VALUE OF LIGNOCELLULOSIC BIOMASS

FIELD OF THE INVENTION

The present invention relates to methods and compositions to improve the nutritional value of lignocellulosic biomass. The present invention relates to methods and compositions which result in lignocellulosic biomass being a suitable replacement to conventionally used starch from grains in animal feed.

BACKGROUND OF THE INVENTION

Approximately 750 million tonnes of cereal grains per year are used for animal nutrition. The replacement of a proportion of these cereal grains with cheaper feed sources would provide a significant benefit to the population as a whole.

The replacement of grain (e.g. corn) for cheaper cellulosic materials (e.g. lignocellulosic biomass) would revolutionize the animal feed markets, and/or increase the stability of the human food and energy markets.

The nutritional value of lignocellulosic biomass has not been sufficient to render it a viable alternative to high energy feedstuffs, such as grain. Monogastric animals like swine and poultry for instance, cannot digest and absorb glucose from lignocellulose of fibrous ingredients, such as agricultural waste, e.g. to an extent that allows replacement of significant amounts of cereal grains in the animal's diet. Animals, particularly monogastric animals, do not have the enzymes needed to digest cellulose and hemicellulose, and even if they did, the retention time in the intestine is a limitation for conversion of cellulose to glucose.

Cellulose is an organic compound with the formula (C ₆ H ₁₀ O ₅ ) _n , a polysaccharide consisting of a linear chain of β(1→4) linked D-glucose units.

Hemicellulose is any of several heteropolymers (matrix polysaccharides) present along with cellulose in almost all plant cell walls. Hemicelluloses include xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. Hemicelluloses contain many different sugar monomers. For instance, besides 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 small amounts of L-sugars as well. Xylose is in most cases the sugar monomer present in the largest amount, although in softwoods mannose can be the most abundant sugar. Not only regular sugars can be found in hemicellulose, but also their acidified form, for instance glucuronic acid and galacturonic acid can be present. Schutte et al (British J. of Nutrition 1991 , 66, 83-93) investigated nutritional implications of D- xylose in pigs. Schutte et al taught that ileal digestibility of D-xylose as well as that of D- glucose was close to 100%. The presence of D-xylose in the diet decreased ileal digesta pH and increased ileal flow of volatile fatty acids, suggesting the occurrence of microbial degradation of D-xylose in the pig small intestine. Schutte suggested that ileal and faecal digestibility of dry matter (DM), organic matter (OM), gross energy (GE), and nitrogen (N), as well as N retention, decreased significantly in pigs fed on very high dosages of D-xylose (e.g. 200g D-xylose/kg diet). Schutte (1991 ) also found that at 10% inclusion in pig diets, 50% of the xylose energy appeared in the urine. Verstegen et al (J. Animal. Physiol, a. Anim. Nutr. 77 (1997) 180-188) conducted a trial in pigs to evaluate xylose as an energy source for pigs. Depending on the calculation method used, when included at 10% in the diet, between 38-64% of the xylose energy in the diet appeared as metabolizable energy (ME), When pigs fed 10% xylose in the diet were compared to similar pigs fed 5% glucose in the diet, the ME of both diets were similar and weight gain was similar between treatments.

Savory et al (British Journal of Nutrition (1992), 67, 103-1 14) studied the metabolic fate of U- C-labelled monosaccharides in fowl, and suggested that xylose was absorbed more slowly than glucose and galactose, but faster than mannose and arabinose. Savory (1992) also observed that xylose and arabinose were metabolised to a less extent than the hexose (C6) sugars evident by the greater recovery of U- ¹⁴ C-labelIed xylose and arabinose in excreta from birds.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the glucose and xylose yield obtained from treating DaCS (dilute ammonium pre-treated corn stover) with Cellulase SC.

Figure 2 shows the release of glucose and xylose from DaCS using Accellerase^Trio™, Cellulase SC or Cellulase SC + endoxylanase.

