METHOD OF PREPARING A FEED ADDITIVE COMPOSITION, USE OF AN ENZYMATIC COMPOSITION IN COMBINATION WITH

专利摘要:
methods and compositions to improve the nutritional value of lignocellulosic biomass. The present invention relates to a method of preparing a feed additive composition comprising: (a) physically and/or chemically and/or biologically treating a lignocellulosic biomass, (b) mixing the physically pretreated lignocellulosic biomass and / or chemically and/or biologically with an enzymatic composition, wherein the enzymatic composition comprises at least the following activities: endoglucanase activity, (beta)-glucosidase activity and endoxylanase activity, and wherein the enzymatic composition comprises no or substantially no activity (beta)-xylosidase and/or no, or substantially no (alpha)-l-arabinofuranosidase activity, (c) incubate the same for at least about 3 to 120 hours and, (d) optionally, dry and/or optionally , pack.
公开号:BR112015031628B1
申请号:R112015031628-0
申请日:2014-06-19
公开日:2021-06-29
发明作者:Luis Fernando Romero Millán；Shukun Yu；Maria Walsh；Suzanne E. Lantz；Colin Mitchinson；Benjamin S. Bower；Susan Lund Arent
申请人:Dupont Nutrition Biosciences Aps.；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[0001] The present invention relates to methods and compositions to improve the nutritional value of lignocellulosic biomass. The present invention relates to methods and compositions that result in a lignocellulosic biomass suitable as a substitute for conventionally used grain starch in animal feed. BACKGROUND OF THE INVENTION
[0002] About 750 million tons of cereal grains per year are used for animal nutrition. Replacing a proportion of these cereal grains with cheaper food sources would provide a significant benefit to the population as a whole.
[0003] The replacement of grains (eg corn) by cheaper cellulosic materials (eg lignocellulosic biomass) could revolutionize animal feed markets, and/or increase the stability of food and energy markets.
[0004] The nutritional value of lignocellulosic biomass was not sufficient to make it a viable alternative to high energy diets such as grains. Monogastric animals, such as swine and poultry, for example, cannot digest and absorb glucose from lignocellulose from fibrous ingredients, such as agricultural waste, for example, to an extent that allows the replacement of significant amounts of cereal grains in the diet of the animal. Animals, especially monogastric animals, do not have the necessary enzymes to digest cellulose and hemicellulose, and even if they do, the retention time in the intestine is a limitation for the conversion of cellulose to glucose.
[0005] Cellulose is an organic compound with the formula(C6H10O5)n, a polysaccharide consisting of a linear chain of β(1^4) D-glucose units linked.
[0006] Hemicellulose is any one of several heteropolymers (matrix polysaccharides) present, together with cellulose, in almost all plant cell walls. Hemicelluloses include xylan, glucuronoxylan, arabinoxylan, glycomannan and xyloglucan. Hemicelluloses contain many different sugar monomers. For example, in addition to glucose, sugar monomers in hemicellulose can include xylose, mannose, galactose, rhamnose and arabinose. Hemicelluloses contain most of the D-pentose (C-5) sugars and occasionally also small amounts of L-sugars. Xylose is, in most cases, the most abundant sugar monomer, although in soft woods, mannose may be the most abundant sugar. Not only can common sugars be found in hemicellulose, but also the acidified form of them, eg glucuronic acid and galacturonic acid can be present.
[0007] Schutte et al (British J. of Nutrition 1991, 66, 83-93) investigated nutritional implications of D-xylose in pigs. Schutte et al taught that the ileal digestibility of D-xylose, like that of D-glucose, was close to 100%. The presence of D-xylose in the diet decreased the pH of the ileal food bolus and increased the ileal flux of volatile fatty acids, suggesting the occurrence of microbial degradation of D-xylose in the small intestine of pigs. Schutte suggested that the ileal and faecal digestibility of dry matter (DM), organic matter (OM), total energy (GE) and nitrogen (N), as well as N retention, decreased significantly in pigs fed with very high doses. high D-xylose (eg on a 200 g D-xylose/kg diet). Schutte (1991) also found that, with a 10% inclusion in pig diets, 50% of the xylose energy appeared in the urine.
[0008] Verstegen et al (J. Animal. Physiol. a. Anim. Nutr. 77 (1997) 180-188) performed a pig trial to evaluate xylose as an energy source for pigs. Depending on the calculation method used, when included in the 10% diet, between 38 and 64% of the xylose energy in the diet appeared as metabolizable energy (ME). When pigs fed 10% xylose diet were compared to similar pigs fed 5% glucose diet, ME of both diets was similar and weight gain was similar between treatments.
[0009] Savory et al (British Journal of Nutrition (1992), 67, 103-114) studied the metabolic fate of U-14C-labeled monosaccharides in birds, and suggested that xylose was absorbed more slowly than glucose and glucose. galactose, but faster than mannose and arabinose. Savory (1992) also found that xylose and arabinose were metabolized to a lesser degree than hexose (C6) sugars, as evidenced by the greater recovery of U-14C-labeled xylose and arabinose in poultry droppings. BRIEF DESCRIPTION OF THE DRAWINGS
[00010] Figure 1 shows the yield of glucose and xylose obtained by treating DaCS (corn stubble pretreated with dilute ammonia) with SC cellulase.
[00011] Figure 2 shows the release of glucose and xylose from DaCS using Accellerase®Trio™, Cellulase SC or Cellulase SC + endoxylanase.
[00012] Figure 3 shows the release of sugars from DaCS by Accellerase®Trio™, Cellulase SC118, Cellulase 151 and SC118 + xylanase.
[00013] Figure 4 shows the hydrolysis of DaCS by Accellerase®Trio™, Cellulase SC and Cellulase SC + 4 different endoxylanases.
[00014] Figure 5 shows an HPLC chromatogram of the hydrolyzate of DaCS produced by Accellerase®Trio™, 30 µL.
[00015] Figure 6 shows an HPLC chromatogram of the hydrolyzate of DaCS produced by cellulase SC and cellulase SC in combination with the endoxylanase SoloC118, 50 µL.
[00016] Figure 7 shows the hydrolysis of DaCS by Accellerase®Trio™, Cellulase SC and Cellulase SC + endoxylanases.
[00017] Figure 8 shows the solubilization of DaCS (DaCS residue weight, g) with or without SC118 cellulose and Danisco's Xylanase™ endoxylanase (see EP1222256, which is incorporated herein by reference) at 50 °C, pH 5 .0 for 40 h.
[00018] Figure 9 shows the solubilization of DaCS (% solubilized material) with or without SC118 cellulose and Danisco's Xylanase™ endoxylanase at 50 °C, pH 5.0, for 40 h.
[00019] Figure 10 shows the release of monosugars from DaCS by Cellulase SC118 and the dose effect of Danisco's Xylanase™ xylanase at 50 °C, pH 5.0, for 40 h.
[00020] Figure 11 shows the release of sugars (monomeric + polymeric forms) from DaCS by Cellulase SC118 and the dose effect of Danisco's Xylanase™ xylanase at 50 °C, pH 5.0 for 40 h.
[00021] Figure 12 shows feed intake and body weight gain in broilers fed with xylose as an energy source.
[00022] Figure 13 shows the body weight at day 21 in broilers fed with xylose as an energy source.
[00023] Figure 14 shows the feed conversion ratio d0-21 in broilers fed with xylose as an energy source.
[00024] Figure 15 shows a nucleotide sequence (SEQ ID NO: 1) encoding an endoxylanase (FoxXyn6). The signal sequence is in bold (uppercase letters).
[00025] Figure 16 shows a nucleotide sequence (SEQ ID NO: 2) encoding an endoxylanase (FoxXyn6).
[00026] Figure 17 shows a polypeptide sequence (SEQ ID NO: 3) of an endoxylanase (FoxXyn6). This is the active form of the enzyme (eg, the mature form of the enzyme).
[00027] Figure 18 shows a nucleotide sequence (SEQ ID NO: 4) encoding an endoxylanase (FoxXyn4). The signal sequence is in bold (uppercase letters).
[00028] Figure 19 shows a nucleotide sequence (SEQ ID NO: 5) encoding an endoxylanase (FoxXyn4).
[00029] Figure 20 shows a polypeptide sequence (SEQ ID NO: 6) of an endoxylanase (FoxXyn4). This is the active form of the enzyme (eg, the mature form of the enzyme).
[00030] Figure 21 shows a nucleotide sequence (SEQ ID NO: 7) encoding an endoglucanase (EG1) from Aspergillus niger (CBS513.88).
[00031] Figure 22 shows a polypeptide sequence (SEQ ID NO: 8) of an endoglucanase (EG1) from Aspergillus niger (CBS513.88).
[00032] Figure 23 shows a nucleotide sequence (SEQ ID NO: 9) encoding an endoglucanase (EG2) from Aspergillus niger CBS513.88.
[00033] Figure 24 shows a polypeptide sequence (SEQ ID NO: 10) of an endoglucanase (EG2) from Aspergillus niger CBS513.88.
[00034] Figure 25 shows a nucleotide sequence (SEQ ID NO: 11) encoding an endoxylanase.
[00035] Figure 26 shows a polypeptide sequence (SEQ ID NO: 12) of an endoxylanase.
[00036] Figure 27 shows a nucleotide sequence (SEQ ID NO: 13) encoding a β-glucosidase from Aspergillus niger CBS513.88.
[00037] Figure 28 shows a polypeptide sequence (SEQ ID NO: 14) of a β-glucosidase from Aspergillus niger CBS513.88.
[00038] Figure 29 shows a nucleotide sequence (SEQ ID NO: 15) encoding a lytic polysaccharide monooxygenase from Aspergillus niger CBS513.88.
[00039] Figure 30 shows a polypeptide sequence (SEQ ID NO: 16) of a lytic polysaccharide monooxygenase from Aspergillus niger CBS513.88.
[00040] Figure 31 shows a nucleotide sequence (SEQ ID NO: 17) encoding an Aspergillus niger CHB1A (CBS513.88).
[00041] Figure 32 shows a polypeptide sequence (SEQ ID NO: 18) of a CHB1A from Aspergillus niger (CBS513.88).
[00042] Figure 33 shows a nucleotide sequence (SEQ ID NO: 19) encoding a CHB1B from Aspergillus niger (CBS513.88).
[00043] Figure 34 shows a polypeptide sequence (SEQ ID NO: 20) of a CHB1B from Aspergillus niger (CBS513.88).
[00044] Figure 35 shows a nucleotide sequence (SEQ ID NO: 21) encoding a CHB1 from Trichoderma reesei.
[00045] Figure 36 shows a polypeptide sequence (SEQ ID NO: 22) of a CHB1 from Trichoderma reesei.
[00046] Figure 37 shows a nucleotide sequence (SEQ ID NO: 23) encoding a CHB2 from Trichoderma reesei.
[00047] Figure 38 shows a polypeptide sequence (SEQ ID NO: 24) of a CHB2 from Trichoderma reesei.
[00048] Figure 39 shows a nucleotide sequence (SEQ ID NO: 25) encoding an endoglucanase (EG1) from Trichoderma reesei.
[00049] Figure 40 shows a polypeptide sequence (SEQ ID NO: 26) of an endoglucanase (EG1) from Trichoderma reesei.
[00050] Figure 41 shows a nucleotide sequence (SEQ ID NO: 27) encoding an endoglucanase (EG2) from Trichoderma reesei.
[00051] Figure 42 shows a polypeptide sequence (SEQ ID NO: 28) of an endoglucanase (EG2) from Trichoderma reesei.
[00052] Figure 43 shows a nucleotide sequence (SEQ ID NO: 29) encoding an endoglucanase (EG3) from Trichoderma reesei.
[00053] Figure 44 shows a polypeptide sequence (SEQ ID NO: 30) of an endoglucanase (EG3) from Trichoderma reesei.
[00054] Figure 45 shows a nucleotide sequence (SEQ ID NO: 31) encoding a lytic polysaccharide monooxygenase from Trichoderma reesei.
[00055] Figure 46 shows a polypeptide sequence (SEQ ID NO: 32) of a lytic polysaccharide monooxygenase from Trichoderma reesei.
[00056] Figure 47 shows a nucleotide sequence (SEQ ID NO: 33) encoding an endoxylanase from Trichoderma reesei.
[00057] Figure 48 shows a polypeptide sequence (SEQ ID NO: 34) of an endoxylanase from Trichoderma reesei.
[00058] Figure 49 shows a nucleotide sequence (SEQ ID NO: 35) encoding a β-glycosidase from Trichoderma reesei.
[00059] Figure 50 shows a polypeptide sequence (SEQ ID NO: 36) of a β-glycosidase from Trichoderma reesei. STATEMENTS OF THE INVENTION
[00060] In a first aspect, the present invention provides a method of preparing feed additive composition, comprising:
[00061] a. pre-treat physically and/or chemically and/or biologically the lignocellulosic biomass,
[00062] b. mixing the physically and/or chemically and/or biologically pretreated lignocellulosic biomass with an enzymatic composition, wherein the enzymatic composition comprises at least the following activities: endoglucanase activity, β-glycosidase activity and endoxylanase activity, and wherein the enzymatic composition comprises no, or substantially no, β-xylosidase activity and/or no, or substantially no, α-L-arabinofuranosidase activity
[00063] c. incubate it for at least about 3 to 120 hours, preferably 6 to 48 hours,
[00064] d. and optionally drying and/or optionally packaging.
[00065] In another aspect, the present invention provides the use of an enzyme composition, in which the enzyme composition comprises at least the following activities: endoglucanase activity, β-glucosidase activity and endoxylanase activity, and in which the enzyme composition comprises none, or substantially no β-xylosidase activity and/or no, or substantially no α-L-arabinofuranosidase activity, in the manufacture of a feed additive composition for improving the nutritional value to an animal of a lignocellulosic biomass.
[00066] Another aspect of the present invention is a feed additive composition obtainable (eg obtained) by a method of the present invention.
[00067] In another aspect, there is provided a feed additive composition or a feed ingredient comprising a hydrolyzed physically and/or chemically and/or biologically pre-treated lignocellulosic biomass with an enzyme composition, wherein the enzyme composition comprises at least the following: an endoglucanase activity, a β-glucosidase activity and an endoxylanase activity, and no or substantially no β-xylosidase activity and/or α-L-arabinofuranosidase activity.
[00068] Another aspect of the present invention provides a feed or feed for animals comprising a feed additive composition or feed ingredient according to the present invention, or a feed additive composition obtainable (preferably obtained) by a method or use of the present invention.
[00069] In another aspect, a premix comprising a feed additive composition or feed ingredient according to the present invention or a feed additive composition obtainable (preferably obtained) by a method or use of the present is provided. invention, and at least one mineral and/or at least one vitamin.
[00070] The present invention further provides a method of preparing an animal feed comprising contacting a feed component with a feed additive composition or feed ingredient according to the present invention, or a feed additive composition. ration obtainable (preferably obtained) by a method or use of the present invention.
[00071] The present invention also provides a method for improving a biophysical characteristic of an animal, which comprises administering to the animal a feed additive composition obtainable (e.g., obtained) by a method of the present invention, or an additive composition of feed according to the present invention, or a premix according to the present invention, or an animal feed according to the present invention, or an animal feed obtainable (e.g. obtained) by a method of the present invention.
[00072] In yet another aspect, the present invention provides the use of a feed additive composition obtainable (e.g., obtained) by a method or use of the present invention, or a feed additive composition according to the present invention , or a premix according to the present invention, or an animal feed according to the present invention, or an animal feed obtainable (e.g., obtained) by a method of the present invention for improving a biophysical characteristic of a animal.
[00073] In a preferred embodiment, the enzyme composition comprises no, or substantially no β-xylosidase activity, and no, or substantially no, α-L-arabinofuranosidase activity.
[00074] Suitably, the enzyme composition may further comprise one or more enzymes selected from the group consisting of: a cellobiohydrolase I and a cellobiohydrolase II.
[00075] Suitably, the enzyme composition may further comprise a lytic polysaccharide monooxygenase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
[00076] The inventors' research (see Example 5) has shown that high levels of C-5 monomeric sugars (eg xylose) in the feed can have negative consequences on the health of an animal that ingests the feed.
[00077] However, the use of high levels of lignocellulosic biomass in foodstuffs can result in an insufficient nutritional value of the feed or, if lignocellulosic biomass is treated in an attempt to make them more nutritionally valuable , a situation may occur where the content of C-5 monomeric sugars (eg xylose) becomes too high in the animal feed and can be harmful to the animal's health.
[00078] Therefore, the inventors have determined that C-5 monomeric sugars should preferably be removed from any lignocellulosic biomass before being used to feed an animal.
[00079] The present inventors have presently discovered a unique method for treating lignocellulosic biomass with an enzymatic composition that can result in a feed or feed additive composition with reduced level of C-5 monomeric sugars (such as xylose), while maintaining high levels of C-6 oligomers and monomeric sugars that are beneficial (eg, nutritional) to animals.
[00080] It was surprising that by removing the C-5 monomeric carbohydrate formation activities, significant formation of xylo-oligomers could be achieved without compromising the efficiency or time of conversion of cellulose to glucose. The resulting composition was much more beneficial in feed applications.
[00081] The present inventors have surprisingly found that eliminating C5 monomeric sugar production and promoting C5 oligomer production along with cellulase activities (eg to produce a C6 oligomer and C-6 monomeric sugars) is a advantageous mix of activities.
[00082] Methods and uses comprising this enzymatic composition will allow the inclusion of higher levels of lignocellulosic biomass in animal feed and in animal diets.
[00083] The present inventors surprisingly found that the nutritional value of lignocellulosic biomass for animals (particularly for monogastric animals) can be significantly improved with the use of an enzyme composition comprising at least the following activities: endoglucanase activity, β- activity glycosidase and endoxylanase activity; wherein one or both of the following enzyme activities are absent or substantially absent in the enzyme composition: β-xylosidase activity and/or α-L-arabinofuranosidase activity in the treatment of lignocellulosic biomass.
[00084] Surprisingly it was found that by reducing or excluding the activities of beta-xylosidase and alpha-L-arabinofuranosidase, the nutritional value of lignocellulosic biomass for animals (particularly for monogastric animals) can be significantly improved.
[00085] It appears from the data that the benefits derive from reducing the amount of monomeric sugars (particularly C-5 monomeric sugars) in the composition, with the concomitant and significant increase in oligomeric sugars (eg, xylo-oligomers) in composition.
[00086] This was surprising because prior to the present invention, it was known that if C6 monomer-forming enzymes were removed from the system, the accumulation of products (eg, cellobiose), the enzyme reaction is progressively delayed due to feedback inhibition of the reaction. Thus, it was understood, prior to the present invention, that the feedback mechanism of the reaction, when the amount of monomeric sugars formed was reduced, resulted in an overall reduction in the enzymatic reaction. Surprisingly, no evident feedback mechanism was observed with the enzyme composition, for example, which had low β-xylosidase activity and/or α-L-arabinofuranosidase activity for use in the present invention.
[00087] The present inventors have surprisingly found that methods using enzymatic mixtures that break down cellulose and hemicellulose, but that do not have monomeric carbohydrate-forming activities, or that have reduced monomer-forming activities (particularly no formation activities of C-5 monomeric carbohydrates can significantly improve the nutritional value of lignocellulosic biomass in animals (particularly, monogastric animals).
[00088] This effect is further enhanced by pre-treatment of lignocellulosic biomass physically and/or chemically and/or biologically. Therefore, the lignocellulosic biomass is physically and/or chemically and/or biologically treated, and the physically, chemically and/or biologically pre-treated lignocellulosic biomass is further treated with an enzymatic composition comprising at least the following activities: endoglucanase activity, β activity -glucosidase and endoxylanase activity; wherein one or both of the following enzyme activities are absent or substantially absent in the enzyme composition: β-xylosidase activity and α-L-arabinofuranosidase activity.
[00089] The present invention means that lignocellulosic biomass can be used as a substitute for conventionally used starch (eg grain) in animal feed. This has significant advantages. For example, the use of the present invention means that a significant proportion of the cereal grains conventionally used for animal nutrition would no longer need to be used in animal feed applications. The present invention enables a reduction in the cost of preparing animal feed. Additionally or alternatively, the present invention eases the pressures on resources in supply chains that have been caused by the larger human population and restrictions on the supply of fossil fuels.
[00090] The presently claimed invention is relevant to both monogastric animals and ruminants, but it is more specifically relevant to monogastric animals.
[00091] In ruminants, food supplements according to the present invention or produced by the present invention can be used to replace cereal grains that are used for intensive production of meat and milk in ruminants, and/or to increase efficiency of the system.
[00092] The present invention relates to a method for preparing a feed additive composition comprising:
[00093] a. pre-treat physically and/or chemically and/or biologically the lignocellulosic biomass,
[00094] b. mixing the physically and/or chemically and/or biologically pretreated lignocellulosic biomass with an enzymatic composition, wherein the enzymatic composition comprises at least the following activities: endoglucanase activity, β-glycosidase activity and endoxylanase activity, and wherein the enzymatic composition comprises no or substantially no β-xylosidase activity and/or no or substantially no α-L-arabinofuranosidase activity
[00095] c. incubate it for at least about 3 to 120 hours, preferably 6 to 48 hours,
[00096] d. and optionally drying and/or optionally packaging.
[00097] The term "absent" or "none" as used herein means that the enzyme composition has no β-xylosidase activity and no α-arabinofuranosidase activity.
[00098] The terms "substantially absent" and "substantially none", as used herein, in reference to β-xylosidase activity and/or α-L-arabinofuranosidase activity, mean an activity less than the activity of 3000 β-xylosidase units/mg (suitably less than 2000 units/mg, suitably less than 1500 units/mg), as determined using the "beta-xylosidase activity assay" taught herein and/or less than 1000 units/mg of activity of α-L-arabinofuranosidase (suitably less than 500 units/mg, suitably less than 450 units/mg) as determined using the "alpha-L-arabinofuranosidase activity assay" taught herein.
[00099] In one embodiment, the terms "substantially absent" and "substantially without" as used herein in reference to β-xylosidase activity and/or α-L-arabinofuranosidase activity mean an activity of less than 1500 units/mg of the β-xylosidase activity, as determined using the "Beta-Xylosidase Activity Assay" taught herein, and/or less than 450 units/mg of α-L-arabinofuranosidase activity, as determined using the "Alpha-Activity Assay" L-Arabinofuranosidase" taught herein.
[000100] In one embodiment, the enzyme composition for use in the present invention has less than 3000 units/mg of β-xylosidase activity (suitably less than 2000 units/mg, suitably less than 1500 units/mg), as determined using the " beta-xylosidase activity assay" taught herein, and less than 1000 units/mg of α-L-arabinofuranosidase activity (suitably less than 500 units/mg, suitably less than 450 units/mg), as determined using the "assay of alpha-L-arabinofuranosidase activity" taught in this document.
[000101] In one embodiment, the terms "substantially absent" and "substantially none" as used herein in reference to β-xylosidase activity and/or α-L-arabinofuranosidase activity mean an activity of less than 1500 units/mg of the β-xylosidase activity, as determined using the "Beta-Xylosidase Activity Assay" taught herein, and less than 450 units/mg of α-L-arabinofuranosidase activity, as determined using the "Alpha-Activity Assay" L-Arabinofuranosidase" taught in this document.
[000102] In a preferred embodiment, the enzyme composition comprises no, or substantially no β-xylosidase activity, and no, or substantially no, α-L-arabinofuranosidase activity.
[000103] In a preferred embodiment, the enzyme composition comprises no, or substantially no β-xylosidase activity, and no, or substantially no, α-L-arabinofuranosidase activity.
[000104] In a preferred embodiment, the lignocellulosic biomass for use in the present invention may be a physically pretreated, chemically pretreated, biologically pretreated, or combinations thereof.
[000105] In another embodiment, the physically, chemically and/or biologically pretreated lignocellulosic biomass can be incubated with the enzyme composition for at least about 6 to 48 hours.
[000106] In another preferred embodiment, the enzyme composition for use in the present invention may consist essentially (or consist) of endoglucanase activity, β-glucosidase activity and endoxylanase activity.
[000107] In one embodiment, the enzyme composition for use in the present invention may further comprise one or both of the following enzyme activities: cellobiohydrolase I activity and cellobiohydrolase II activity.
[000108] In one embodiment, the enzyme composition for use in the present invention may further comprise the lytic polysaccharide monooxygenase activity.
[000109] In some embodiments, the enzyme composition for use in the present invention may comprise β-glucosidase activity and endoxylanase activity, and one or more of the following enzymatic activities: cellobiohydrolase I activity, cellobiohydrolase II activity or polysaccharide activity lytic monooxygenase.
[000110] In some embodiments, the enzyme composition for use in the present invention may consist essentially (or consist) of endoglucanase activity, β-glucosidase activity and endoxylanase activity, and one or more of the following enzymatic activities: cellobiohydrolase I activity, activity cellobiohydrolase II or lytic polysaccharide monooxygenase activity.
[000111] In some embodiments, the enzyme composition for use in the present invention may comprise endoglucanase activity, β-glucosidase activity and endoxylanase activity, and two or more (appropriately three) of the following enzymatic activities: cellobiohydrolase I activity, cellobiohydrolase II activity or lytic polysaccharide monooxygenase activity.
[000112] In some embodiments, the enzyme composition for use in the present invention may comprise (or consist essentially of, or consist of) endoglucanase activity, β-glucosidase activity and endoxylanase activity, and two or more (appropriately three) of the following enzymatic activities : cellobiohydrolase I activity, cellobiohydrolase II activity or lytic polysaccharide monooxygenase activity.
[000113] In one embodiment, the enzyme composition for use in the present invention can be characterized by its enzymatic activity.
[000114] In one embodiment, the enzyme composition for use in the present invention comprises (or consists essentially of, or consists of) at least the following activities: endoglucanase activity, as determined by the "Endoglucanase Activity Assay", β-glucosidase activity, as determined using the "Beta-Glycosidase Activity Assay" and endoxylanase activity as determined using the "Endoxylanase Activity Assay".
[000115] The ranges in which these activities may be present in said enzyme composition are defined in the tables below and in the subsequent paragraphs:

