start reaction.This reacts on 50 DEG C, vibrate under 180rpm rotating speed 40h.Collect the fluid sample (120 μ l) during reaction time 6h, use 120 μ lMilliQ water dilute samples, in order to analyzing at being stored in 5 DEG C.At the end of enzyme reaction, in each sample of collecting, add 240 μ lmilliQ water again, then in 100 DEG C of water-baths, heat 5min, afterwards centrifugal 10min under 10000 × g.Filter 250 μ l supernatants subsequently, and analyze in 10 μ l filtrate sample introductions to HPLC.Filter the glucose and xylose reference material of 1% (w/v), use and carry out HPLC analysis with the described identical condition of " materials and methods " part and sample size.
table 7.
be the commercial enzyme (C2730) that can derive from Sigma-Aldrich, there is some cellulase activity, such as endoglucanase activity and activity of beta-glucosidase.
DaniscoXylanase tMbe derive from Dansico (now belonging to DuPont) commercial enzyme and there is endo-xylanase activity.
bG tMbe can derive from Danisco (now belonging to DuPont) commercial enzyme and there is activity of beta-glucosidase.
result
Result display is added in the pretreated maize straw of weak aqua ammonia and causticity delignification switchgrass daniscoXylanase tMwith bG tMto the release of glucose, there is cooperative effect, because serving as reasons (0.1ml/gDM), DaniscoXylanase tM(3.5 μ l/gDM) and bG tM(0.15ml/gDM) glucose that combination discharges is more than the glucose discharged separately by often kind of enzyme or only combined the glucose (table 8) discharged by two kinds of enzymes.In this example, by adding daniscoXylanase tMwith bG tMand the endoglucanase activity provided, endo-xylanase are active and the combination of activity of beta-glucosidase obtain with trio tMsimilar glucose release. daniscoXylanase tMwith bG tMcombination wood sugar is discharged significantly reduce because with trio tMcompare, in the combination of this enzyme, there is not a large amount of α-L-arabinofuranosidase activity and xylobiase activity.
table 8 uses trio tM , daniscoXylanase tM and bG tM hydrolysis is through the pretreated maize straw of weak aqua ammonia (DAcs) and causticity delignification switchgrass (DLswg) time xylose and glucose release.
1dAcs-through the pretreated maize straw of weak aqua ammonia
2dLswg-causticity delignification switchgrass
N.d.-do not detect
Example 8 use from aspergillus niger cellulase (SigmaC1184) and bG tM hydrolysis is through rare the maize straw (DaCS) of WITH AMMONIA TREATMENT and causticity delignification switchgrass (DLswg)
Be add 1gDaCS and 5mlMilliQ water in the plastic tube of 12ml respectively to 6 volumes, mixing.Equally, be add 1gDLswg and 5mlMilliQ water in the plastic tube of 12ml to other 8 volumes, mixing.By all pipes at 5 DEG C hold over night to guarantee DaCS and DLswg well-hydrated.Within second day, add 150 μ l1NHCl, pH is adjusted to 4.6 to 5.1.Add enzyme (listed in following table 9), start reaction.This reacts on 50 DEG C, vibrate under 180rpm rotating speed 40h.Collect the fluid sample (120 μ l) during reaction time 6h, use 120 μ lMilliQ water dilute samples, in order to analyzing at being stored in 5 DEG C.At the end of enzyme reaction, in each sample of collecting, add 240 μ lmilliQ water again, then in 100 DEG C of water-baths, heat 5min, afterwards centrifugal 10min under 10000 × g.Filter 250 μ l supernatants subsequently, and analyze in 10 μ l filtrate sample introductions to HPLC.Filter the glucose and xylose reference material of 1% (w/v), use and carry out HPLC analysis with the described identical condition of " materials and methods " part and sample size.
table 9
Cellulase from aspergillus niger is the commercial enzyme (C1184) that can derive from Sigma-Aldrich, and has some cellulase activity, such as endoglucanase activity, and endo-xylanase is active.
bG tMbe can derive from Danisco (now belonging to DuPont) commercial enzyme and there is activity of beta-glucosidase.
result
Result show to add in the pretreated maize straw of weak aqua ammonia and causticity delignification switchgrass from aspergillus niger cellulase and bG tMto the release of glucose, there is cooperative effect because by from aspergillus niger cellulase and bG tMthe glucose of coupling release is more than the glucose (table 10) discharged separately by often kind of enzyme.In this example, by add from aspergillus niger cellulase and bG tMand the endoglucanase activity provided, endo-xylanase are active and the combination of activity of beta-glucosidase obtain with trio tMsimilar glucose release.From aspergillus niger cellulase and bG tMcombination wood sugar is discharged significantly reduce because with trio tMcompare, in the combination of this enzyme, there is not a large amount of α-L-arabinofuranosidase activity and xylobiase activity.
table 10 uses trio tM , from aspergillus niger cellulase and bG tM hydrolysis when the pretreated maize straw of weak aqua ammonia (DAcs) and causticity delignification switchgrass (DLswg), xylose and glucose releases put.