Figure 3 shows the release of sugars from DaCS by Accellerase ^® Trio™, Cellulase SC118, Cellulase 151 and SC1 8 + xylanase. Figure 4 shows hydrolysis of DaCS by Accellerase ^® Trio™, Cellulase SC and Cellulase SC + 4 different endoxylanases.

Figure 5 shows a HPLC chromatogram of DaCS hydrolyzate produced by Accellerase ^® Trio™, 30μΙ.

Figure 6 shows a HPLC chromatogram of DaCS hydrolyzate produced by Cellulase SC and Cellulase SC in combination with endoxylanase (SoloC1 18, 50μ!_.

Figure 7 shows hydrolysis of DaCS by Accellerase ^® Trio™, Cellulase SC and Cellulase SC + endoxylanases.

Figure 8 shows solubilization of DaCS (weight of DaCS residue, g) with or without cellulose SC1 18 and endoxylanase Danisco Xylanase™ (see EP1222256 which is incorporated herein by reference) at 50°C pH5.0 for 40h.

Figure 9 shows solubilization of DaCS (% solubilized material) with or without cellulose SC1 18 and endoxylanase Danisco Xylanase™ at 50°C pH5.0 for 40h.

Figure 10 shows release of monosugars from DaCS by Cellulase SC1 18 and the dose effect of xylanase Danisco Xylanase™ at 50°C pH5.0 for 40h.

Figure 11 shows release of sugars (monomeric+polymeric forms) from DaCS by Cellulase SC1 18 and the dose effect of xylanase Danisco Xylanase™ at 50°C pH5.0 for 40h.

Figure 12 shows feed intake and body weight gain in broilers fed xylose as an energy source.

Figure 13 shows body weight at day 21 in broilers fed xylose as an energy source.

Figure 14 shows feed conversion ratio dO-21 in broilers fed xylose as an energy source. Figure 15 shows a nucleotide sequence (SEQ ID No. 1 ) encoding an endoxylanase (FoxXyn6). The signal sequence is shown in bold (upper case).

Figure 16 shows a nucleotide sequence (SEQ ID No. 2) encoding an endoxylanase (FoxXyn6).

Figure 17 shows a polypeptide sequence (SEQ ID No. 3) of an endoxylanase (FoxXyn6). This is the active form of the enzyme (e.g. the mature form of the enzyme).

Figure 18 shows a nucleotide sequence (SEQ ID No. 4) encoding an endoxylanase (FoxXyn4). The signal sequence is shown bold (upper case).

Figure 19 shows a nucleotide sequence (SEQ ID No. 5) encoding an endoxylanase (FoxXyn4).

Figure 20 shows a polypeptide sequence (SEQ ID No. 6) of an endoxylanase (FoxXyn4). This is the active form of the enzyme (e.g. the mature form of the enzyme).

Figure 21 shows a nucleotide sequence (SEQ ID No. 7) encoding an endoglucanase (EG1 ) from Aspergillus niger (CBS513.88). Figure 22 shows a polypeptide sequence (SEQ ID No. 8) of an endoglucanase (EG1 ) from Aspergillus niger (CBS513.88).

Figure 23 shows a nucleotide sequence (SEQ ID No. 9) encoding an endoglucanase (EG2) from Aspergillus niger CBS513.88.

Figure 24 shows a polypeptide sequence (SEQ ID No. 10) of an endoglucanase (EG2) from Aspergillus niger CBS513.88.

Figure 25 shows a nucleotide sequence (SEQ ID No. 1 1) encoding an endoxylanase.

Figure 26 shows a polypeptide sequence (SEQ ID No. 12) of an endoxylanase.

Figure 27 shows a nucleotide sequence (SEQ ID No. 13) encoding a β-glucosidase from Aspergillus niger CBS513.88.

Figure 28 shows a polypeptide sequence (SEQ ID No. 14) of a β-glucosidase from Aspergillus niger CBS513.88.

Figure 29 shows a nucleotide sequence (SEQ ID No. 15) encoding a lytic polysaccharide monooxygenase from Aspergillus n/o/er CBS513.88.

Figure 30 shows a polypeptide sequence (SEQ ID No. 16) of a lytic polysaccharide monooxygenase from Aspergillus niger CBS513.88.