[000116] 1A unit of CMC activity releases 1 μmol of reducing sugars (expressed as glucose equivalents) in one minute at 50°C and pH 4.8.
[000117] 2A pNPG unit represents 1 μmol of nitrophenol released from para-nitrophenyl-B-D-glycopyranoside per minute at 50°C and at pH 4.8.
[000118] 3An ABX unit is defined as the amount of enzyme needed to generate 1 μmol of xylose reducing sugar equivalent per minute at 50°C and pH 5.3.
[000119] In one embodiment, the enzyme composition for use in the present invention may comprise at least 500 CMC U/g of endoglucanase activity (suitably at least 1000 CMC U/g of activity, suitably at least 2000 CMC U/g of activity or suitably at least 3000 CMC U/g activity) as determined using the "Endoglucanase Activity Assay".
[000120] In another embodiment, the enzyme composition for use in the present invention may comprise at least 200 pNPG U/g of β-glycosidase activity (suitably at least 1000 pNPG U/g of activity, suitably at least 1500 pNPG U/g of activity, or suitably at least 2500 pNPG U/g activity), as determined using the "Beta-glucosidase Activity Assay".
[000121] In another embodiment, the enzyme composition for use in the present invention may comprise at least 1500 ABX U/g of endoxylanase activity (suitably at least activity equal to 3000 ABX U/g, suitably activity equal to at least 4000 ABX U/ g or suitably at least 4500 ABX U/g activity, suitably at least 5000 ABX U/g) as determined using the "Endoxylanase Activity Assay".
[000122] Suitably, the enzyme composition for use in the present invention may comprise (or consist essentially of, or consist of) at least 500 CMC U/g of endoglucanase activity (suitably at least 1000 CMC U/g of activity, suitably at least 2000 CMC U/g activity or suitably at least 3000 CMC U/g activity), as determined using the "Endoglucanase Activity Assay" at least 200 pNPG U/g β-glucosidase activity (suitably at least 1000 pNPG activity U/g, suitably at least 1500 pNPG U/g activity or suitably at least 2500 pNPG U/g activity), as determined using the "Beta-Glucosidase Activity Assay", and at least 1500 ABX U/g activity endoxylanase (suitably at least 3000 ABX U/g activity, suitably at least 4000 ABX U/g activity or suitably at least 4500 ABX U/g activity, suitably at least 5000 ABX U/g) as determined using the "Activity Test Endoxylanase".
[000123] In one embodiment, the enzyme composition for use in the present invention may comprise at least about 500 CMC U/g to about 4000 CMC U/g of endoglucanase activity (suitably at least 1000 CMC U/g to about 3500 CMC U /g activity, suitably at least about 1500 CMC U/g at about 3000 CMC U/g activity or suitably at least about 1500 CMC U/g at about 2500 CMC U/g activity), as determined using the "Endoglucanase Activity Assay".
[000124] In another embodiment, the enzyme composition for use in the present invention may comprise at least about 200 pNPG U/g to about 3500 pNPG U/g of β-glycosidase activity (suitably at least about 300 pNPG U/g to about from 3000 pNPG U/g activity, suitably at least about 500 pNPG U/g activity to about 2500 pNPG U/g activity, or suitably at least about 1000 pNPG U/g at about 2000 pNPG U/g of activity) as determined using the "Beta-glucosidase Activity Assay".
[000125] In another embodiment, the enzyme composition for use in the present invention may comprise at least about 1500 ABX U/g to about 6000 ABX U/g of endoxylanase activity (suitably at least about 2000 ABX U/g to about 5000 ABX U/g activity, suitably at least about 2500 ABX U/g at about 4500 ABX U/g activity, or suitably at least about 3000 ABX U/g at about 4000 ABX U/g activity), as per determined using the "Endoxylanase Activity Assay".
Suitably, the enzyme composition for use in the present invention may comprise (or consist essentially of, or consist of) about 500 CMC U/g to about 4000 CMC U/g of endoglucanase activity (suitably at least about 1000 CMC U/g about 3500 CMC U/g activity, suitably at least about 1500 CMC U/g at about 3000 CMC U/g activity, or suitably at least about 1500 CMC U/g at about 2500 CMC U/ g activity), as determined using the "Endoglucanase Activity Assay", about 200 pNPG U/g about 3500 pNPG U/g β-glycosidase activity (suitably at least about 300 pNPG U/g about 3000 pNPG U/g activity, suitably at least about 500 pNPG U/g activity to about 2500 pNPG U/g activity, or suitably at least about 1000 pNPG U/g about 2000 pNPG U/g activity) , as determined using the "Beta-Glucosidase Activity Assay", and at least about 1500 ABX U/ga ce about 6000 ABX U/g endoxylanase activity (suitably at least about 2000 ABX U/g activity to about 5000 ABX U/g activity, suitably at least about 2500 ABX U/g about 4500 ABX U activity /g activity, or suitably at least about 3000 ABX U/g and about 4000 ABX U/g activity) as determined using the "Endoxylanase Activity Assay".
[000127] In a preferred embodiment, the enzyme composition for use in the present invention may comprise (or consist essentially of, or consist of) about 1000 CMC U/g to about 3500 CMC U/g of endoglucanase activity, as determined using the "Endoglucanase Activity Assay", about 300 pNPG U/g about 3000 pNPG U/g of β-glycosidase activity, as determined using the "Beta-glycosidase Activity Assay" and about 2000 ABX U/g about 5000 ABX U/g of endoxylanase activity as determined using the "Endoxylanase Activity Assay".
[000128] In another embodiment, the enzyme composition for use in the present invention may further comprise one or more of the following enzymatic activities: protease activity (for example, serine protease activity (EC 3.4.21) and/or alkaline subtilisin protease activity (EC 3.4.21.62)), pectinase activity (EC 3.2.1.15), α-glucuronidase activity (EC 3.2.1.139), β-glucuronidase activity (EC3.2.1.31) or esterase activity (EC 3.1.1.73).
[000129] The esterase activity can be the activity of feruloyl esterase (E.C. 3.1.1.73).
[000130] The endoglucanase activity, as referred to in the present invention, may be the endo-1,4-β-D-glucanase activity. An endoglucanase is one that catalyzes the endohydrolysis of (1^4) - β-D-glucosidic bonds in cereal cellulose, lichenin and β-D-glucans. In other words, endoglucanase activity, as defined herein, means an enzyme that endohydrolyzes (1^4)-β-D-glycosidic bonds in cereal cellulose, lichenin and β-D-glucans. The endoglucanase activity can be classified under E.C. classification with E.C. number 3.2.1.4. Another name for endoglucanase is β-glucanase.
[000131] An endoglucanase for use in the enzyme composition for use in the present invention may be one or more endoglucanases encoded by a nucleic acid comprising (or consisting of, or consisting essentially of) one or more nucleotide sequences selected from the group consisting of in: SEQ ID No. 29, SEQ ID No. 27, SEQ ID No. 25, SEQ ID No. 9 and SEQ ID No. 7.
[000132] An endoglucanase for use in the enzyme composition for use in the present invention may be one or more endoglucanases comprising (or consisting of, or consisting essentially of) one or more polypeptide sequences selected from the group consisting of: SEQ ID No 30, SEQ ID No. 28, SEQ ID No. 26, SEQ ID No. 10 and SEQ ID No. 8. "Endoglucanase Activity Assay" (CMC U/g)
[000133] Pipette 1 mL of 1% sodium carboxymethylcellulose (CMC) saline solution (prepared with 0.05 M sodium acetate buffer) into the sample and blank tubes. Incubate tubes in a 50°C water bath for 10 minutes. Pipette 1 mL of the enzyme dilution at 15 second intervals into the sample tubes. Mix tubes after each addition. After 10 minutes, add 3 mL of 3,5-dinitrosalicylic acid 1% sodium salt (DNS) in the same order and time as the enzyme addition to the sample tubes. Add 3 mL DNS to sample blank tubes. After adding the DNS, remove the test tubes to another rack that is not in the 50°C water bath. Add 1 mL of the diluted enzyme to the corresponding blank sample. Cap the tubes and boil for exactly 5 minutes. Remove from the 100°C water bath and place in an ice bath for 10 minutes. Leave at room temperature for 10 to 15 minutes. Transfer to 3 mL cuvettes. Using the reagent blank to zero the spectrophotometer, each sample is read at 540 nm against deionized water.
[000134] The activity in this procedure is measured against a standard enzyme with assigned CMC units. One unit of CMC activity releases 1 μmol of reducing sugars (expressed as glucose equivalents) in one minute at 50°C and pH 4.8.
[000135] In one embodiment, a lytic polysaccharide monooxygenase for use in the present invention can be one taught in Levasseur et al Biotechnology for Biofuels 2013, 6: 41 and Kittle et al Biotechnology for Biofuels 2012, 5: 79, these references are incorporated herein by reference.
[000136] A lytic polysaccharide monooxygenase for use in the enzyme composition for use in the present invention may be one or more lytic polysaccharide monooxygenase encoded by a nucleic acid comprising (or consisting of, or consisting essentially of) one or more sequences of nucleotides selected from the group consisting of: SEQ ID No. 31 and SEQ ID No. 15.
[000137] A lytic polysaccharide monooxygenase for use in the enzyme composition for use in the present invention may be one or more lytic polysaccharide monooxygenases comprising (or consisting of, or consisting essentially of) one or more of the polypeptide sequences selected from the group consisting of in: SEQ ID No. 32 and SEQ ID No. 16.
[000138] The cellobiohydrolase (CBH) activity can be a class I CBH activity (CBH I) or a class II CBH activity (CBH II), or a combination of both CBH I and CBH II. Suitably, cellobiohydrolase can hydrolyze (1^4)-β-D-glycosidic bonds in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains. Another term for cellobiohydrolase activity may be exo-cellobiohydrolase activity or cellulose 1,4-β-cellobiohydrolase activity. Cellobiohydrolase II activity can be classified under the E.C. classification with the E.C. number 3.2.1.91. Cellobiohydrolase I activity can be classified under E.C. classification with E.C. number 3.2.1.176.
[000139] A cellobiohydrolase (CBH) for use in the enzyme composition for use in the present invention may be one or more cellobiohydrolases encoded by a nucleic acid that comprises (or consists of, or consists essentially of) one or more sequences of nucleotides selected from the group consisting of: SEQ ID No. 23, SEQ ID No. 21, SEQ ID No. 19 and SEQ ID No. 17.
[000140] A cellobiohydrolase (CBH) for use in the enzyme composition for use in the present invention may be one or more cellobiohydrolases comprising (or consisting of, or consisting essentially of) one or more of the polypeptide sequences selected from the group consisting of : SEQ ID No. 24, SEQ ID No. 22, SEQ ID No. 20 and SEQ ID No. 18.
[000141] Endoxylanase activity - may be endo-1,4-beta-xylanase activity. Preferably, the endoxylanase endohydrolyzes the (1,4)-β-D-xylosidic bond in the xylans. Preferably, the endoxylanase is classified as E.C. 3.2.1.8.
[000142] An endoxylanase for use in the enzyme composition for use in the present invention may be one or more endoxylanases encoded by a nucleic acid that comprises (or consists of, or consists essentially of) one or more nucleotide sequences selected from the group consisting of: SEQ ID No. 33, SEQ ID No. 11, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 2 and SEQ ID No. 1.
[000143] An endoxylanase for use in the enzyme composition for use in the present invention may be one or more endoxylanases comprising (or consisting of, or consisting essentially of) one or more polypeptide sequences selected from the group consisting of: SEQ ID No. 34 , SEQ ID No. 12, SEQ ID No. 6 and SEQ ID No. 3. "Endoxylanase Activity Assay" (ABX U/g)
[000144] Pipette 1.8 mL of 1% Birchwood 4-O-Methyl Glucuronoxylan Substrate Solution into each test tube. Incubate for 10 to 15 minutes, allowing to equilibrate to 50°C. Pipette 0.2 ml of the enzyme dilution using positive displacement pipettes or equivalent. Whirl until blended. Incubate each sample at 50°C for exactly 5 minutes. Add 3 ml of 3.5-nitrosalicylic acid (DNS) sodium salt solution 1% and mix. Cover the top of the test tubes with lids to prevent evaporation. Place test tubes in a boiling water bath for exactly 5 minutes. Cool test tubes for 10 minutes in an ice/water bath. Incubate the test tube for 10 minutes at room temperature. Transfer the contents of the test tubes to cuvettes and measure at 540 nm against deionized water. Correct the absorbance for the background color by subtracting the corresponding enzyme bank. This assay measures the release of reducing sugars by the action of endoxylanase in a birch wood xylan substrate. The reducing sugar release rate, measured with the DNS, is proportional to the enzymatic activity.
[000145] An ABX unit is defined as the amount of enzyme needed to generate 1 μmol of xylose reducing sugar equivalent per minute at 50°C and pH 5.3.
[000146] The β-glucosidase activity, as defined in this document, is the hydrolysis of non-reducing terminal β-D-glycosyl residues with the release of β-D-glucose. β-glucosidase activity can be classified according to E.C. classification with E.C. number 3.2.1.21.
[000147] A β-glycosidase for use in the enzyme composition for use in the present invention may be one or more β-glycosidases encoded by a nucleic acid that comprises (or consists of, or consists essentially of) one or more nucleotide sequences selected from the group consisting of: SEQ ID No. 35 and SEQ ID No. 13.
[000148] A β-glycosidase for use in the enzyme composition for use in the present invention may be one or more β-glycosidases comprising (or consisting of, or consisting essentially of) one or more polypeptide sequences selected from the group consisting of: SEQ ID No. 36 and SEQ ID No. 14."Beta-glucosidase activity assay" (pNPG U/g)
[000149] Pipette 1 mL of 3% nitrophenyl-beta-D-glycopyranoside (pNPG) solution (prepared with 0.05 M sodium acetate buffer) into test tubes in duplicate for each sample and control. Place in a 50°C water bath for 5 minutes. Add 200 μl of control or sample to their respective tubes in duplicate at 15 to 30 second intervals. To the reagent blank tube, add 200 μL of sodium acetate buffer. Whirl each tube after adding the sample. Allow tubes to incubate for 10 minutes. After 10 minutes of incubation, add 500 μL of 1M sodium carbonate solution to stop the reaction. Whirl each tube after addition and place the tube on a shelf outside the water bath. Add 10 mL of milli-Q water to each tube and vortex to mix. Using the reagent blank as zero on the spectrophotometer, the concentration of 4-nitrophenol is measured by reading each sample at 400 nm.
[000150] One pNPG unit represents 1 μmol of nitrophenol released from para-nitrophenyl-B-D-glycopyranoside per minute at 50°C and pH 4.8.
[000151] The β-xylosidase activity can hydrolyze successive xylose residues from the non-reducing terminal of (1^3)-β-D-xylans, for example, the β-xylosidase can be a 1,3 β-D-xylosidase. 1,3 β-D-xylosidases can be classified according to EC classification, with EC number 3.2.1.72, or they can catalyze the hydrolysis of (1^4)-β-D-xylans to remove successive residues of D -xylose of the non-reducing or reducing terminals, for example, the β-xylosidase can be a 1,4-β-xylosidase. 1,4-β-xylosidases can be classified according to E.C. classification with E.C. number 3.2.1.37."Beta-xylosidase activity assay"
[000152] The protocol for the beta-xylosidase enzyme assay is similar to that previously described (Ruttersmith, LD, Daniel RM 1993. Thermostable β-glucosidase and β-xylosidase from Thermotoga sp. strain FjSS3-B.1. Biochim. Biophys. Acta. 1156: 167-172, the teaching of which is incorporated herein by reference), with some modifications.
[000153] The beta-xylosidase activity assay, according to the present invention, is performed as follows: the substrate for beta-xylosidase is p-nitrophenyl-β-D-xylopyranoside (pNβxp) (Sigma-N2132) . A dose-response curve is created for each enzyme being tested. Each enzyme is tested in 7 doses (100, 50, 25, 12.5, 6.25, 3.13 and 1.56 ppm) with a non-enzymatic control containing only substrate and assay buffer. All solutions are made up in 50 mM sodium acetate buffer. The standard reaction mixture contains 280 μL of 0.1 M sodium acetate buffer at pH 5.0 (final concentration 50 mM) and 80 μL of substrate in 50 mM sodium acetate buffer (1 mM final concentration), and enzyme, in various dose concentrations in 50 mM sodium acetate buffer is then added to swell the reaction mixture to 400 µL. The reaction mixture (buffer and substrate) is placed in a water bath at 70°C to preheat before adding the enzyme. Enzyme is added to start the reaction. The sample mixture is incubated for 10 min, after which 0.8 ml of 0.1 M Na2CO3 is added at the end of the reaction. The concentration of p-nitrophenol released is calculated using the molar extinction coefficient, ε 400 = 18.300 M-1 cm-1. Control is subtracted from enzyme-containing wells and absorbance values (A400) are converted to concentration as described in the protocol. A unit of activity is defined as the formation of 1 µmol of p-nitrophenol from pNβxp per minute under the test conditions.
[000154] α-L-arabinofuranosidases (E.C. 3.2.1.55) can hydrolyze arabinan to L-arabinose."Alpha-L-arabinofuranosidase activity assay"
[000155] The protocol is similar to that previously described for α-L-arabinofuranosidase (Miyazaki, K. 2005). Hyperthermophilic α-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein. Extremophiles 9(5):399-406, the teaching of which is hereby incorporated by reference), with some modifications.
[000156] The assay of alpha-L-arabinofuranosidase activity, according to the present invention, is performed as follows: the substrate for α-L-arabinofuranosidase was p-nitrophenyl-α-L-arabinofuranoside (pNαLaf) (Sigma - N3641). A dose-response curve is created for each enzyme being tested. Each enzyme is tested in 7 doses (100, 50, 25, 12.5, 6.25, 3.13 and 1.56 ppm) with a non-enzymatic control containing only substrate and assay buffer. All solutions are made up in 50 mM sodium acetate buffer. The standard reaction mixture contains 280 μL of 0.1 M sodium acetate buffer at pH 5.0 (final concentration 50 mM) and 80 μL of substrate in 50 mM sodium acetate buffer (1 mM final concentration), and enzyme, in various dose concentrations in 50 mM sodium acetate buffer are then added to swell the reaction mixture to 400 µL. The reaction mixture (buffer and substrate) is placed in a 70°C water bath to preheat before adding the enzyme. Enzyme is added to start the reaction. The reaction mixture is incubated for 10 minutes, after which it is terminated by the addition of 0.8 mL of 0.1 M Na2CO3. The concentration of p-nitrophenol released is calculated using the molar extinction coefficient at 400 nm, ε = 10,500 M-1 cm-1 (Miyazaki, 2005). Control is subtracted from enzyme-containing wells and absorbance values (A400) are converted to concentration as described in the protocol. A unit of enzyme activity is defined as the formation of 1 μmol of p-nitrophenol from the substrate per minute under the test conditions.
[000157] The term "consists essentially of" or "consists essentially of", as used in the context of the activity of the enzyme composition for use in the present invention, means that the enzyme composition has the characterized activity or activities, but no other enzyme and /or no other enzymatic activity that is capable of digesting lignocellulosic biomass.
[000158] By way of example only, the following enzymes may be appropriately used in accordance with the present invention:



[000159] In one embodiment, the enzyme composition for use in the present invention may be a single enzyme or a combination of enzymes (for example, a mixture of enzymes).
[000160] In a preferred embodiment, the enzyme composition according to the present invention is an enzyme mixture.
[000161] In one embodiment, each of the different enzymatic activities defined in the present invention are preferably provided by a separate protein. In other words, each enzyme activity is a different enzyme protein. Preferably, the enzyme activity is the primary (or only) activity of the protein. In other words, preferably the defined activity is not a secondary activity of a protein. biomass
[000162] The term "lignocellulosic" refers to a composition that includes both lignin and cellulose. The lignocellulosic material may also comprise hemicellulose.
[000163] The term "cellulosic" refers to a composition comprising cellulose and additional components, including hemicellulose.
[000164] In one embodiment, lignocellulosic biomass is any cellulosic or lignocellulosic material. The term "lignocellulosic biomass" refers to any lignocellulosic material and includes materials that comprise cellulose. The lignocellulosic biomass may optionally further comprise hemicellulose, lignin, starch, oligosaccharides and monosaccharides.
[000165] Biomass can also comprise additional components such as protein, lipid, ash and/or extractable substances. Biomass may be derived from a single source, or biomass may comprise a mixture derived from more than one source; for example, the biomass may comprise a mixture of corn cobs and corn husks, or a mixture of grass and leaves.
[000166] Preferably, the lignocellulosic biomass comprises at least 15% of cellulose.
[000167] In one embodiment, the lignocellulosic biomass comprises at least 20% cellulose.
[000168] Lignocellulosic biomass can be any cellulosic or lignocellulosic material, such as agricultural waste, bioenergy crops, industrial solid waste, municipal solid waste, sludge derived from paper manufacturing, garden waste, wood waste, forest waste and combinations thereof .
[000169] In one embodiment, lignocellulosic biomass can be selected from the group consisting of corn cobs, crop residues such as corn husks, corn stubble, grasses, beet pulp, wheat straw, wheat chaff, straw of oat, wheat bran, wheat shorts, rice bran, rice husk, wheat bran, oat husk, palm kernel, citrus pulp, cotton, lignin, barley straw, hay, rice straw, rice husk, yellow millet (switchgrass), miscanthus, esparto, spotted reed, paper waste, sugarcane bagasse, sorghum bagasse, sorghum forage, sorghum stubble, soybean stubble, soy, components obtained from the grinding of trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits and flowers.
[000170] In one embodiment, lignocellulosic biomass can be selected from the group consisting of wet cake, corn fiber, corn germ meal, corn meal, hominy meal, corn gluten meal, gluten meal, corn particles germ, wheat bran, wheat bran, distillery dry grains (DDG) and distillery dry grain solubles (DDGS) based, for example, on corn, wheat, sorghum or combinations thereof.
[000171] In a preferred modality, lignocellulosic biomass can be selected from the group consisting of corn stubble, wheat straw, corn cobs, sugarcane bagasse, yellow millet, sorghum forage and rice straw.
[000172] In another embodiment, lignocellulosic biomass can be selected from the group consisting of corn stubble, wheat straw, corn cobs, sugarcane bagasse, yellow millet, sorghum forage and rice straw.
[000173] In one embodiment, biomass that is useful for the invention includes biomass that has a relatively high carbohydrate value, is relatively dense and/or is relatively easy to collect, transport, store and/or manipulate.
[000174] In one embodiment, the lignocellulosic biomass may comprise less than 50% starch, preferably less than 40% starch.
[000175] In one embodiment, the lignocellulosic biomass may comprise less than 30% starch, preferably less than 10% starch.
[000176] In one embodiment, lignocellulosic biomass may comprise less than 3% starch.
[000177] In one embodiment, lignocellulosic biomass may comprise less than 1% starch.
[000178] Suitably, lignocellulosic biomass does not comprise starch.
[000179] In one embodiment, the lignocellulosic biomass does not comprise a cereal grain. Pre-treatment
[000180] In one embodiment, the lignocellulosic biomass is pretreated by any pretreatment process known in the art that is capable of disrupting the compact structure of the lignocellulosic biomass to expose the cellulose fibers, and/or resulting in a decreasing biomass crystallinity (eg, cellulose crystallinity) and/or increasing accessible surface area for enzymatic activity.
[000181] Several pretreatment processes have been developed over the last few decades. Generally, these pretreatments can be divided into mechanical/physical, physicochemical, chemical and biological pretreatments, or combinations thereof. An overview of the various pretreatments can be found, for example, in X Zhao, L Zhang, D Liu Review: Fundamentals of different pretreatments to increase the enzymatic digestibility of lignocelluloses, Biofuels, Bioprod. Bioref. 6:561-579 (2012), the teaching of which is hereby incorporated by reference.
[000182] The pretreatment can be any treatment known in the art that results in low crystallinity of cellulose, as measured by the Segal method (Segal et al "An Emprical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer", Textile Research Journal, Oct 1959, Vol. 29, No. 10, 786-794), the teaching of which is hereby incorporated by reference, or which increases accessible surface area for enzymatic activity, or a combination both.
[000183] In one embodiment, the pretreatment is a pretreatment that reduces the crystallinity of cellulose in lignocellulosic biomass.
[000184] In one embodiment, pretreatment is a pretreatment that reduces the crystallinity of cellulose in lignocellulosic biomass by at least about 20% when measured by the Segal method (supra).
[000185] In one embodiment, pretreatment is a pretreatment that increases the surface area accessible for enzymatic activity in lignocellulosic biomass.
[000186] In one embodiment, pretreatment is a pretreatment that increases the accessible surface area by at least 20%, suitably by at least about 30%, and suitably by at least about 40% in lignocellulosic biomass. Accessible surface area can be determined by BET (Brunauer, Emmett and Teller) as taught in Guo et al Bioresource Technology 99 (2008) 6046-6053, the reference of which is incorporated herein by reference.
[000187] The available surface of lignocellulosic biomass can be measured using Brunauer, Emmett and Teller (BET) analysis, where said method comprises (i) weighing 0.5 g of sample; (ii) degassing with high purity nitrogen (99.999 %) at room temperature (eg 25°C) overnight; and (iii) perform the BET analysis using a surface area analyzer (Quantachrome NOVA2000).
[000188] In one modality, lignocellulosic biomass can undergo (or undergo) a physical and/or chemical and/or biological treatment. This can be referred to in the present invention as a physical and/or chemical and/or biological pre-treatment.
[000189] In one embodiment, the physical and/or chemical pretreatment according to the present invention may be a physical treatment or a chemical treatment, or a physicochemical treatment.
[000190] The term physical and chemical treatment is used in this document interchangeably with physical-chemical treatment.
[000191] In one modality, lignocellulosic biomass can undergo (or go through) a physical and/or chemical pre-treatment.
[000192] In one modality, lignocellulosic biomass can undergo (or go through) a physical pre-treatment.
[000193] In one modality, lignocellulosic biomass can undergo (or go through) a chemical pre-treatment.
[000194] In one modality, lignocellulosic biomass can undergo (or go through) a physicochemical pre-treatment.
[000195] The physical and/or chemical and/or biological treatment can be any physical and/or chemical and/or biological treatment (e.g., pre-treatment) known in the art. Mechanical/physical pre-treatment
[000196] Mechanical/physical pretreatment refers to the process of mechanical comminution that occurs by a combination of chipping, grinding, dry grinding, wet grinding, vibrating ball grinding and spinning ball grinding. The raw material size generally ranges from 10 to 30 mm after chipping and from 0.2 to 2 mm after grinding or crushing, and the crystallinity of the cellulose is reduced and/or the accessible surface area is increased. Pre-treatment of solid biomass with hot water, acids or sodium bisulfate to soften the raw material can make the milling process less energy intensive. In one embodiment of the invention, biomass is pretreated with a process for mechanical decrystallization.
[000197] The term physical pretreatment is used interchangeably in the present invention with mechanical pretreatment. Physicochemical pre-treatments
[000198] The physicochemical pretreatments combine the chemical modification of biomass compositions with the physical fracture of the cell wall structure. A useful physicochemical pretreatment is steam explosion. In this pre-treatment, the biomass is treated with saturated steam under high pressure (high pressure and high temperature), optionally including the addition of acids, bases or other chemicals, and then the pressure is rapidly reduced, causing the materials undergo an explosive decompression.
[000199] In one modality, the physical and/or chemical treatment (for example, pre-treatment) can be hydrothermolysis or wet oxidation, and comprises: high pressure and/or high temperature with liquid water and/or steam , optionally including the addition of acids, bases or other chemicals.
[000200] In one modality, the pressure is in the range of 2.1 to 4.1 MPa (300 to 600 psi), preferably from 2.4 to 3.8 MPa (350 to 550 psi), preferably from 2, 8 to 3.4 MPa (400 to 500 psi).
[000201] In one embodiment, high temperature means temperatures in the range from about 100 to 300°C, preferably from about 140 to 240°C, such as from about 170 to 200°C.
[000202] By way of example only, the pre-treatment can be a physical treatment comprising steam explosion.
[000203] In terms of cellulose crystallinity, after the vapor explosion, the crystallinity index (CrI) of the substrate increases. The crystallinity index (CrI) is taught in Segal (above).
[000204] One more example of a physicochemical pretreatment is the ammonia fiber explosion (AFEX) where the lignocellulosic material is permeated with liquid ammonia, followed by increasing the temperature up to about 90°C. Without wishing to stick to theory, the ammonia gas formed interacts with the biomass under pressure (for example, from 1.7 to 2 MPa (17 to 20 bar) for 5 to 10 minutes) and the pressure then is then rapidly released, which may result in decrystallization of cellulose, pre-hydrolysis of hemicellulose and alteration of the lignin structure and/or increase in accessible surface area.
[000205] The crystallinity index (CrI) can be influenced by the AFEX pretreatment conditions, since small differences in the pretreatment conditions can result in the formation of different cellulose crystalline structures.
[000206] Another useful physicochemical pre-treatment process is the pre-treatment with hot liquid water, which uses water as a means to pre-treat the biomass under pressure to keep the water in a liquid state at elevated temperatures. Pretreatment is also commonly referred to as hydrothermolysis or hydrothermal pretreatment. It can solubilize about 40 to 60% of the total biomass with 4 to 22% of the cellulose and almost all of the hemicelluloses to form liquid soluble oligosaccharides. As a result, an increase in accessible surface area can be observed. Additionally, or alternatively, a decrease in the crystallinity of cellulose, an inferior association of cellulose with lignin and depolymerization of cellulose can be observed, which contributes to the improvement of the accessibility of the cellulose.
[000207] In one embodiment, the pretreatment can be a physicochemical pretreatment, comprising a hydrothermolysis or a hydrothermal treatment.
[000208] In one embodiment, the lignocellulosic biomass is wheat straw and the pretreatment comprises the use of a hydrothermal treatment. Pretreatment in accordance with the present invention may suitably include hydrothermal treatment as set forth in as taught in WO2011/125056 (IBICON), the teaching of which is incorporated herein by reference.
[000209] Radiation pretreatments involve processes of pretreatment of lignocellulosic biomass with Y-irradiation, ultrasound, electron beam or microwave. Radiation pretreatments can also be coupled with other pretreatments to further increase the accessibility of cellulose. Without wishing to be bound by theory, at a high irradiation dose, substrates become fragile due to radiation degradation of cellulose, hemicellulose and lignin. The crystallinity of cellulose can be destroyed to some extent by Y-radiation. Microwave pretreatment is usually carried out in the presence of water, in an organic solvent, alkali or in a dilute acidic solution. Chemical pre-treatments
[000210] Chemical pretreatments involve processes using various chemicals to pretreat biomass under various conditions. The mechanisms of these pretreatments vary depending on the chemicals used and the pretreatment conditions. Examples of suitable chemical pretreatment processes include, but are not limited to: dilute acid pretreatment, alkali pretreatment, sulfite pretreatment, oxidative pretreatment such as wet oxidation , cellulose solvent pretreatment, ammonia percolation (APR) and organosolv pretreatment.
[000211] In one embodiment, the pretreatment may be a chemical treatment comprising the use of an acid or alkaline treatment.
[000212] By way of example, the physical and/or chemical treatment can be the addition of basic catalyst, or other methods known in the art.
[000213] In one embodiment, lignocellulosic biomass is corn stubble is physical and/or chemical treatment comprises the use of an acid treatment.
[000214] In one embodiment, the pretreatment is a pretreatment with dilute acid. For example, lignocellulosic biomass material can be mixed with dilute acid, typically H2SO4 and water to form a slurry, heated by steam to the desired temperature (usually between 160 and 220°C) and, after a residence time, evaporated at atmospheric pressure.
[000215] In a specific modality, the pre-treatment can comprise the steps of: a) impregnating the lignocellulosic biomass (for example, wheat straw) with 0.2% H2SO4, for example, by immersion; b) press the material until it reaches a dry matter content between 40 and 50% and c) pre-treat with steam for 10 minutes at 190°C. Optionally press the resulting slurry to filter out the liquid and optionally wash the solid residue 2 to 3 times and press until a final solids content of about 40 to 50% is obtained.
[000216] In another specific modality, the pre-treatment can comprise the steps of: a) impregnating the lignocellulosic biomass (for example, wheat straw) with 1% acetic acid, by immersion; b) press the material until it reaches a dry matter content between 40 and 50% and c) pre-treat with steam for 10 minutes at 200°C. Optionally press the subsequent slurry to filter out the liquid and optionally wash the solid residue 2 to 3 times and press until a final solids content of about 40 to 50% is obtained.
[000217] Alkaline pretreatment may involve treating lignocellulosic biomass with various alkalis or bases such as NaOH, KOH, CaOH2, aqueous ammonia, peroxide and lime (using CaOH2) to pretreat the lignocellulosic biomass. Without wishing to be bound by theory, it is believed that during alkaline pretreatment, the intermolecular ester bonds that cross-link the xylan hemicellulose and lignin are saponified, thus resulting in delignification of the biomass. It has been described that alkalis are suitable agents for swelling cellulose and altering crystalline cellulose polymorphs, which indicates an alteration of the crystalline network of hydrogen bonds, thus affecting the digestibility of cellulose.
[000218] In one modality, the physical and/or chemical treatment can be a chemical treatment that comprises the use of an alkaline treatment (for example, with ammonia).
[000219] In one modality, the pretreatment is pretreatment with anhydrous ammonia.
[000220] In a specific modality, the pretreatment can be a pretreatment with anhydrous ammonia, comprising the steps of 1) placing lignocellulosic biomass material (for example, corn stubble) in a flask, which is evacuated under pressure to enable better penetration of anhydrous ammonia; b) contacting the biomass material with an aqueous solution, comprising 12% ammonia, at 140°C; c) pretreatment with ammonia can last up to 25 h, but optimal release of glucose and xylose can be observed after a shorter period of time (eg 15 minutes); d) remove additional ammonia solution by applying vacuum to achieve a final solids content of 50 to 60% dry matter.
[000221] The physicochemical treatment may alternatively be the pretreatment method using ammonia (e.g. dilute ammonia) described in WO 2006/110891, WO 2006/11899, WO 2006/11900 and WO2006/110901 (DuPont), the teaching of these documents being incorporated herein by way of reference.
[000222] In one modality, the pretreated biomass is corn stubble pretreated with dilute ammonia (DaCS).
[000223] In one modality, the pretreatment is caustic delignification.