1dAcs-through the pretreated maize straw of weak aqua ammonia
2dLswg-causticity delignification switchgrass
The all publications mentioned in above description are incorporated to herein by reference.Those skilled in the art should understand, and without departing from the scope and spirit in the present invention, can carry out difference amendment and change to the method for the invention and system.Although in conjunction with specific preferred embodiment, invention has been described, should be appreciated that, the present invention for required protection should not be confined to these specific embodiments undeservedly.In fact, also should in following Claims scope for biochemistry and biotechnology or the apparent different modification for implementing described pattern of the present invention of those skilled in the relevant art.

Claims (15)

1. prepare a method for feed addictive composition, described method comprises:
A. physics and/or chemical and/or biological method preprocessing lignocellulose living beings are used;
B. will through physics and/or chemistry and/or the pretreated lignocellulose biomass of biological method mix with enzymatic compositions, wherein said enzymatic compositions at least has following activity: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, and wherein said enzymatic compositions does not have or substantially do not have xylobiase activity, and/or it is active not have or substantially do not have α-L-arabinofuranosidase;
C. by the mixture incubation of described lignocellulose biomass and described enzymatic compositions at least about 3 to 120 hours;
D. and optionally carry out drying and/or optionally pack.
2. the purposes of an enzymatic compositions, wherein manufacturing for improving lignocellulose biomass in the process of the feed addictive composition of the nutritive value of animal, described enzymatic compositions at least has following activity: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, and wherein said enzymatic compositions does not have or substantially do not have xylobiase activity, and/or it is active not have or substantially do not have α-L-arabinofuranosidase.
3. according to method according to claim 1 or claim 2 or purposes, wherein said enzymatic compositions also has the one or both in following enzymatic activity: cellobiohydrolase I is active and cellobiohydrolase II is active.
4., according to method in any one of the preceding claims wherein or purposes, wherein said enzymatic compositions also has dissolubility polysaccharide monooxygenase activity.
5., according to method in any one of the preceding claims wherein or purposes, wherein said method also comprises to feed addictive composition described in detoxification.
6. according to method in any one of the preceding claims wherein or purposes, wherein said lignocellulose biomass is any cellulose or ligno-cellulosic materials, such as agricultural residue, bioenergy crops, industrial solid wastes, municipal solid waste, sludge from papermaking, yard waste, timber refuse, forestry waste and their combination.
7. according to method in any one of the preceding claims wherein or purposes, wherein said lignocellulose biomass is selected from corn ear, crop residue is maize peel such as, maize straw, grass, beet pulp, wheat stalk, wheat shell, oat straw, wheat time powder (wheatmiddlings), wheat prostitute (wheatshorts), rice bran, rice husk, wheat bran, oat shell, palm kernel, citrus pulp, cotton, lignin, Barley straw, hay, rice straw, rice husk, switchgrass, Chinese silvergrass, Value of Spartina Anglica, reed canary grass, paper discarded object, bagasse, Chinese sorghum bagasse, forage sorghum, broomcorn straw, soybean stalk, soybean, pulverize the component that trees obtain, branch, root, leaf, wood chip, sawdust, shrub and bushes, vegetables, fruit and flower.
8. a feed addictive composition, described feed addictive composition can obtain (such as, obtaining) by method according to any one of claim 1 to 7 or purposes.
9. a feed addictive composition or feed ingredient, described feed addictive composition or feed ingredient comprise by enzymatic compositions be hydrolyzed through physics and/or chemistry and/or the pretreated lignocellulose biomass of biological method, wherein said enzymatic compositions at least has following activity: endoglucanase activity, activity of beta-glucosidase and endo-xylanase are active, and do not have or substantially not there is xylobiase activity, and/or it is active not have or substantially do not have α-L-arabinofuranosidase.
10. a feed or feedstuff, described feed or feedstuff comprise feed addictive composition according to claim 9 or feed ingredient, or can be obtained the feed addictive composition of (preferably obtaining) by the method according to any one of claim 1 to 7 or purposes.
11. 1 kinds of pre-compositions, described pre-composition comprises feed addictive composition according to claim 9 or feed ingredient or can obtain the feed addictive composition of (preferably obtaining) and at least one mineral matter and/or at least one vitamin by the method according to any one of claim 1 to 7 or purposes.
12. method according to any one of claim 1 to 7 or purposes, wherein said method or purposes also comprise the step that feed ingredient is contacted with described feed addictive composition.
13. 1 kinds of methods preparing feedstuff, described method comprises makes feed ingredient and feed addictive composition according to claim 9 or feed ingredient, or can be obtained the feed addictive composition contact of (preferably obtaining) by the method according to any one of claim 1 to 7 or purposes.
14. 1 kinds for improving the method for the biophysical properties of animal, described method is comprised and can be obtained (such as by the method according to any one of claim 1 to 7 or purposes, obtain) feed addictive composition or feed addictive composition according to claim 9 or pre-composition according to claim 11 or feedstuff according to claim 10 or the feedstuff that can obtain (such as, obtaining) by method according to claim 13 be applied to animal.
15. can be obtained (such as by method according to any one of claim 1 to 7 or purposes, obtain) feed addictive composition or feed addictive composition according to claim 9 or pre-composition according to claim 11 or feedstuff according to claim 10 or the feedstuff of (such as, obtaining) can be obtained for improving the purposes of the biophysical properties of animal by method according to claim 1.
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