Figure 31 shows a nucleotide sequence (SEQ ID No. 17) encoding a CHB1A from Aspergillus niger (CBS513.88).

Figure 32 shows a polypeptide sequence (SEQ ID No. 18) of a CHB1A from Aspergillus n/ger (CBS513.88).

Figure 33 shows a nucleotide sequence (SEQ ID No. 19) encoding a CHB1 B from Aspergillus niger (CBS513.88).

Figure 34 shows a polypeptide sequence (SEQ ID No. 20) of a CHB1 B from Aspergillus /?/ger (CBS513.88).

Figure 35 shows a nucleotide sequence (SEQ ID No. 21 ) encoding a CHB1 from Trichoderma reesei.

Figure 36 shows a polypeptide sequence (SEQ ID No. 22) of a CHB1 from Trichoderma reesei.

Figure 37 shows a nucleotide sequence (SEQ ID No. 23) encoding a CHB2 from Trichoderma reesei.

Figure 38 shows a polypeptide sequence (SEQ ID No. 24) of a CHB2 from Trichoderma reesei.

Figure 39 shows a nucleotide sequence (SEQ ID No. 25) encoding an endoglucanase (EG1) from Trichoderma reesei.

Figure 40 shows a polypeptide sequence (SEQ ID No. 26) of an endoglucanase (EG1) from Trichoderma reesei. Figure 41 shows a nucleotide sequence (SEQ ID No. 27) encoding an endoglucanase (EG2) from Trichoderma reesei.

Figure 42 shows a polypeptide sequence (SEQ ID No. 28) of an endoglucanase (EG2) from Trichoderma reesei.

Figure 43 shows a nucleotide sequence (SEQ ID No. 29) encoding an endoglucanase (EG3) from Trichoderma reesei.

Figure 44 shows a polypeptide sequence (SEQ ID No. 30) of an endoglucanase (EG3) from Trichoderma reesei.

Figure 45 shows a nucleotide sequence (SEQ ID No. 31) encoding a lytic polysaccharide monooxygenase from Trichoderma reesei.

Figure 46 shows a polypeptide sequence (SEQ ID No. 32) of a lytic polysaccharide monooxygenase from Trichoderma reesei.

Figure 47 shows a nucleotide sequence (SEQ ID No. 33) encoding an endoxylanase from Trichoderma reesei.

Figure 48 shows a polypeptide sequence (SEQ ID No. 34) of an endoxylanase from Trichoderma reesei.

Figure 49 shows a nucleotide sequence (SEQ ID No. 35) encoding a β-glucosidase from Trichoderma reesei.

Figure 50 shows a polypeptide sequence (SEQ ID No. 36) of a β-glucosidase from Trichoderma reesei.

STATEMENTS OF THE INVENTION

In a first aspect the present invention provides a method of preparing a feed additive composition comprising:

a. physically and/or chemically and/or biologically pre-treating lignocellulosic biomass, b admixing the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass with an enzyme composition, wherein the enzyme composition comprises at least the following activities: endoglucanase activity, β-glucosidase activity and endoxylanase activity and wherein the enzyme composition comprises no, or substantially no, β-xylosidase activity and/or a- L-arabinofuranosidase activity c, incubating same for at least about 3-120 hours, preferably 6-48 hours, d and optionally drying and/or optionally packaging In another aspect the present invention provides a use of an enzyme composition, wherein the enzyme composition comprises at least the following activities: endoglucanase activity, β- glucosidase activity and endoxylanase activity and wherein the enzyme composition comprises no, or substantially no, β-xylosidase activity and/or a-L-arabinofuranosidase activity in the manufacture of a feed additive composition for improving the nutritional value to an animal of a lignocellulosic biomass.

A further aspect of the present invention is a feed additive composition obtainable (e.g. obtained) by a method of the present invention. In another aspect, there is provided a feed additive composition or a feed ingredient comprising a physically and/or chemically and/or biologically pre-treated lignocellulosic biomass hydrolysed with an enzyme composition, wherein the enzyme composition comprises at least the following: an endoglucanase, a β-glucosidase and an endoxylanase activity and no, or substantially no, β-xylosidase activity and/or a-L-arabinofuranosidase activity.