[000224] In one embodiment, the pretreatment may be a caustic delignification pretreatment comprising the steps of 1) treating the biomass with between 0.5 and 3% nucleophilic base (e.g., sodium hydroxide (NaOH) , lithium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide or combinations thereof) for between 0.25 and 20 h at 25 to 200°C at pH in the range of 9 to 11; 2) recover the pretreated biomass filtration and wash with deionized water to remove any excess alkali and dissolved by-products.
[000225] In a specific modality, the pretreatment can be a caustic delignification pretreatment, comprising the steps of 1) treating the biomass with between 0.5 and 3% sodium hydroxide (NaOH) for between 0, 25 and 1.5 h at around 121°C, at a pH in the range of 9 to 11; 2) recover pretreated biomass filtration and wash with deionized water to remove any excess alkali and dissolved by-products.
[000226] One way to perform caustic delignification is taught in Xu et al (2011) Bioresources 6(1) 707-720 (which reference is incorporated herein by way of reference). In one embodiment, the pretreated biomass can be a biomass treated by the caustic delignification process taught in Xu et al (2011) above.
[000227] In one embodiment, the pretreated biomass is caustic delignified corn stubble (DLcs) and/or caustic delignified yellow millet (DLswg).
[000228] In one embodiment, the pretreated biomass is caustic delignified corn stubble (DLcs) and/or caustic delignified yellow millet (DLswg).
[000229] In one embodiment, the sulfite pretreatment or sulfite pulping process comprises sulfite treatment of the biomass material under acidic conditions, followed by mechanical size reduction, using disk refinement. Sulfite pretreatment results in considerable removal of hemicellulose, a decrease in DP cellulose and sulfonation of lignin with increased hydrophilicity of lignin.
[000230] Another chemical pretreatment can be an oxidative pretreatment, which refers to processes with oxidants used to remove lignin and the reducing substance. Oxidants commonly used for oxidative delignification include ozone, hydrogen peroxide, oxygen and peracetic acids. This treatment can optionally be combined with other chemical or hydrothermal treatments. The mechanisms of lignin oxidative degradation vary according to the oxidants used and the reaction condition, such as pH. For ozonolysis, wet oxidation and delignification with peracids, the degradation of aromatic and olefinic structures involves the initial electrophilic attack by oxidants, whereas during alkaline-pretreatment with H2O2, these structures are destroyed by the nucleophilic attack of hydroperoxide anions.
[000231] In one modality, the physical and/or chemical treatment is a wet oxidation.
[000232] In a useful embodiment, wet oxidation is one that involves high pressure and/or high temperature with liquid water and/or steam, optionally including the addition of acids, bases or chemicals.
[000233] High pressure can mean pressure in the range of 2.1 to 4.1 MPa (300 to 600 psi), preferably from 2.8 to 3.4 MPa (400 to 500 psi), as well as about 3, 1 MPa (450 psi).
[000234] High temperature can mean temperatures in the range of from about 100 to 300°C, preferably from about 180 to 200°C, for 5 to 15 minutes (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151, whose teaching is here incorporated by reference). In one modality, the high temperature can be around 140 to 235°C.
[000235] Suitably, wet explosion is a modification of the wet oxidation pretreatment method, where wet oxidation and steam explosion, as described above, are combined. In wet explosion, the oxidizing agent is introduced during the pre-treatment after a certain residence time. The treatment is then terminated by vaporization at atmospheric pressure (WO 2006/032282, which is incorporated herein by way of reference).
[000236] Just as an example, in one modality, the physical and/or chemical pre-treatment may involve high pressure and/or high temperature with liquid water, where the water exists as a mixture of liquid and steam, optionally including the addition of acids, bases or other chemicals. High pressure, in this mode, can be in the range of 0.3 to 3.4 MPa (50 to 300 psi), suitably 0.7 to 1.4 MPa (100 to 200 psi), for example, about 1, 0 MPa (150 psi). High temperature, in this embodiment, may be in the range of about 100 to 300°C, suitably about 170 to 220°C, as well as about 170 to 200°C.
[000237] Another useful chemical pretreatment is organosolv pretreatment, which delignifies cellulosic biomass material by extraction using aqueous ethanol (eg 40 to 60% ethanol) at 160 to 200°C for 30 to 60 minutes. Sulfuric acid can be added as a catalyst. In organosolv pretreatment, most of the hemicellulose can be removed.
[000238] Solvent-based cellulose pretreatment is also a chemical pretreatment for the treatment of lignocellulosic biomass based on cellulose solvents such as phosphoric acid (CPA) and ionic liquids (IL). Biological pre-treatment
[000239] In one modality, the lignocellulosic biomass can undergo a biological treatment (for example, a biological pre-treatment). Such biological treatment can be any biological treatment known in the art.
[000240] In biological pretreatments, microorganisms can be used for the pretreatment of biomass, to increase the enzymatic digestibility of the remaining solids. The microorganisms used are normally capable of degrading lignin and carbohydrate polymers. For example, some types of fungi can produce lignocellulolytic enzymes, which work synergistically to degrade the plant cell wall, and other types can produce hydrogen peroxide.
[000241] After biological pretreatment, the accessible surface area can be increased, resulting in increased digestibility of cellulose.
[000242] Biological pretreatment may, by way of example only, include treatment with a white rot fungus. Several white rot fungi, such as Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Phlebia subserialis, and Pleurotous ostreatus, are known to efficiently metabolize lignin in various lignocellulosic materials (see, for example, Singh D and Chen S. 2008. The white-rote fungus Phanerotus. chrysosporium: conditions for the production of lignin-degrading enzymes. Applied Microbiology Biotechnology 81,399-417, the teaching of which is incorporated herein by reference).
[000243] The cellulosic material will also be subjected to particle size reduction, pre-dip, humidification, washing and/or conditioning before pre-treatment (for example, before physical and/or chemical and/or biological pre-treatment ) using methods known in the art.
[000244] The method of the present invention may further comprise a step of placing a feed component in contact with the feed additive composition, according to the present invention.
[000245] The present invention also relates to methods and uses for improving a biophysical characteristic of an animal.
[000246] Preferably, an animal is fed with a feed additive composition obtainable (eg obtained) by the method of the present invention or a feed additive composition according to the present invention, or a premix according to the present invention, or an animal feed according to the present invention, or an animal feed obtainable (e.g. obtained) by a method of the present invention.
[000247] The biophysical trait may be selected from the group consisting of one or more of the following: animal performance, animal growth performance, feed conversion ratio (FCR), ability to digest a raw material (for example, nutrient digestibility, including starch, fat, protein and fiber digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass, feed efficiency, body fat percentage, fat distribution body, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, eg manure production and/or nitrogen excretion.
[000248] In the present invention, the lignocellulosic biomass physically and/or chemically and/or biologically pretreated is incubated with the enzymatic composition.
[000249] Suitably, the duration of such incubation is from 6 to 120 hours, preferably from 10 to 60 hours, preferably from 20 to 50 hours, preferably from 35 to 50 hours and preferably from 40 to 48 hours.
[000250] In some modalities, the incubation can last from 4 h to 72 h. In one modality, the incubation period is from 6 h to 48 h. In one embodiment, the incubation period is at least about 6 h. In another modality, the incubation period is less than 72 h. A person skilled in the art will understand that the incubation period will be influenced by the dry matter inclusion rate, and that as the dry matter inclusion rate increases, the incubation time must increase proportionately to achieve the same effect.
[000251] Suitably, the incubation is such that about 5 to 20% (preferably from about 10 to 13%) (w/w) of the pentoses (xylose and arabinose) and about 1 to 10% (preferably, of about 1 to 4%) (w/w) of the hexoses in the biomass are in oligo- and polymer form at the end of the incubation period.
[000252] Suitably, the incubation is such that about 10 to 29% (preferably about 16 to 21% (w/w)) glucose and about 1 to 10% (preferably about 4 to 7%) of xylose are in monomeric units at the end of the incubation period.
[000253] The duration of incubation may depend on the enzymes used and/or the concentration of enzyme used. The purpose of the incubation period is to allow the enzymes to degrade the biomass sufficiently. By way of example, sufficient biomass degradation can be determined by a person skilled in the art, for example, by weight analysis to measure the reduction of insoluble biomass after enzymatic treatment.
[000254] In one embodiment, the product and/or the feed additive composition, and/or the premix and/or the animal feed, according to the present invention, are dried.
[000255] The term "drying" means the water content (% by weight) of the product and/or the feed additive composition, and/or the premix and/or the feed, in accordance with the present invention, is reduced to less than 15%, preferably less than 10%.
[000256] Therefore, the present invention further provides a dry product or a substantially dry product, and/or a feed additive composition and/or a premix and/or an animal feed, according to the present invention, its water content (% by weight) being less than 30%, preferably less than 15%, preferably less than 10% and preferably less than 5%.
[000257] In some embodiments, the present invention can provide a slurry product or a slurry product. A semi-liquid product or a slurry product, according to the present invention, is a product whose water content (% by weight) is less than 90%, preferably less than 80%, preferably less than 70% or, more preferably less than 60%.
[000258] In one embodiment, the product and/or feed additive composition, and/or premix and/or animal feed, according to the present invention, are packaged and/or stored in a state dry or in a substantially dry state.
[000259] The terms "dry" or "dry state", as used in the present invention, mean that the product and/or the feed additive composition, and/or the premix and/or the feed, of according to the present invention, do not contain or contain only a very small amount of water. In other words, the term "dry" or "dry state", as used in the present invention, can mean that the product and/or the feed additive composition, and/or the premix and/or the animal feed , according to the present invention, comprises less than 5%, and preferably less than 1%, water content (% by weight).
[000260] The term "substantially dry state", as used in the present invention, means the product and/or the feed additive composition, and/or the premix and/or the animal feed, in accordance with the present invention, contains only a very small amount of water. In other words, the term "substantially dry" as used in the present invention can mean that the product and/or the feed additive composition, and/or the premix and/or the feed, in accordance with with the present invention, comprises less than 30%, preferably less than 15%, and preferably less than 10% water content (% by weight).
[000261] In one embodiment, the product and/or feed additive composition, and/or premix and/or feed for animals, according to the present invention, comprises less than 20% by weight, of moisture content.
[000262] In another embodiment, the product and/or feed additive composition, and/or premix and/or feed for animals, according to the present invention, comprises less than 15% by weight of content of moisture.
[000263] In another embodiment, the product and/or feed additive composition, and/or premix and/or feed for animals, according to the present invention, comprises less than 10% by weight of content of moisture.
[000264] In another embodiment, the product and/or the feed additive composition, and/or the pre/mix and/or the animal feed, according to the present invention, comprises less than 5% by weight of content of moisture.
[000265] Preferably, the product and/or the feed additive composition according to the present invention comprises at least 70%, preferably at least 60% of the xylose units present in the material, such as oligomers or polymers.
[000266] The enzyme composition for use in the present invention preferably generates soluble oligomers and soluble polymers with sugar units, including xylose, rhamnose, glucose, mannose, galactose, glucuronate, galacturonate (such as xylo-oligomers, arabinoxylan oligomers) in the product and/or in the feed additive composition according to the present invention.
[000267] In a preferred embodiment, the enzymatic composition does not release, or does not generate or release significant amounts of C-5 sugars (eg, xylose) in monomer form.
[000268] Preferably, less than 50% of the total C-5 sugars (eg xylose) in the feed additive composition and/or feed ingredient will be present in monomer form.
[000269] The inventors have found that C-5 sugars (for example, xylose and/or arabinose and/or arabinofuranose) can be kept at a low level in the feed additive composition, according to the present invention, using a composition enzyme that is absent or substantially absent in β-xylosidase activity and/or α-arabinofuranosidase activity.
[000270] The method according to the present invention may further comprise feeding an animal with the feed additive composition.
[000271] In one embodiment, it is envisioned that the method of the present invention further comprises mixing a feed component with the feed additive composition, for example, to thereby provide a feed or feed for animals.
[000272] The present invention relates to uses and methods for improving the biophysical characteristics of an animal by administering to an animal an effective amount of a feed additive composition, according to the present invention, or an additive composition of feed produced by a method of the present invention, or an animal feed comprising such a feed additive composition.
[000273] As used in the present invention, the term "biophysical characteristics", as used herein, means one or more of the groups selected from the following: animal performance, animal growth performance, feed conversion ratio (FCR ), ability to digest a raw material (eg nutrient digestibility, including starch, fat, protein and fiber digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass , feeding efficiency, body fat percentage, body fat distribution, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, eg manure production and/or nitrogen excretion .
[000274] The term "improving", as used in the present invention, means improved compared to feeding an animal with lignocellulosic biomass that has not been treated in accordance with the present invention.
[000275] The enzyme(s) can be dosed in an adequate concentration in the biomass to ensure the efficient degradation of the lignocellulosic biomass. In some embodiments, the enzyme may be in the range of about 2.5 g to 20 kg of active protein per ton of pretreated biomass, suitably approximately 5 g to 14 kg of active protein per ton of pretreated biomass.
[000276] Just as an example, assuming the inclusion of 5 to 60% of biomass per metric ton (MT) of feed, the active protein per MT of feed would be 0.125 g - 12 Kg per MT of feed.
[000277] In one embodiment, the amount of enzyme in the feed can be between about 0.125 g to about 12 kg (suitably from about 500 g to about 10 kg, suitably from about 1 kg to about 8 kg) per TM of feed.
[000278] It is anticipated that the amount of biomass treated in the feed will vary. In one modality, the amount of biomass treated in the feed is less than 90% w/w of the total feed, preferably less than 80%, suitably less than 70%, suitably less than 60%, suitably less than 50% and, properly, less than 40%. Preferably, the amount of biomass treated in animal feed is less than 60%.
[000279] In one modality, the amount of biomass treated in the feed is greater than 30% w/w of the total feed, preferably greater than 5%, preferably greater than 10%, preferably greater than 20%, preferably greater than 30 %, preferably greater than 40%, suitably greater than 50%, and suitably greater than 60%. Preferably, the amount of biomass treated in the animal feed is greater than 5% or 10%.
[000280] In another modality, the amount of biomass treated in the feed is in the range of about 5 to 70%, suitably in the range of about 5 to 60%, suitably from 5 to 50%, and suitably from 10 to 40 % w/w of total feed.
[000281] In another modality, the amount of biomass treated in the feed is in the range of 10 to 70%.
[000282] In another modality, the amount of biomass treated in the feed is in the range of 10 to 40%.