A further aspect of the present invention provides a feed or feedstuff 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.

In a further aspect there is provided 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 invention, and at least one mineral and/or at least one vitamin.

The present invention yet further provides a method of preparing a feedstuff comprising contacting a feed component with 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.

The present invention also provides a method for improving a biophysical characteristic of an animal which method comprises administering to an animal a feed additive composition obtainable (e.g. obtained) by a method of the present invention or a feed additive composition according to the present invention or a premix according to the present invention or a feedstuff according to the present invention or a feedstuff obtainable (e.g. obtained) by a method of the present invention. In a yet further aspect the present invention provides 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 a feedstuff according to the present invention or a feedstuff obtainable (e.g. obtained) by a method of the present invention for improving a biophysical characteristic of an animal.

In a preferable embodiment the enzyme composition comprises no, or substantially no, β- xylosidase activity and no, or substantially no, a-L-arabinofuranosidase activity. Suitably the enzyme composition may further comprise one or more enzyme selected from the group consisting of: a cellobiohydrolase I and a cellobiohydrolase II.

Suitably the enzyme composition may further comprise a lytic polysaccharide monooxygenase.

DETAILED DISCLOSURE OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The inventors research (See Example 5) has shown that high levels of C-5 monomer sugars (e.g. xylose) in feed can have negative consequences on the health of an animal ingesting the feed.

However using high levels of lignocellulosic biomass in feedstuffs can either result in not enough nutritional value in the feedstuff or if the lignocellulosic biomass are treated in an attempt to render them more nutritionally valuable this can lead to a situation where the C-5 monomer sugars (e.g. xylose) becomes too high in the feedstuff and can be detrimental on the health of the animal.

Therefore the inventors determined that the C-5 monomer sugars should preferably be removed from any lignocellulosic biomass prior to feeding to an animal. The present inventors have now found a unique method for treatment of lignocellulosic biomass with an enzyme composition which can result in a feedstuff or a feed additive composition having a reduced level of C-5 monomer sugars (such as xylose) whilst maintaining high levels of C-6 oligomer and monomer sugars which are beneficial (e.g. nutritional) to the animals. It was surprising that if one removed the C-5 carbohydrate monomer forming activities that a significant formation of xylooligomers could be achieved without compromising the efficiency or time of conversion of cellulose to glucose. The resultant composition was much more beneficial in feed applications.

The present inventors have surprisingly found that the elimination of C5-monomer sugar production and promotion of C5-oligomer production together with cellulase activities (e.g. to produce C6-oligomer and C-6 monomer sugars) is an advantageous mix of activities. Methods and uses comprising this enzyme composition will allow the inclusion of higher levels of lignocellulosic biomass into animal feed and into animal diets.

The present inventors have surprisingly found that the nutritional value of lignocellulosic biomass to animals (particularly monogastric animals) can be significantly improved by using an enzyme composition comprising at least the following activities: endoglucanase activity, β- glucosidase activity 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 a-L-arabinofuranosidase activity in the treatment of lignocellulosic biomass. Surprisingly it has been found that by reducing or excluding beta-xylosidase and alpha-L- arabinofuranosidase activities the nutritional value of lignocellulosic biomass to animals (particularly monogastric animals) can be significantly improved.

It would appear from the data that the benefits are derived from reducing the amount of monomer sugars (particularly monomer C-5 sugars) in the composition whilst significantly increasing the oligomer sugars (e.g. xylooligomers) in the composition.

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 (e.g. cellobiose), the enzyme reaction is progressively slowed down since due to a feedback inhibition of the reaction. Thus it had been understood prior to the present invention that the feedback mechanism of the reaction when the amount of monomer sugars formed was reduced resulted in an overall decrease in the enzyme reaction. Surprisingly no apparent feedback mechanism was observed with the enzyme composition, e.g. which was low in β- xylosidase activity and/or a-L-arabinofuranosidase activity for use in the present invention. The present inventors surprisingly found methods using enzyme mixtures which breakdown cellulose and hemicellulose but which lack carbohydrate monomer forming activities or reduced monomer forming activities (particularly lacking C-5 carbohydrate monomer forming activities) can significantly improve the nutritional value of lignocellulosic biomass in animals (particularly monogastric animals).

This effect is further enhanced by pre-treating the 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 and/or chemically and/or biologically pre-treated lignocellulosic biomass is subsequently treated with an enzyme composition comprising at least the following activities: endoglucanase activity, β-glucosidase activity 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 a-L- arabinofuranosidase activity.

The present invention means that lignocellulosic biomass can be used as a replacement to conventionally used starch (e.g. from grains) in animal feed. This has significant advantages. For example, the use of the present invention means that a significant proportion of cereal grains conventionally used for animal nutrition would no longer need to be used in animal feed applications. The present invention allows a reduction in the cost of preparing the animal feed. In addition or alternatively, the present invention eases the resource pressures in the supply chains that have been caused by increased human population size and constraints on the supply of fossil fuels. The presently claimed invention is relevant for both monogastric animals and ruminants, but is most specifically relevant for monogastric animals.

In ruminants, feed supplements in accordance with the present invention or produced by the present invention can be used to replace the cereal grains that are used for intensive production of meat and milk in ruminants, and/or for increasing the efficiency of the system.

The present invention relates to a method of preparing a feed additive composition comprising:

a. physically and/or chemically and/or biologically pre-treating lignocellulosic biomass, b admixing the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass with an enzyme composition, wherein the enzyme composition comprises at least the following activities: endoglucanase activity, β-glucosidase activity and endoxylanase activity and wherein the enzyme composition comprises no, or substantially no, β-xylosidase activity and/or a- L-arabinofuranosidase activity, c, incubating same for at least about 3-120 hours, preferably 6-48 hours, d and optionally drying and/or optionally packaging

The term "absent" or "no" as used herein means that the enzyme composition has no β- xylosidase activity and no a-arabinofuranosidase activity.

The terms "substantially absent" and "substantially no" as used herein in reference to β- xylosidase activity and/or α-L-arabinofuranosidase activity mean an activity of less than 3000 unit/mg 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/or less than 1000 units/mg α-L-arabinofuranosidase activity (suitably less than 500 units/mg, suitably less than 450 units/mg) as determined using the "Alpha-L-arabinofuranosidase Activity Assay" taught herein.

In one embodiment the terms "substantially absent" and "substantially no" as used herein in reference to β-xylosidase activity and/or α-L-arabinofuranosidase activity mean an activity of less than 1500 units/mg β-xylosidase activity as determined using the "Beta-Xylosidase Activity Assay" taught herein and/or less than 450 units/mg α-L-arabinofuranosidase activity as determined using the "Alpha-L-arabinofuranosidase Activity Assay" taught herein.

In one embodiment the enzyme composition for use in the present invention has less than 3000 unit/mg 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 α-L-arabinofuranosidase activity (suitably less than 500 units/mg, suitably less than 450 units/mg) as determined using the "Alpha-L-arabinofuranosidase Activity Assay" taught herein.

In one embodiment the terms "substantially absent" and "substantially no" as used herein in reference to β-xylosidase activity and/or α-L-arabinofuranosidase activity mean an activity of less than 1500 units/mg β-xylosidase activity as determined using the "Beta-Xylosidase Activity Assay" taught herein and less than 450 units/mg a-L-arabinofuranosidase activity as determined using the "Alpha-L-arabinofuranosidase Activity Assay" taught herein. In a preferred embodiment, the enzyme composition may comprise no, or substantially no, β- xylosidase activity and no, or substantially no, α-L-arabinofuranosidase activity.

In a preferred embodiment, the enzyme composition may comprise no β-xylosidase activity and no α-L-arabinofuranosidase activity.

In a preferred embodiment the lignocellulosic biomass for use in the present invention may be physically pre-treated, chemically pre-treated, biologically pre-treated, or combinations thereof lignocellulosic biomass. In another embodiment the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass may be incubated with the enzyme composition for at least about 6- 48 hours.