[000283] In one embodiment, "mixing", as used in the present invention, includes any method for mixing, such as mixing, blending, sprinkling, etc.
[000284] In one embodiment, the enzyme composition may be in a dry enzyme formulation (for example, in the form of granules or in a vehicle (such as in a wheat vehicle)) before mixing with lignocellulosic biomass (such as lignocellulosic biomass physically and/or chemically and/or biologically pretreated). In another embodiment, the enzyme composition may be in a liquid formulation prior to mixing with lignocellulosic biomass (such as physically and/or chemically and/or biologically pretreated lignocellulosic biomass).
[000285] When the enzyme is in a liquid formulation before mixing with lignocellulosic biomass (such as physically and/or chemically and/or biologically pre-treated lignocellulosic biomass), the enzyme can be mixed by spraying the enzyme formulation or dipping the lignocellulosic biomass (such as physically and/or chemically and/or biologically pretreated lignocellulosic biomass), for example, in the enzyme formulation.
[000286] The biomass physically and/or chemically and/or biologically pretreated (before or after treatment with enzyme) and/or the dry solid fraction can be ground and/or pulverized, and/or transformed into a bran. Animal
[000287] The term "animal", as used in the present invention, means an animal to which a feed additive composition according to the present invention will be administered or administered, or an animal feed comprising said feed additive composition according to the present invention.
[000288] Preferably, the animal is a mammal, a bird, fish or crustacean including, for example, cattle or a domesticated animal (for example, a pet).
[000289] In one modality, the "animal" is cattle.
[000290] The term "cattle", as used in the present invention, refers to any farm animal. Preferably, livestock comprises one or more of cows or bulls (including calves), pigs (including piglets, pigs), poultry (including broilers, laying hens, chickens and turkeys), birds, fish (including water fish sweet, such as salmon, cod, trout and carp, for example, Chinese carp and marine fish such as sea bass), crustaceans (such as shrimp, mussels and scallops), horses (including racehorses), sheep (including lambs).
[000291] In another embodiment, the "animal" is a domesticated animal or pet, or an animal kept in a zoo environment.
[000292] The term "domesticated animal or pet or animal kept in a zoo environment", as used herein, refers to any relevant animal, including canines (eg dogs), felines (eg cats), rodents (eg guinea pigs, rats and mice), birds, fish (including freshwater fish and marine fish) and horses.
[000293] In one embodiment, the animal is a monogastric animal. In a preferred embodiment, the monogastric animals can be poultry or pigs (or a combination thereof).
[000294] In another modality, the animal is a ruminant animal. packaging
[000295] In one embodiment, the enzymatic composition and/or the feed additive composition, and/or the feed ingredient and/or the premix, and/or the feed or animal feed, in accordance with the present invention, is packaged.
[000296] In a preferred embodiment, the enzyme composition and/or the feed additive composition, and/or the feed ingredient and/or the premix, and/or the feed or animal feed is packaged in a pouch , as in a paper bag.
[000297] In an alternative embodiment, the enzyme composition and/or the feed additive composition, and/or the feed ingredient and/or the premix, and/or the feed or animal feed, may be sealed in a container. Any suitable container can be used. Ration
[000298] The feed additive composition of the present invention can be used as - or in the preparation of - a feed.
[000299] The term "feed" is used synonymously in the present invention with "animal feed".
[000300] The feed can be in the form of a solution or as a solid - depending on the use and/or the mode of application, and/or the mode of administration.
[000301] When used as - or in the preparation of - a feed - as a functional feed - the composition of the present invention may be used together with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient , a nutritionally acceptable adjuvant or a nutritionally active ingredient.
[000302] In a preferred embodiment, the feed additive composition of the present invention is mixed with a feed component to form an animal feed.
[000303] The term "feed component" as used in the present invention means all or part of the feed. Part of the feed can mean one feed constituent or more than one feed constituent, for example 2 or 3 or 4. In one embodiment, the term "feed component" encompasses a premix or constituents of the premix.
[000304] Preferably, the feed can be a forage, or a premix thereof, a compound feed or a premix thereof. In one embodiment, the feed additive composition according to the present invention can be mixed with a compound feed, a compound feed component or with a premix of a compound feed or with a forage, a forage component. or a premix of a forage.
[000305] The term forage, as used in the present invention, means any food that is supplied to an animal (rather than the animal having to search for itself). Forage encompasses plants that have been cut down.
[000306] The term forage includes hay, straw, silage, compacted and pelleted feed, oils and mixed feed, and also germinated grains and legumes.
[000307] The forage can be obtained from one or more of the plants selected from: alfalfa (lucerne), barley, gherkin, brassicas, Chau moellier, kale, rapeseed (canola), rutabaga (swedish turnip), turnip, clover, hybrid clover , red clover, subterranean clover, white clover, grass, false oat grass, fescue, Bermuda grass, bromus, Danthonia decumbens, meadow grasses (from naturally mixed meadow pastures, ryegrass grass, ryegrass, grass meadows, maize (maize), millet, oats, sorghum, soybeans, trees (pruned tree shoots for hay trees), wheat and vegetables.
[000308] The term "compound feed" means a commercial feed in the form of a bran, a pellet, nuts, a pie or a crumbled. Compound feeds can be mixed from various raw materials and additives. These mixtures are formulated according to the specific requirements of the target animal.
[000309] Compound rations can be complete rations that provide all the nutrients needed daily, concentrates that provide a portion of the ration (protein, energy) or supplements that provide only additional micronutrients such as vitamins and minerals.
[000310] The main ingredients used in compound feeds are feed grains, which include corn, soybeans, sorghum, oats and barley.
[000311] Suitably, a premix, as referred to in the present invention, can be a composition composed of micro-ingredients, such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential ingredients. Premixes are generally compositions suitable for blending into commercial feeds.
[000312] Any feedstock of the present invention may comprise one or more feed materials selected from the group consisting of a) cereals, such as small grains (e.g. wheat, barley, rye, oats and combinations thereof) and/or large grains such as corn or sorghum; b) plant by-products (eg cereals) such as wet cake, dry distillery grains (DDG), eg distillery dry grain solubles (DDGS), corn fiber, corn germ meal, corn bran, hominy ration, corn gluten ration, germ particles, wheat bran, wheat bran or combinations thereof (preferably by-products of the methods according to the present invention); c) protein obtained from sources such as soy, sunflower, peanuts, lupine, peas, fava beans, cotton, canola, fish meal, dry plasma protein, meat and bone meal, potato protein, whey, copra, Sesame; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins.
[000313] An animal feed of the present invention may contain at least 30%, at least 40%, at least 50% or at least 60% by weight of corn and soybean meal or corn, and whole soybean, or corn meal. wheat or sunflower bran.
[000314] Additionally or alternatively, an animal feed of the present invention may comprise at least one high fiber material and/or at least one by-product of at least one high fiber material to provide an animal feed with high fiber content. Examples of high fiber feed materials include: wheat, barley, rye, oats, plant by-products (eg cereals) such as wet cake, distillery dry grain (DDG) and distillery dry grain solubles ( DDGS), corn fiber, corn germ meal, corn bran, hominy ration, corn gluten ration, fine wheat bran, wheat bran or combinations thereof. Some protein sources can also be considered to have high fiber content: protein obtained from sources such as sunflower, lupine, fava beans and cotton.
[000315] In the present invention, the feed can be one or more of the following: a compound feed and premix, including pellets, nuts or pie (cattle); a crop or crop residues: corn, soybeans, sorghum, oats, barley, corn husks, copra, straw, mill, sugar beet residues; Fish's flour; freshly cut grass and other forage plants; meat and bone meal; molasses; oil pie and mill pie; oligosaccharides; conserved forage plants: hay and silage; seaweed; seeds and grains, whole or prepared by crushing, grinding, etc.; sprouted grains and legumes; Yeast extract.
[000316] The term "feed" in the present invention also encompasses, in some embodiments, food for pets. A pet food is a material of animal or plant origin intended for consumption by pets, such as dog food or cat food. Pet food, such as dog or cat food, can be in a dry form, such as kibble pellets for dogs, or in a moist canned form. Cat food may contain the amino acid taurine.
[000317] The term "feed" in the present invention also encompasses, in some embodiments, fish feed. A fish feed typically contains the macronutrients, trace elements and vitamins needed to keep captive fish in good health. Fish feed can be in the form of a flake, pellet or tablet. Pelleted shapes, some of which sink quickly, are often used for larger fish or for feeding bottom-dwelling species. Some fish feeds also contain additives, such as beta-carotene or sex hormones, to artificially increase the color of ornamental fish.
[000318] The term "feed" in the present invention also encompasses, in some embodiments, bird feed. Bird food includes foods that are used both in bird feeders and to feed pet birds. Typically, bird food comprises a variety of seeds, but it can also include tallow (beef or mutton fat).
[000319] As used in the present invention, the term "contact" refers to the direct or indirect application of the composition of the present invention to the product (for example, feed). Examples of application methods that can be used include, but are not limited to, treating the product in a material comprising the feed additive composition, directly applying by mixing the feed additive composition to the product, spraying the composition. of feed additive on the surface of the product or immersion of the product in a preparation of the feed additive composition.
[000320] In one embodiment, the feed additive composition of the present invention is preferably mixed with the product (e.g., feed). Alternatively, the feed additive composition can be included in the emulsion or raw ingredients of an animal feed.
[000321] For some applications, it is important that the composition is made available on or on the surface of a product to be affected/treated. This allows the composition to confer one or more of the following favorable characteristics: biophysical characteristics, for example, where the biophysical characteristic is selected from the group consisting of one or more of the following: animal performance, animal growth performance, ratio conversion ratio (FCR), ability to digest a raw material (eg, nutrient digestibility, including starch, fat, protein and fiber digestibility), nitrogen retention, carcass yield, growth rate, weight gain , body weight, mass, feeding efficiency, body fat percentage, body fat distribution, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, eg manure production and /or nitrogen excretion.
[000322] The feed additive compositions of the present invention can be applied to intercalate, coat and/or impregnate a product (e.g., an animal feed or raw ingredients of an animal feed) with a controlled amount of enzyme(s). ).
[000323] Preferably, the enzyme composition and/or feed additive composition of the present invention will be thermally stable to heat treatment up to about 70°C; up to about 85°C; or up to about 95°C. The heat treatment can be carried out for up to about 1 minute; up to about 5 minutes; up to about 10 minutes; up to about 30 minutes; or up to about 60 minutes. The term thermally stable means that at least about 75% of the enzyme components that were present/active in the additive prior to heating to the specified temperature were still present/active after cooling to room temperature. Preferably, at least about 80% of the enzyme components that were present and active in the additive prior to heating to the specified temperature are still present and active after cooling to room temperature.
[000324] In a particularly preferred embodiment, the enzyme composition and/or the feed additive composition is homogenized to produce a powder.
[000325] In a preferred alternative embodiment, the enzyme composition and/or the feed additive composition is formulated into granules, as described in WO2007/044968 (referred to as TPT granules), incorporated herein by reference.
[000326] In another preferred embodiment, when the enzyme composition and/or the feed additive composition is formulated into granules, the granules comprise a hydrated barrier salt coated on the protein core. The advantage of such a salt coating is better thermotolerance, improved storage stability and protection against other feed additives that would otherwise have an adverse effect on the enzyme.
[000327] Preferably, the salt used for salt coating has a water activity greater than 0.25, or constant humidity greater than 60% at 20°C.
[000328] Preferably, the salt coating comprises a Na2SO4.
[000329] The method of preparing a feed additive composition may also comprise the additional step of pelletizing the powder. The powder can be mixed with other components known in the art. The powder, or the mixture comprising the powder, can be forced through a mold, and the strips produced are cut into suitable pellets of varying length.
[000330] Optionally, the pelletizing step can include a steam treatment, or a conditioning stage, before forming the pellets. The mixture comprising the powder can be placed in a conditioner, for example in a mixer with steam injection. The mixture is heated in the conditioner to a specified temperature, such as 60 to 100°C, with typical temperatures being 70°C, 80°C, 85°C, 90°C or 95°C. Residence time can range from seconds to minutes, and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minutes, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.
[000331] It will be understood that the feed additive composition of the present invention is suitable for addition to any suitable feed material.
[000332] As used in the present invention, the term feed material refers to the basic feed material to be consumed by an animal. It will be further understood that it may comprise, for example, at least one or more unprocessed grains, and/or processed plant and/or animal material such as soy meal or bone meal.
[000333] As used in the present invention, the term "animal feed" refers to a feed material to which one or more feed additive compositions have been added.
[000334] It will be understood, by the person skilled in the art, that different animals require different feeds for animals, and even the same animal may require different feeds depending on the purpose for which the animal is raised.
[000335] Preferably, the feed can comprise feed raw materials comprising corn (maize) or corn (corn), wheat, barley, triticale, rye, rice, tapioca, sorghum and/or any of the by-products as well. as protein-rich components such as soybean meal, rapeseed meal, canola meal, cottonseed meal, sunflower seed meal, animal by-product meal and mixtures thereof. More preferably, the animal feed may comprise animal fats and/or vegetable oils.
[000336] Optionally, the animal feed can also contain additional minerals such as, for example, additional calcium and/or vitamins.
[000337] Preferably, the animal feed is a mixture of corn bran and soybeans.
[000338] In another aspect, a method for producing an animal feed is provided. Animal feeds are normally produced in feed mills, where the raw materials are first ground to a suitable particle size and then mixed with appropriate additives. Animal feed can then be produced as a dough or in pellets; the latter typically involve a method whereby the temperature is raised to a target level and then the feed is passed through a mold to produce pellets of a certain size. The pellets are cooled. Subsequently, liquid additives such as fat and enzymes can be added. The production of animal feeds may also involve an additional step which includes extrusion or expansion prior to pelleting - in particular, by suitable techniques which may at least include the use of steam.
[000339] The animal feed can be a feed for a monogastric animal, such as poultry (for example, broiler, laying hens, turkeys, ducks, geese, waterfowl), pigs (all age categories), a pet (eg dogs, cats) or fish, and preferably the pet food is for poultry.
[000340] Just as an example, a feed for chickens, for example, broilers, may comprise one or more of the ingredients listed in the table below, for example, in the % for age indicated in the table below:


[000341] By way of example only, the dietary specification for chickens, such as a broiler, may be as set out in the Table below:

[000342] By way of example only, feed for laying hens may comprise one or more of the ingredients listed in the table below, for example, in the % for age indicated in the table below:


[000343] By way of example only, the dietary specification for laying hens may be as set out in the Table below:

[000344] By way of example only, feed for turkeys may comprise one or more of the ingredients listed in the table below, for example in the % for age indicated in the table below:

[000345] By way of example only, the dietary specification for turkeys may be as set out in the Table below:

[000346] By way of example only, feed for piglets may comprise one or more of the ingredients listed in the table below, for example, in the % for age indicated in the table below:

[000347] By way of example only, the dietary specification for piglets may be as set out in the Table below:


[000348] By way of example only, rations for growing/ready for slaughter pigs may comprise one or more of the ingredients listed in the table below, for example, in the % for age indicated in the table below:

[000349] By way of example only, the dietary specification for growing/ready to slaughter pigs may be as set out in the Table below:

Shapes
[000350] The enzyme composition of the present invention and/or the feed additive composition of the present invention, and other components and/or the animal feed comprising the same can be used in any suitable form.
[000351] The enzyme composition of the present invention and/or the feed additive composition of the present invention can be used in the form of solid or liquid preparations, or alternatively thereto. Examples of solid preparations include powders, pastes, cakes, capsules, pellets, tablets, powders and granules which can be wettable, spray dried or freeze dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions.
[000352] In some applications, the feed additive composition of the present invention can be mixed with feed or administered in drinking water.
[000353] Suitable examples of forms include one or more of: powders, pastes, cakes, pellets, tablets, pills, capsules, eggs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate, delayed, modified release applications , sustained, pulsed or controlled.
[000354] By way of example, if the composition of the present invention is used in a solid form, for example, pelleted, it may also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate , dibasic calcium phosphate and glycine; disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
[000355] Examples of nutritionally acceptable carriers for use in preparing the forms include, for example, water, saline, alcohol, silicone, waxes, petroleum gel, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose , magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, essential oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
[000356] Preferred excipients for the forms include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols.
[000357] For aqueous suspensions and/or elixirs, the composition of the present invention can be combined with various sweetening or flavoring agents, dyes or tinctures, with emulsifying and/or suspending agents and with diluents such as water, propylene glycol and glycerin, and combinations of the same. Combination with other components
[000358] The feed additive composition, or the feed ingredient, or feed, animal feed or premix of the present invention can be used together with other components.
[000359] The combination of the present invention, the feed additive composition or the feed ingredient, or feed, animal feed or premix of the present invention, and another component that is suitable for animal consumption and is capable of providing a medical or physiological benefit to the consumer.
[000360] In one embodiment, the "other component" may be one or more enzymes.
[000361] Additional enzymes suitable for use in the present invention may be one or more of the enzymes selected from the group consisting of: endoglucanases (E.C. 3.2.1.4); cellobiohydrolases (EC 3.2.1.91), β-glycosidases (EC 3.2.1.21), cellulases (EC 3.2.1.74), lichenases (EC 3.1.1.73), lipases (EC 3.1.1.3), lipid acyltransferases (generally classified as EC 2.3.1.x), phospholipases (EC 3.1.1.4, EC 3.1.1.32 or EC 3.1.1.5), phytases (eg 6-phytase (EC 3.1.3.26) or a 3-phytase (EC 3.1.3.8 ), alpha-amylases (EC 3.2.1.1), xylanases (EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.37, EC 3.1.1.72, EC 3.1.1.73), glycoamylases (EC 3.2.1.3), proteases ( for example, subtilisin (EC 3.4.21.62) or a bacillolysin (EC 3.4.24.28) or an alkaline serine protease (EC 3.4.21.x) or a keratinase (EC3.4.xx)) and/or mannanases (eg, a β-mannanase (EC 3.2.1.78)).
[000362] In one embodiment (particularly for feed applications), the other component may be one or more of the enzymes selected from the group consisting of xylanases (EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.37, EC 3.1.1.72 , EC 3.1.1.73), an amylase (including α-amylases (EC 3.2.1.1), G4-forming amylases (EC 3.2.1.60), β-amylases (EC 3.2.1.2) and Y-amylases (EC 3.2.1.3. ); and/or a protease (for example, subtilisin (EC 3.4.21.62) or a bacillolysin (EC 3.4.24.28) or an alkaline serine protease (EC 3.4.21.x) or a keratinase (EC 3.4.xx)).
[000363] In one embodiment (particularly for feed applications), the other component can be a combination of an amylase (eg α-amylases (EC 3.2.1.1)) and a protease (eg subtilisin (EC 3.4) .21.62)).
[000364] In one modality (particularly for feed applications), the other component can be a β-glucanase, eg an endo-1,3(4)-β-glucanase (E.C.) 3.2.1.6).
[000365] In one modality (particularly for feed applications), the other component may be a mannanase (eg a β-mannanase (E.C. 3.2.1.78)).
[000366] In one modality (particularly for feed applications), the other component can be a lipase (EC 3.1.1.3), a lipid acyltransferase (generally classified as EC 2.3.1.x) or a phospholipase (EC 3.1.1.4 , EC 3.1.1.32 or EC 3.1.1.5), suitably a lipase (EC 3.1.1.3).
[000367] In one modality (particularly for feed applications), the other component can be a protease (eg subtilisin (EC 3.4.21.62) or a bacillolysin (EC 3.4.24.28) or an alkaline serine protease (EC 3.4. 21.x) or a keratinase (EC3.4.xx)).
[000368] In one embodiment, the additional component may be a stabilizer or an emulsifier, or a binder, or a vehicle, or an excipient, or a diluent or a disintegrant.
[000369] The term "stabilizer", as used in the present invention, is defined as an ingredient or as a combination of ingredients that prevents a product (eg a feed product) from being modified over time.
[000370] The term "emulsifier" as used in the present invention refers to an ingredient (eg a feed ingredient) that prevents the separation of emulsions. Emulsions are two immiscible substances, one present in the form of droplets, contained within the other. Emulsions may consist of the oil-in-water type, where the droplet or dispersed phase is oily and the continuous phase is water; or water-in-oil, where water becomes the dispersed phase and the continuous phase is oily. Foams, which are gas in liquid, and suspensions, which are solid in liquid, can also be stabilized through the use of emulsifiers.
[000371] As used in the present invention, the term "binder" refers to an ingredient (eg a feed ingredient) that holds the product together through physical or chemical reactions. During "gelling", for example, water is absorbed, providing a binding effect. However, binders can absorb other liquids, such as oils, keeping them inside the product. In the context of the present invention, binders could typically be used in solid or low moisture products, for example in bakery products; puff pastries, donuts, breads and others. Examples of granulation binders include one or more of: polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.
[000372] "Vehicles" means materials suitable for administering the enzyme, and include any material known in the art, such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer or the like, that is non-toxic and non-interacting with any components of the composition in a harmful way.
[000373] The present invention provides a method for preparing a composition (e.g., a feed additive composition) comprising mixing the feed additive of the present invention (and preferably, corn or a corn by-product) with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a component of wheat, sucrose, starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate and mixtures thereof.
[000374] Examples of "excipients" include one or more of: microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols.
[000375] Examples of "disintegrants" include one or more of: starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates.
[000376] Examples of "diluents" include one or more of: water, ethanol, propylene glycol and glycerin, and combinations thereof.
[000377] The other components can be used simultaneously (for example, when they are mixed together or even when they are administered by different routes) or sequentially (for example, they may be administered by different routes) to the feed additive of the present invention.
[000378] In one embodiment, preferably, the feed additive composition, the feed ingredient, the feed, the animal feed or the premix, according to the present invention, does not comprise chromium or organic chromium.
[000379] In one embodiment, preferably, the feed additive composition, the feed ingredient, the feed, the animal feed or the premix according to the present invention does not contain sorbic acid. biophysical characteristic
[000380] As used in the present invention, "biophysical characteristic" means any biophysical property of an animal that improves its health, and/or its performance and/or yield. By way of example, the biophysical trait of interest may be one or more from the selected group of: animal performance, animal growth performance, feed conversion ratio (FCR), ability to digest a raw material (eg, nutrient digestibility, including starch, fat, protein and fiber digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass, feed efficiency, body fat percentage, fat distribution body, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, eg manure production and/or nitrogen excretion.
[000381] In a preferred embodiment, the biophysical characteristic arising from a method or use according to the present invention may be selected from the group consisting of one or more of the following: animal performance, animal growth performance, ratio of feed conversion (FCR), ability to digest a raw material (eg nutrient digestibility including starch, fat, protein and fiber digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass, feeding efficiency, body fat percentage, body fat distribution, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, eg manure production and/ or nitrogen excretion.
[000382] In one modality, the biophysical characteristic of the animal represents the performance of the animal. Performance
[000383] As used in the present invention, "animal performance" can be determined by the feed efficiency, and/or the weight gain of the animal, and/or the feed conversion ratio and/or the digestibility of a nutrient in a feed (eg amino acid digestibility) and/or by digestible energy or metabolizable energy in a feed and/or by nitrogen retention.
[000384] Preferably, the "animal performance" is determined by the feed efficiency, and/or the weight gain of the animal and/or the feed conversion ratio.
[000385] By "improved animal performance" is meant that there is greater feed efficiency, and/or greater weight gain, and/or reduced feed conversion ratio, better digestibility of nutrients or energy in a feed and/or better retention of nitrogen in the animal, resulting from the use of the feed additive composition of the present invention compared to feeding the animal with lignocellulosic biomass that has not been treated in accordance with the present invention.
[000386] Preferably, by "improved animal performance" is meant that there is greater feed efficiency and/or greater weight gain and/or reduced feed conversion ratio.
[000387] As used in the present invention, the term "feed efficiency" refers to the amount of weight gain in an animal that occurs when the animal is fed ad libitum, or with a specific amount of food over a period of time.
[000388] By "improved feed efficiency" is meant that the use of a feed additive composition according to the present invention in the feed results in a greater weight gain per unit of feed intake compared to an animal fed with lignocellulosic biomass that has not been treated in accordance with the present invention. Feed Conversion Ratio (FCR)
[000389] As used in the present invention, the term "feed conversion ratio" refers to the amount of feed supplied to an animal to increase the animal's weight by a specified amount.
[000390] An improved feed conversion ratio represents a lower feed conversion ratio.
[000391] By "lower feed conversion ratio" or "improved feed conversion ratio" is meant that the use of a feed additive composition in the feed results in a smaller amount of feed needed to be fed to an animal to increase the weight of the animal in a specified amount compared to the amount of feed needed to increase the animal weight by the same amount when lignocellulosic biomass, which has not been treated in accordance with the present invention, is used in or as the feed. Nutrient digestibility
[000392] Nutrient digestibility, as used in the present invention, means the fraction of a nutrient that disappears from the gastrointestinal tract or from a specific segment of the gastrointestinal tract, for example, the small intestine. Nutrient digestibility can be measured as the difference between what is given to the animal and what comes out in the animal's feces, or between what is given to the animal and what remains in the bolus in a specific segment of the gastrointestinal tract, by example, in the ileum.
[000393] Nutrient digestibility, as used in the present invention, can be measured by the difference between the intake of a nutrient and the nutrient excreted through the total collection of excreta over a period of time; or with the use of an inert marker that is not absorbed by the animal and that allows the researcher to calculate the amount of nutrients that have disappeared in the entire gastrointestinal tract, or in a segment of the gastrointestinal tract. Such an inert marker can be titanium dioxide, chromic oxide or acid-insoluble ash. Digestibility can be expressed as a percentage of nutrients in the feed, or as mass units of digestible nutrients per mass units of nutrients in the feed.
[000394] Nutrient digestibility, as used herein, encompasses starch digestibility, fat digestibility, protein digestibility and amino acid digestibility.
[000395] Energy digestibility, as used in the present invention, means the gross energy of the feed consumed minus the gross energy of feces or the gross energy of the feed consumed minus the gross energy of the bolus remaining in a specified segment of the gastrointestinal tract of the animal, for example, in the ileum. Metabolizable energy as used in the present invention refers to apparent metabolizable energy and means the total energy of the feed consumed minus the total energy contained in faeces, urine and gaseous products of digestion. Energy digestibility and metabolizable energy can be measured as the difference between total energy intake and total energy excreted in faeces or bolus present in the specified segment of the gastrointestinal tract, using the same methods to measure nutrient digestibility, with appropriate corrections for nitrogen excretion to calculate the metabolizable energy of the feed. Nitrogen retention
[000396] Nitrogen retention, as used in the present invention, means the ability of an animal to retain dietary nitrogen as body mass. A negative nitrogen balance occurs when nitrogen excretion exceeds daily intake and is often seen when muscle wasting occurs. A positive nitrogen balance is often associated with muscle growth, particularly in growing animals.
[000397] Nitrogen retention can be measured as the difference between nitrogen intake and nitrogen excreted through the total collection of excreta and urine over a period of time. Excreted nitrogen is understood to include undigested feed proteins, endogenous proteinaceous secretions, microbial protein, and urinary nitrogen. Carcass yield and meat yield
[000398] The term carcass yield, as used in the present invention, means the amount of carcass as a proportion of live body weight, after a commercial or experimental slaughter process. The term carcass means the body of an animal that has been slaughtered for food, with the head, entrails, limbs, and feathers or skin removed. The term meat yield, as used in the present invention, means the amount of edible meat as a proportion of live body weight, or the amount of a cut of meat as a proportion of live body weight. Weight gain
[000399] The present invention further provides a method of increasing weight gain in an animal, for example in poultry or swine, which comprises feeding said animal with an animal feed comprising a feed additive composition according to the present invention.
[000400] A "greater weight gain" refers to an animal with greater weight gain being fed a feed comprising a feed additive composition, compared to an animal being fed a feed comprising or consisting of lignocellulosic biomass, which has not been treated in accordance with the present invention.
[000401] Unless defined otherwise, all technical and scientific terms used in this document have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th ed., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the scholar in the art a general dictionary of many of the terms used in the present disclosure.
[000402] The present disclosure is not limited to the exemplary methods and materials disclosed in the present invention, and any methods and materials similar or equivalent to those described in the present invention may be used in practicing or testing the embodiments of this disclosure. Numeric ranges include the numbers that define the range. Unless otherwise noted, any nucleic acid sequences are written from left to right, in 5' to 3' orientation; and the amino acid sequences are written from left to right in orientation from amino group to carboxyl group, respectively.
[000403] The headings provided herein are not limitations on the various aspects or modalities of this disclosure, which may be adopted by reference to the descriptive report as a whole. In this sense, the terms defined immediately below are more fully defined by reference to the descriptive report as a whole.
[000404] Amino acids are referred to in this document using the amino acid name, the three letter abbreviation or the single letter abbreviation.
[000405] The term "protein", as used herein, includes proteins, peptides and polypeptides.
[000406] As used herein, the term "sequence of amino acids" is synonymous with the term "polypeptide" and/or the term "protein". In some cases, the term "sequence of amino acids" is synonymous with the term "peptide". In some cases, the term "sequence of amino acids" is synonymous with the term "enzyme".
[000407] The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional one-letter and three-letter codes representing amino acid residues can be used. The 3-letter code for amino acids is defined in accordance with the Joint Commission on Biochemical Nomenclature IUPACIUB (JCBN). It is also understood that a polypeptide can be encoded by more than one nucleotide sequence due to the degeneration of the genetic code.
[000408] It should be noted that as used herein and in the appended claims, the singular forms "a", "an", "the" and "a" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such candidate agents, and reference to "feed" includes reference to one or more feeds and equivalents thereof known to those of skill in the art.
[000409] The publications discussed herein are provided solely by their disclosure prior to the filing date of this application. Nothing in this document should be construed as an admission that such publications constitute prior art to the appended claims on this subject.
[000410] The invention will now be described, by way of example only, with reference to the Figures and Examples below. ExamplesSummary
[000411] Due to population growth and improved quality of life in BRIC countries, food and feed prices have increased. The present invention aims to provide alternative feed ingredients for animal production.
[000412] The present inventors have surprisingly found that the elimination of the production of the C5 monomeric sugar and the promotion of the production of the C5 oligomer, together with the cellulase activities (for example, to produce a C6 oligomer and C-6 monomeric sugars) is advantageous in the production of plant materials (eg lignocellulosic biomass) for use in or as animal feed. Materials and methods
[000413] The enzymes tested were:
[000414] • Cellulase SC is an enzymatic composition comprising the following enzymatic activities: endoglucanase activity, β-glucosidase activity and endoxylanase activity, and which has no, or substantially no β-xylosidase activity and α-L-arabinofuranosidase activity. Cellulase SC was kept at -20°C and thawed prior to use.
[000415] • Endoxylanase (endo-1,4-β-xylanase) - Xylanase™ 40000Lda Danisco (synonymous: Xylanase Y5 or Y5) available from Danisco Animal Nutrition, DuPont A/S - this has been diluted to 9293 U/g.
[000416] • Endoxylanase (endo-1,4-β-xylanase) - Econase XT™, available from ABVista. This was in purified form, at 0.51 mg protein/ml. Econase XT™ is a variant of the actinomycete Nonomuraea flexuosa xylanase (Leskinen et al. Appl Microbiol Biotechnol (2005) 67: 495 - 505).
[000417] • Endoxylanase (endo-1,4-β-xylanase) - (FoxXyn6), as taught in PCT/CN2012/079655 (which is incorporated herein by reference) which is encoded by the nucleotide sequence SEQ ID No. 1 or SEQ ID No. 2, and has the polypeptide sequence SEQ ID No. 3.
[000418] • Endoxylanase (endo-1,4-β-xylanase) - (synonym: FveABC5,FoxXyn4, FveXyn4), as taught in PCT/CN2012/079650 (which is hereby incorporated by reference), which is encoded by nucleotide sequence SEQ ID No. 4 or SEQ ID No. 5, and has the polypeptide sequence SEQ ID No. 6.
[000419] • Accellerase®Trio™ is a comparative enzymatic composition comprising endoglucanase, β-glucosidase and endoxylanase activity, as well as enzymatic activities that form C-5 monomers, such as β-xylosidase activity and α-L-arabinofuranosidase activity. The recommended dosage of Accellerase®Trio™ is 0.05 to 0.3 ml per gram of cellulose, or approximately 0.03 to 0.16 ml per gram of biomass (depending on biomass composition). Accellerase®Trio™ was kept at -20°C prior to use.
[000420] Pretreated biomass: Corn stubble pretreated with diluted ammonia (DaCS) was obtained from DuPont (batch no. HP90-HP93). It is ~65% solids and contains low level of acetamide. It was produced in accordance with the DuPont patent application (WO 2006/110901 A2 - which is incorporated herein by reference).
[000421] Sugar analysis using Aminex HPX-87P column (300 x 7.8 mm), from Biorad (Hercules, CA USA). The release of sugars and oligomers is indicative of biomass hydrolysis. According to the manufacturer, the following sugars can be well separated: 1. Cellobiose, 0.1%, 2. Glucose, 10%, 3. Xylose, 0.1%, 4. Galactose, 0.1%, 5. Arabinose, 0.1%, 6. Mannose, 0.5%. The analysis conditions used were: fixed column oven temperature at 80°C, flow rate defined at 0.5 mL of milliQ water/min. The HPLC was Dionex Summit with P580 pump and Shodex IR detector. Glucose, xylose and arabinose (1% (w/v)) standards were used. Injection volumes were 10 or 20 µL.
[000422] The analysis of glucose, xylose, arabinose and oligosaccharides was performed using the Dionex CarboPac column, on a Dionex HPLC.
[000423] The released sugars, oligomers and polymers were also analyzed on a CarboPac column, on a Dionex HPLC.
[000424] For the determination of free monosaccharides: the sample was centrifuged for 5 min at 16000 x g and the supernatant was reserved. A strong anion exchanger (SPE SAX) solid-phase extraction column (available from Sigma Fine Chemicals) (500 mg adsorbent) was conditioned with 2 mL methanol and 2 mL water. 0.1 ml of the sample solution was applied to the column, and the sample was vacuum sucked into a test tube. The column was washed with 2 ml of water in the same test tube. Each sample was diluted to 10 mL with water (or other suitable volume), depending on the concentration of monosaccharides. For the determination of total sugars after acid hydrolysis: 0.100 ml of the sample, 0.100 ml of water and 0.200 ml of 2M H2SO4 were added to a 35 ml test tube. The tube was closed with a buffer and left to stand at 100°C for 3 h. The tube was allowed to cool naturally and 29.6 mL of water was added. The sample was then centrifuged for 5 min at 13000 x g and filtered with 0.45 µm filter paper. It was further diluted with water depending on the sugar concentration. The amount of polymeric sugars was calculated as follows:
[000425] Process samples were analyzed on a Dionex HPLC using the CarboPacTM PA1 column (4 x 250 mm) with the CarboPacTM PA1 pre-column (4 x 40 mm) (available from Dionex), with column oven: 30 °C. Mobile phase: A: HPLC grade water, B: 0.2 M NaOH, injection volume: 25 L. flow rate of 1 mL/min. The gradient program used was: 0 min, 10% B, 1.1 min, 10% B, 5.0 min, 0% B, 33 min, 0% B, 34 min, 100% B , 41 min, 100% B, 42 min, 10% B, 56 min, 10% B. The PED detector (Pulsed Electrochemical Detector) was used. The sugar standards used were: L(+) Arabinose (Merck 1488), D-Galactose (Merck 4058), D(+)-Xylose (Merck 8689), D(+) Glucose (Merck 8337), D(+) Mannose (Merck 5984). Polymeric sugars (mg/L) = total sugars after acid hydrolysis (mg/L) - free monosaccharides (mg/L). Example 1. Hydrolysis of cellulase SC from corn stubble treated with dilute ammonia (DaCS).
[000426] Corn stubble treated with diluted ammonia (DaCS): to a 50 mL Falcon tube, add 15 g of DaCS, tap water and adjust the pH to 5.0 using 2N HCl. 1.33 mL of cellulase SC (Product Lot No. 118, (protein concentration 76.6 g/L) referred to in the present invention as Cellulase SC118) was added to initiate the reaction. The final concentration of DaCS biomass was equal to 18.4% (w/v). Cellulase SC118: The biomass ratio (w/w) was 1.08:100.
[000427] The reaction was carried out at 50°C with stirring at 200 rpm for 70 h. Liquid samples were taken at 21.5 h, 45.5 h, 62.5 h and at the end of the reaction, 70.5 h after a brief centrifugation of the biomass in the Falcon tube. The sample size at time intervals was equal to 60 μL, which was then mixed with 60 μL of water used to wash the tips of the pipettes used to suck the samples. The collected samples were kept at 5°C before analysis by HPLC. Each of the samples was then further mixed with 120 µL of milliQ water, heated to 100°C for 5 min and filtered. The 10 μL filtered were injected into the HPLC for analysis of released sugars. To each of the 3 Falcon tubes, 14 ml of milliQ water was added to wash away the residual biomass obtained after centrifugation. The residual washed biomass was dried at 80°C for 20 h and weighed. Their weights were used to compare the weights of biomass treated in the absence of the enzyme composition. Result of HPLC analysis of Example 1
[000428] Figure 1 shows the yields of glucose and xylose obtained by treating DaCS with SC cellulase.
[000429] Figure 1 is based on the data detailed in the table (Table 1) below. As can be seen from the table, the xylose released corresponds to less than 1/3 of the glucose released from DaCS at 50°C at pH 5.0, for up to 70.5 hours.
Results of the weight analysis of Example 1
[000430] From Table 2, under the in vitro conditions, it can be noted that 56% of the DaCS was converted. The improvement can be achieved by further optimizing the mix.Table 2:. Hydrolysis of corn stubble biomass by cellulase Cellulase SC at 50°C and 70.5 h