In another preferred embodiment the enzyme composition for use in the present invention may consist essentially of (or consists of) endoglucanase activity, β-glucosidase activity and endoxylanase activity.

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.

In one embodiment the enzyme composition for use in the present invention may further comprise lytic polysaccharide monooxygenase activity. In some embodiments the enzyme composition for use in the present invention may comprise endoglucanase activity, β-glucosidase activity and endoxylanase activity and one or more of the following enzyme activities: cellobiohydrolase I activity, cellobiohydrolase II activity or lytic polysaccharide monooxygenase activity. In some embodiments the enzyme composition for use in the present invention may consist essentially of (or consists of) endoglucanase activity, β-glucosidase activity and endoxylanase activity and one or more of the following enzyme activities: cellobiohydrolase I activity, cellobiohydrolase II activity or lytic polysaccharide monooxygenase activity.

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 (suitably three) of the following enzyme activities: cellobiohydrolase I activity, cellobiohydrolase II activity or lytic polysaccharide monooxygenase activity.

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 (suitably three) of the following enzyme activities: cellobiohydrolase I activity, cellobiohydrolase II activity or lytic polysaccharide monooxygenase activity. In one embodiment the enzyme composition for use in the present invention may be characterised by its enzyme activity.

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-glucosidase Activity Assay" and endoxylanase activity as determined using the "Endoxylanase Activity Assay".

The ranges in which these activities may be present in said enzyme composition are set out in the tables below and the paragraphs thereafter:

Range of activity in Units of each enzyme activity in the composition

endoglucanase activity 500-4000 CMC U/g

β-glucosidase activity 200-3500 pNPG U/g ²

endoxylanase activity 1500-6000 ABX U/g ³

Range of activity in Units of each enzyme activity in the composition

endoglucanase activity 1000-3500 CMC U/g ¹ (preferably 1500-3000

CMC U/g)

β-glucosidase activity 300-3000 pNPG U/g ² (preferably 500-2500 pNPG U/g)

endoxylanase activity 2000-5000 ABX U/g ³ (preferably 3000-4000

ABX U/g)

One CMC unit of activity liberates 1 μιηοΙ of reducing sugars (expressed as glucose equivalents) in one minute at 50°C and pH 4.8.

² One pNPG unit denotes 1 mol of nitro-phenol liberated from para-nitrophenyl-B-D- glucopyranoside per minute at 50°C and pH 4.8.

³ One ABX unit is defined as the amount of enzyme required to generate 1 pmol of xylose reducing sugar equivalents per minute at 50°C and pH 5.3.

In one embodiment the enzyme composition for use in the present invention may comprise at least 500 CMC U/g endoglucanase activity (suitably at least 1000 CMC U/g 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".

In another embodiment the enzyme composition for use in the present invention may comprise at least 200 pNPG U/g β-glucosidase activity (suitably at least 1000 pNPG U/g activity, 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".

In a further embodiment the enzyme composition for use in the present invention may comprise at least 1500 ABX U/g endoxylanase activity (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 "Endoxylanase Activity Assay".

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 endoglucanase activity (suitably at least 1000 CMC U/g 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 U/g activity, 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 endoxylanase activity (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 "Endoxylanase Activity Assay". 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 endoglucanase activity (suitably at least about 1000 CMC U/g to about 3500 CMC U/g activity, suitably at least about 1500 CMC U/g to about 3000 CMC U/g activity or suitably at least about 1500 CMC U/g to about 2500 CMC U/g activity) as determined using the "Endoglucanase Activity Assay".