[000431] The results indicate that the most efficient cellu ase for the hydrolysis of biomass is the Accellerase®Trio™. The products are glucose, xylose and a smaller amount of arabinose, as seen in the HPLC chromatograms.
[000432] The Cellulase SC mixture hydrolyzed DaCS, but unlike the comparative enzyme composition Accellerase®Trio™ (Figure 2), Cellulase SC was less efficient in general and also produced less C5 sugars of xylose and arabinose. The formation of fewer C5 sugars is desirable, since monogastric animals are inefficient in terms of the use of C5 sugars. This result indicates that Cellulase SC has low (or no) β-xylosidase activity and α-L-arabinofuranosidase activity.
[000433] It was found that cellulase SC, with an endoxylanase (Xylanase™ from Danisco), promotes the release of glucose and xylo-oligosaccharides. This was found to be highly beneficial for use in animal feed.Example 2. Hydrolysis with Cellulase SC of corn stubble treated with dilute ammonia (DaCS) and the effect of adding an endoxylanase.
[000434] To each of the 9 plastic tubes with a volume of 12 mL, 1 g of DaCS and 5 mL of MilliQ water were added and mixed, and the tubes were left to stand at 5°C for one day another to ensure the DaCS was well hydrated. The next day, 150 µl of 1N HCl was added to adjust the pH to 4.6 to 5.1. Enzymes (listed in Table 3, below) were added to start the reaction. The reaction was carried out at 50°C with stirring speed of 180 rpm for 40 h. Liquid samples (120 μL) were collected at a reaction time of 6 h, 19 h and 40 h, diluted with 120 μL of MilliQ water and stored at 5°C, awaiting analysis. At the end of the enzymatic reaction, 240 μL of MilliQ water was added to each of the collected samples, which were then heated in a water bath at 100°C for 5 min, followed by centrifugation at 10000 g for 10 minutes. 250 μL of the supernatant was then filtered, and 10 μL of the filtrate was injected for analysis by HPLC. Glucose and 1% xylose (w/v) standards were filtered and analyzed by HPLC, under the same conditions with the same injection volume as the hydrolyzate samples collected.
[000435] Biomass weight reduction analysis: For the residual biomass in the 9 tubes obtained after centrifugation, after removing the supernatant, 4 mL of water was added and shaken to wash any soluble substance from the precipitate. The suspension was centrifuged again to obtain the washed biomass residue, which was then dried at 60°C for 48 h.
Results of HPLC analysis of Example 2
[000436] Cellulase Cellulase SC (cellulase SC 118 and cellulase SC 151) produced a much lower level of xylose than Accellerase®Trio™ (see Figure 2 and Figure 3). The glucose produced by Cellulase SC preparations was also lower than that of Accellerase®Trio™. This is also true for the entire reaction process, from 6, 19 and 40 h (Figure 2 below).
[000437] Cellulase SC, together with an endoxylanase (FoxXyn4), promoted the release of glucose and also promoted the release of xylose throughout the reaction time. The xylose (eg C5 sugar) released by Cellulase SC, together with the xylanase, was much lower than that released by Accellerase®Trio™.
[000438] The hydrolysates generated in Table 2 were further analyzed using the CarboPac column on a Dionex HPLC. The results were shown in Figure 3. From Figure 3, it can be seen that the results are similar to those in Figure 2, in relation to the total amount of monosugars produced. In addition, Figure 3 shows that for total soluble polymeric sugars, Cellulase SC or Cellulase SC plus endoxylanase treatment resulted in an amount greater than that of Accellerase®Trio™ treatment.
[000439] From Figure 3, it can be seen that the total amount of soluble sugar in polymeric form released by Cellulase SC, with or without the addition of xylanase, is 4 times greater than that of Accellerase®Trio™, which had beta-xylosidase and alpha-L-arabinofuranosidase activities. The sugars were analyzed using the Dionex CarboPac column.Example 3. Additional study of hydrolysis by Cellulase SC of corn stubble treated with dilute ammonia (DaCS) and the effect of addition of various xylanases.
[000440] Place 1 g of DaCS in each of the tubes listed in Table 3, with a volume of 12 mL, add 4.6 mL of water and 0.15 mL of 1N HCl, and mix. The measured pH was equal to pH 5.03 to 5.17. Then, the enzymes Accellerase®Trio™, Cellulase SC 118 or Cellulase SC 118 plus 10 to 120 uL of the 5 different xylanases were added to start the reaction at 50°C, stirring at 200 rpm for 45 h. Tubes 22 to 24 were controls as no enzymes were added. The 3k, 6k, 12k, 15k, 18k and 21k tubes are the white ones. Table 3. "-" means no addition of the relevant component




[000441] *Pure Econase XT is a more purified version of Econase XT, which shows only 1 band on the SDS-PAGE gel corresponding to xylanase
[000442] At the end of the reaction, all tubes were centrifuged at 3500 rpm for 10 minutes, and the supernatant was collected. The precipitate was washed twice with 4 ml and 3.5 milliQ water, respectively, and centrifugation was repeated. All supernatants were pooled and made up to 12 mL. 0.3 mL of these were heated at 100°C for 7 min, filtered and 10 µL were injected for sugar analysis by HPLC. Arabinose, xylose and glucose (w/v) standards were also filtered and 10 μL were injected. Two HPLC analyzes were performed for each sample.
[000443] Analysis of biomass weight: Final residual biomass precipitates were placed at 80°C for 48 h of drying, and weighed to obtain the net weight. Result of HPLC analysis of Example 3
[000444] As can be seen from Figure 4, a combination of Cellulase SC with all the different xylanases increased, in all cases, the glucose production by about 30 to 40%, which is in agreement with the yield of the glucose produced by Accellerase®Trio™. Xylose production has also increased, but its level is much lower than that achieved by Accellerase®Trio™.
[000445] A typical HPLC chromatogram of the DaCS hydrolyzate produced by Accellerase®Trio™ shows the monosaccharide peak of glucose, xylose and arabinose (see Figure 5), with rather flat peaks in the polymer area (peak 2 with retention of 9.22 min).
[000446] A typical HPLC chromatogram of the DaCS hydrolyzate produced by Cellulase SC (low sign) and Cellulase SC in combination with Danisco's Xylanase™ (upper sign) shows the glucose, xylose and arabinose peaks (see Figure 6), compared to the much larger and wider peaks of the oligomers and polymers in Figure 5 with Accellerase®Trio™.
[000447] Figure 5 and Figure 6 show that Cellulase SC and Cellulase SC together with a xylanase, generated a greatly reduced xylose peak, and also a reduced glucose peak. On the other hand, it has high contents of oligo- or polymers in the region between the retention time of 6.5 and 11.5 min.
[000448] The inventors have surprisingly found that the reduced amount of xylose (and even glucose) and the increased amount of oligomers and polymers are advantageous when the hydrolyzate is used as a feed ingredient, particularly for monogastric animals.
[000449] The inventors found that xylose is not an efficient source of energy for monogastric animals, since about 50% of consumed xylose is excreted in the urine. Oligomers and polymers, on the other hand, can be fermented by the microbiota that occupy the large intestine into short-chain fatty acids, which can be absorbed into the bloodstream and used by animals as an energy source. Results of weight analysis of Example 3
[000450] From Figure 7 below, it can be seen that, in the presence of Cellulase SC, the addition of an endoxylanase (eg Danisco's Xylanase™, Econase XT™, FoxXyn4 or FoxXyn6, or the more highly purified forms of Danisco's Xylanase™, Econase XT™), increased hydrolysis in all cases, based on the reduced weight of residual biomass obtained after the enzymatic reaction. The hydrolysis depicted in Figure 7 was performed using non-industrial processing conditions, and for shorter time periods to accommodate bench scale testing constraints. Therefore, the degree of total hydrolysis was of inferior quality, but still reflected the improved hydrolysis that occurs when Cellulase SC and an endoxylanase are used together. Example 4. Further study of Cellulase SC hydrolysis of corn stubble treated with dilute ammonia (DaCS) and the dose effect of Cellulase SC and endoxylanase (Danisco's Xylanase™)
[000451] 1 g of DaCS was placed in each of the tubes listed in Table 4, and water was added to a volume of 12 ml, together with 0.15 ml of 1N HCl and mixed. Then, the enzymes Cellulase SC 118 50 - 200 μL of Cellulase SC 118 50-200 μL with 10 uL of Xylanase™ from Danisco were added to start the reaction at 50°C, with stirring at 250 rpm for 40 h. After the reaction, the tubes were centrifuged and the supernatant was collected. The precipitate was washed twice with 4 ml and 3.5 ml of water, respectively, by resuspension and centrifugation. The supernatants were pooled and made up to 12 ml with the addition of more water. Pooled supernatants (2 ml) were placed at 100°C for 10 min to inactivate enzymes, then centrifuged and filtered. The filtrate was analyzed by means of HPLC, with an injection volume of 10 μL. The washed precipitates were dried at 80°C for 48 h.
Weight Analysis Results of Example 4
[000452] From Figure 8, it can be seen that the DaCS had a dry matter content equal to 63.3% (column 1 on the left). By incubating the DaCS at 50°C for 40 h in water pH adjusted to pH 5 using HCl, 16.0% of the dry matter was solubilized (left column 2 of Figure 9). Addition of endoxylanase to 10 µL (93 units) further increased the solubility of DaCS up to 26.9%. The addition of cellulase only Cellulase SC118 increased the solubility up to 41.7%. With a combination of 10 μL of endoxylanase and 50 μL of Cellulase SC118, the solubility reached about 51.7%, indicating that xylanase and Cellulase SC have an additive effect. To achieve the level of solubility achieved by 10 μL of xylanase and 50 μL of Cellulase SC, it is necessary to add 3 times or more Cellulase SC alone (150 μL and 200 μL). The conclusion is that, for the solubilization of DaCS, a combination of 50 μL of cellulase SC with 10 μL of endoxylanase is superior to Cellulase SC alone, at 50, 100 and 150 μL. 50 μL of Cellulase SC plus 10 μL of endoxylanase is ideal with a 10% (w/v) DaCS dry matter loading because 100, 150 and 200 μL of Cellulase SC plus 10 μL endoxylanase did not incrementally increase solubility . It is generally accepted that the greater the solubility, the greater the in vivo digestibility. Result of HPLC analysis of Example 4
[000453] From Figure 10, it can be seen that there is no free glucose in the soluble fraction of DaCS, without added enzyme or with Danisco's Xylanase™ at 10 uL (93 units). This indicates that 26.9% of the soluble matter released by the addition of Danisco's Xylanase™ is primarily oligomers and polymers alone (Figure 9). With the addition of Cellulase SC in doses of 50, 100, 150 to 200 μL in the reaction, there is a greater release of glucose and xylose, but the increase is not linear in relation to the dose of Cellulase SC (Figure 10).
[000454] From Figure 10, it can also be noted that the amount of glucose released is greater for the combination of 50 μL of Cellulase SC with 10 μL of Xylanase™ from Danisco than for 100 μL of Cellulase SC alone; 100 μL of Cellulase SC with 10 μL of Danisco's Xylanase™ is approximately equal to 150 μL of Cellulase SC only, and 150 μL of Cellulase SC with 10 μL of Danisco's Xylanase™ is approximately equal to 200 μL of Cellulase SC only.
[000455] In conclusion, it is evident that Danisco's Xylanase™ and Cellulase SC exhibit a synergy in the release of glucose and xylose, as the glucose released by a combination of 50 μL of Cellulase SC and 10 μL of Xylanase™ from Danisco is greater than the sum of glucose released only by Danisco's Xylanase™ (which is basically zero) and 50 μL of Cellulase SC (Figure 10).
[000456] Figure 11 shows the concentration of total soluble sugars in the 12 mL of supernatant obtained after enzymatic hydrolysis of DaCS, followed by centrifugation and washing. It can be seen that the main sugars are glucose, xylose and arabinose; the secondary components are galactose and mannose. Conclusions
[000457] Various cellulase preparations for sugar release have been studied. For animal nutrition, commercial enzyme combinations, particularly those used for the breakdown of lignocellulosic biomass in the bioethanol industry, are not ideal for use in animal nutrition, mainly because such compositions produce too much xylose. It was found that xylose is a sugar that monogastric animals cannot efficiently metabolize and use.
[000458] It was found that enzymatic compositions comprising at least the following activities: endoglucanase activity, β-glucosidase activity and endoxylanase activity; wherein one or both of the following enzymatic activities are absent or substantially absent in the enzymatic composition: β-xylosidase activity and α-L-arabinofuranosidase activity, are the most effective compositions for degrading lignocellulosic biomass, while producing a product that was more suitable for use as an animal feed composition or as a feed for animals.Example 5: Xylose as an energy source for broilers
[000459] A total of 240 male broilers (1 day old) of the Ross 308 lineage were used in this study. The chicks were individually weighed when leaving the egg and were divided into 12 groups of 5 birds, balanced in terms of body weight. They were then weighed as a group, and each group was housed in a cage in electrically heated Petersime brooders. The brooder and ambient temperatures were set at 32 and 29°C, respectively, during the first week. After that, the heat supply in the brooder was turned off and the temperature was maintained at 29°C throughout the experiment. The study duration was 21 days, and during this period the birds were fed one of the following four treatments: 1: basal diet (diet based on corn bran and soy) + 25% corn starch, 0% D-xylose, 2: basal diet + 20% cornstarch, 5% D-xylose, 3: basal diet + 10% cornstarch, 15% D-xylose and 4: basal diet + 0% corn starch, 25% D-xylose. Feed and fresh water were made available to all chicks for ad libitum consumption throughout the study. During the study period, feed intake and bird body weight were monitored weekly to calculate body weight gain, voluntary feed intake and feed conversion efficiency.
[000460] The duration of the study was 21 days, however, after 14 days of treatment, birds on treatment 4 were removed due to high mortality rates and signs of disease. The basal diet was a diet based on corn bran and soybean.
[000461] During the first 14 days of the study, there was a significant decrease in feed intake and weight gain in birds fed diets containing 25% xylose compared to birds fed all other treatments (P<0.05 ) (see Figure 12). On day 14 of the study, all birds on treatment 4 were removed from the study. By this time, mortality in the treatment group (25% xylose) had reached 10% and many more birds were showing symptoms of disorientation, blindness and body tremors.
[000462] After 21 days of feeding the treatment diets, birds fed 10% cornstarch and 15% xylose in the diet showed significantly lower body weight gain and lower feed efficiency compared to birds fed 25% starch and 0% xylose (P<0.05) (see Figure 13 and Figure 14). Birds fed 5% xylose and 20% cornstarch showed intermediate weight gain and FCR compared to the other two treatments, but not significantly different from either of them.
[000463] In conclusion, the replacement of 25% of the energy source in the diet by xylose is harmful to the health of broilers. Broiler chickens appear to tolerate up to 5% xylose in the diet before demonstrating significant adverse effects on growth performance, however, even with the inclusion of 5% xylose in the diet, there is a numerical reduction in growth performance compared to with the control diet.Example 6. Hydrolysis with Accellerase®1500™ and Econase®XT™ of corn stubble treated with dilute ammonia (DaCS) and caustic delignified yellow millet (DLswg)
[000464] In each of 8 plastic tubes with a volume of 12 mL, 1 g of DaCS and 5 mL of MilliQ water are added and mixed. Likewise, 1 g of DLswg and 5 mL of MilliQ water are added to another 8 plastic tubes with a volume of 12 mL, and mixed. All tubes are left to rest at 5°C overnight to ensure that the DaCS and DLswg are well hydrated. The next day, 150 µL of 1N HCl was added to adjust the pH to 4.6 to 5.1. Enzymes (listed in Table 5, below) are added to start the reaction. The reaction is carried out at 50°C with stirring speed of 180 rpm for 40 h. Liquid samples (120 μL) were collected at a reaction time of 6 h, diluted with 120 μL of MilliQ water and stored at 5°C, until the analysis was performed. At the end of the enzymatic reaction, 240 μL of MilliQ water is added to each of the collected samples, which are then heated in a water bath at 100°C for 5 min, followed by centrifugation at 10000 xg for 10 minutes. 250 μL of the supernatant was then filtered, and 10 μL of the filtrate was injected for analysis by HPLC. Glucose and 1% (w/v) xylose standards are filtered and analyzed on HPLC under the same conditions with the same injection volume described in the Materials and Methods section.



[000465] Accellerase®1500 is a commercial enzyme available from Danisco (now part of DuPont) and contains certain cellulase activities such as endoglucanase activity, beta-glucosidase and certain hemicellulases such as endoxylanase activity.
[000466] Econase® XT5P is a commercial enzyme available from AB Vista, and has endo 1-4 Beta xylanase activity. Results
[000467] The results indicate that the addition of Accellerase®1500™ and Econase®XT 5P to dilute ammonia-pretreated corn stubble and caustic delignified yellow millet have a synergy in glucose release, since the amount of glucose released by the combination of Accellerase®1500™ (0.3 mL/g) and Econase®XT 5P (1.4 mg/g DM) is greater than the amount of glucose released by each enzyme individually (Table 6) . The combination of endoglucanase, endoxylanase and β-glycosidase activities, as predicted in this example, by the addition of Accellerase®1500™ and Econase®XT 5P, resulted in substantial glucose release similar to that of Accellerase®Trio™. The combination of Accellerase®1500™ and Econase®XT 5P resulted in significantly less release of xylose due to the absence of significant amounts of α-L-arabinofuranosidase and β-xylosidase activity present in this enzyme combination compared to Accellerase®Trio™ .Table 6. Release of xylose and glucose from corn stubble pretreated with dilute ammonia (DAcs) and caustic delignified yellow millet (DLswg) using Accellerase®Trio™, Accellerase®1500™ and Econase®XT 5P.
1DAcs - Dilute Ammonia Pretreated Corn Stubble 2Example 7. Hydrolysis of Dilute Ammonia Treated Corn Stubble (DaCS) and Caustic Delignified Yellow Millet (DLswg) with , Celluclast®, Xylanase™ from Danisco and Accellerase®BG™
[000468] In each of 12 plastic tubes with a volume of 12 mL, 1 g of DaCS and 5 mL of MilliQ water are added and mixed. Likewise, 1 g of DLswg and 5 ml of MilliQ water are added to another 12 plastic tubes with a volume of 12 ml, and mixed. All tubes are left to rest at 5°C overnight to ensure that the DaCS and DLswg are well hydrated. The next day, 150 µL of 1N HCl was added to adjust the pH to 4.6 to 5.1. Enzymes (listed in Table 7, below) are added to start the reaction. The reaction is carried out at 50°C with stirring speed of 180 rpm for 40 h. Liquid samples (120 μL) were collected at a reaction time of 6 h, diluted with 120 μL of MilliQ water and stored at 5°C, until the analysis was performed. At the end of the enzymatic reaction, 240 μL of MilliQ water is added to each of the collected samples, which are then heated in a water bath at 100°C for 5 min, followed by centrifugation at 10000 xg for 10 minutes. 250 μL of the supernatant was then filtered, and 10 μL of the filtrate was injected for analysis by HPLC. Glucose and 1% (w/v) xylose standards are filtered and analyzed on HPLC, under the same conditions with the same injection volume described in the Materials and Methods section.




[000469] Celluclast® is a commercial enzyme available from Sigma-Aldrich (C2730) and contains certain cellulase activities such as endoglucanase activity and beta-glucosidase activity.
[000470] Danisco's Xylanase™ is a commercial enzyme available from Danisco (now part of DuPont) that has endoxylanase activity.
[000471] Accellerase®BG™ is a commercial enzyme available from Danisco (now part of DuPont) that has β-glucosidase activity. Results
[000472] The results indicate that the addition of Celluclast®, Xylanase™ from Danisco, and Accellerase®BG™ to corn stubble pretreated with dilute ammonia and caustic delignified yellow millet have a synergy in glucose release as they the amount of glucose released by the combination of Celluclast® (0.1 mL/g DM), Danisco's Xylanase™ (3.5 uL/g DM) and Accellerase®BG™ (0.15 mL/g DM) is greater than than the amount of glucose released by each enzyme, alone or in combination with just 2 enzymes (Table 8). The combination of endoglucanase, endoxylanase and β-glycosidase activities, as predicted in this example, by the addition of Celluclast®, Xylanase™ from Danisco and Accellerase®BG™, resulted in similar glucose release to that of Accellerase®Trio™. The combination of Celluclast®, Danisco's Xylanase™ and Accellerase®BG™ resulted in significantly less release of xylose due to the absence of significant amounts of α-L-arabinofuranosidase and β-xylosidase activity present in this enzyme combination compared to Accellerase® Trio™. Table 8. Release of xylose and glucose from corn stubble pretreated with dilute ammonia (DAcs) and caustic delignified yellow millet (DLswg) using Accellerase®Trio™, Celluclast®, Xylanase™ from Danisco and Accellerase®BG™
1DAcs - corn stubble pretreated with dilute ammonia 2DLswg - caustic delignified yellow millet na - not detectedExample 8. Hydrolysis of diluted ammonia treated corn stubble (DaCS) and caustic delignified yellow millet (DLswg) with Aspergillus niger Cellulase ( Sigma C1184) and Accellerase®BG™
[000473] In each of the 6 plastic tubes with a volume of 12 mL, 1 g of DaCS and 5 mL of MilliQ water are added and mixed. Likewise, 1 g of DLswg and 5 mL of MilliQ water are added to another 8 plastic tubes with a volume of 12 mL, and mixed. All tubes are left to rest at 5°C overnight to ensure that the DaCS and DLswg are well hydrated. The next day, 150 µL of 1N HCl was added to adjust the pH to 4.6 to 5.1. Enzymes (listed in table 9, below) are added to start the reaction. The reaction is carried out at 50°C with stirring speed of 180 rpm for 40 h. Liquid samples (120 μL) were collected at a reaction time of 6 h, diluted with 120 μL of MilliQ water and stored at 5°C, until the analysis was performed. At the end of the enzymatic reaction, 240 μL of MilliQ water is added to each of the collected samples, which are then heated in a water bath at 100°C for 5 min, followed by centrifugation at 10000 xg for 10 minutes. 250 μL of the supernatant was then filtered, and 10 μL of the filtrate was injected for analysis by HPLC. Glucose and 1% (w/v) xylose standards are filtered and analyzed on HPLC under the same conditions with the same injection volume described in the Materials and Methods section.



[000474] Aspergillus niger cellulase is a commercial enzyme available from Sigma-Aldrich (C1184) and contains certain cellulase activities such as endoglucanase activity as well as endoxylanase activity.
[000475] Accellerase®BG™ is a commercial enzyme available from Danisco (now part of DuPont) that has β-glucosidase activity. Results
[000476] The results indicate that the addition of A. niger Cellulase and Accellerase®BG™ to corn stubble pretreated with dilute ammonia and caustic delignified yellow millet has a synergy in the release of glucose, since the amount of glucose released by the combination of A. niger Cellulase and Accellerase®BG™ is greater than the amount of glucose released by each enzyme individually (Table 10). The combination of endoglucanase, endoxylanase and β-glycosidase activities, as predicted in this example, by the addition of cellulase from A. niger and Accellerase®BG™, resulted in similar glucose release to that of Accellerase®Trio™. The combination of A. niger cellulase and Accellerase®BG™ resulted in significantly lower release of xylose due to the absence of significant amounts of α-L-arabinofuranosidase and β-xylosidase activity present in this enzyme combination, compared to Accellerase®Trio ™.Table 10. Release of xylose and glucose from corn stubble pretreated with dilute ammonia (DAcs) and delignified caustic yellow millet (DLswg) using Accellerase®Trio™, A. niger cellulase and Accellerase®BG™
1DAcs - corn stubble pretreated with dilute ammonia2 2DLswg - caustic delignified yellow millet
[000477] All publications mentioned in the above descriptive report are hereby incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to one skilled in the art, without departing from the scope and spirit of the present invention. While the invention has been described in conjunction with specific preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to persons versed in the fields of biochemistry and biotechnology, or in related fields, are intended to fall within the scope of the following claims.

权利要求:
Claims (10)
[0001]
1. Method of preparing a feed additive composition, characterized in that it comprises: a. pre-treat physically, chemically and/or biologically the lignocellulosic biomass, b. mixing the physically, chemically and/or biologically pretreated lignocellulosic biomass with an enzymatic composition, wherein the enzymatic composition comprises one or more of the following activities: endoglucanase activity, β-glycosidase activity and endoxylanase activity, and wherein the enzymatic composition comprises none or less than 1500 units/mg of β-xylosidase activity and/or none or less than 450 units/mg of α-L-arabinofuranosidase activity,c. incubate it for 3 to 120 hours.
[0002]
2. Method according to claim 1, characterized in that it further comprises drying and/or packaging the lignocellulosic biomass.
[0003]
3. Use of an enzymatic composition in combination with physical and/or chemical and/or biological pretreatment, wherein the enzymatic composition comprises one or more of the following activities: endoglucanase activity, β-glucosidase activity and endoxylanase activity, and in which the enzyme composition comprises none, or less than 1500 units/mg of β-xylosidase activity and/or none, or less than 450 units/mg of α-L-arabinofuranosidase activity, characterized by the fact that it is in the manufacture of a feed additive composition to reduce the level of C-5 monomer sugars and maintain high levels of C-6 monomer and oligomer sugars for a lignocellulosic biomass animal.
[0004]
4. Method or use, according to any one of claims 1 to 3, characterized in that the enzymatic composition further comprises one or both of the following enzymatic activities: cellobiohydrolase I activity and cellobiohydrolase II activity.
[0005]
5. Method or use, according to any one of claims 1 to 4, characterized in that the enzymatic composition further comprises the lytic activity of polysaccharide mono-oxygenase.
[0006]
6. Method or use, according to any one of claims 1 to 5, characterized in that the method further comprises feeding the feed additive composition to an animal.
[0007]
7. Method or use, according to any one of claims 1 to 6, characterized in that lignocellulosic biomass is any cellulosic or lignocellulosic material, for example, agricultural waste, bioenergy crops, solid industrial waste, solid urban waste, sludge derived from the manufacture of paper, garden waste, wood waste, forest waste and combinations thereof.
[0008]
8. Method or use according to any one of claims 1 to 7, characterized in that the lignocellulosic biomass is selected from the group consisting of corn cobs, crop residues such as corn husks, corn stubble, grasses , beet pulp, wheat straw, wheat chaff, oat straw, wheat bran, wheat shorts, rice bran, rice husk, wheat bran, oat husk, palm kernel, citrus pulp, cotton, lignin, barley straw, hay, rice straw, rice husk, yellow millet, miscanthus, esparto, spotted reed, paper waste, sugarcane bagasse, sorghum bagasse, forage of sorghum, sorghum stubble, soybean stubble, soybean, components obtained from the grinding of trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits and flowers.
[0009]
9. Method or use, according to any one of claims 1 to 8, characterized in that the method or use further comprises a step of contacting a feed component with the feed additive composition.
[0010]
10. Method of preparing a feed, characterized in that it comprises contacting a feed component with a feed additive composition or feed ingredient, preferably with a feed additive composition obtainable (preferably obtained) by the method or use as defined in any one of claims 1 to 8.

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公开号 | 公开日

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BR112015031628A2|2017-11-07|

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AU2014283210A1|2015-12-24|

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WO2014202716A1|2014-12-24|

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CN105517450A|2016-04-20|

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EP3013155A1|2016-05-04|

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AR096670A1|2016-01-27|

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法律状态:
2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-05| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-12-01| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-04-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-29| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 19/06/2014, OBSERVADAS AS CONDICOES LEGAIS. |

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US201361837748P| true| 2013-06-21|2013-06-21|

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US61/837,748|2013-06-21|

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PCT/EP2014/062935|WO2014202716A1|2013-06-21|2014-06-19|Methods and compositions to improve the nutritional value of lignocellulosic biomass|

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