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 β-glucosidase activity (suitably at least about 300 pNPG U/g to 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 to about 2000 pNPG U/g activity) as determined using the "Beta-glucosidase Activity Assay". in a further 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 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 to about 4500 ABX U/g activity or suitably at least about 3000 ABX U/g to about 4000 ABX U/g activity) as 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 endoglucanase activity (suitably at least about 1000 CMC U/g to about 3500 CMC U/g activity, suitably at least about 1500 CMC U/g to about 3000 CMC U/g activity or suitably at least about 1500 CMC U/g to about 2500 CMC U/g activity) as determined using the "Endoglucanase Activity Assay", about 200 pNPG U/g to about 3500 pNPG U/g β-glucosidase activity (suitably at least about 300 pNPG U/g to 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 to about 2000 pNPG U/g activity) as determined using the "Beta-glucosidase Activity Assay" and at least about 1500 ABX U/g to about 6000 ABX U/g 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 to about 4500 ABX U/g activity or suitably at least about 3000 ABX U/g to about 4000 ABX U/g activity) as determined using the "Endoxylanase Activity Assay".

In a preferred embodiment, the enzyme composition for use in the present invention may comprise (or consist essentially of, or consists of) about 1000 CMC U/g to about 3500 CMC U/g endoglucanase activity as determined using the "Endoglucanase Activity Assay", about 300 pNPG U/g to about 3000 pNPG U/g β-glucosidase activity as determined using the

"Beta-glucosidase Activity Assay" and about 2000 ABX U/g to about 5000 ABX U/g endoxylanase activity as determined using the "Endoxylanase Activity Assay". In another embodiment the enzyme composition for use in the present invention may further comprise one or more of the following enzyme activities: protease activity (e.g. serine protease activity (E.C. 3.4.21) and/or alkaline subtilisin protease activity (E.C. 3.4.21.62)), pectinase activity (E.C. 3.2.1.15), a-glucuronidase activity (E.C. 3.2.1.139), β-glucuronidase activity (E.C. 3.2.1.31) or esterase activity (E.C. 3.1.1.73).

The esterase activity may be feruloyl esterase activity (E.C. 3.1.1.73).

Endoglucanase activity as referred to herein may be endo-1 ,4-p-D-glucanase activity. An endoglucanase is one which catalyses the endohydrolysis of (1→4)-p-D-glucosidic linkages in cellulose, lichenin and cereal β-D glucans. In other words endoglucanase activity as defined herein means and enzyme which endohydrolyses (1→4)-p-D-glucosidic linkages in cellulose, lichenin and cereal β-D glucans. Endoglucanase activity can be classified under E.C. classification E.C. 3.2.1.4. Another name for endoglucanase is β-glucanase.

An endoglucanase for use in the enzyme composition for use in the present invention may be one or more endoglucanase(s) encoded by a nucleic acid comprising (or consisting of, or consisting essentially of) one or more nucleotide sequence(s) selected from the group consisting of: SEQ ID No. 29, SEQ ID No. 27, SEQ ID No. 25, SEQ ID No. 9 and SEQ ID No. 7.

An endoglucanase for use in the enzyme composition for use in the present invention may be one or more endoglucanase(s) comprising (or consisting of, or consisting essentially of) one or more of the 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/q)

Pipette 1 ml of 1% carboxylmethyl cellulose sodium salt (CMC) solution (prepared with 0.05M sodium acetate buffer) into sample and blank tubes. Incubate tubes in a 50°C water bath for 10 minutes. Pipette 1 ml of enzyme dilution at 15 second intervals to the sample tubes. Mix tubes after each addition. After 10 minute, add 3 ml of 1 % 3,5 dinitrosalicylic acid sodium salt (DNS) in the same order and timing as the enzyme addition to the sample tubes. Add 3 ml of DNS to the sample blank tubes. After adding the DNS remove the test tubes to another rack not in the 50°C water bath. Add 1 ml of diluted enzyme to the corresponding sample blank. 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 10-15 minutes. Transfer to 3 ml cuvettes. Using the reagent blank to zero the spectrophotometer, each sample is read at 540 nm against de-ionised water.

The activity in this procedure is measured relative to an enzyme standard with assigned CMC units. One CMC unit of activity liberates 1 μηηοΙ of reducing sugars (expressed as glucose equivalents) in one minute at 50°C and pH 4.8. In one embodiment a lytic polysaccharide monooxygenase for use in the present invention may 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.

A lytic polysaccharide monooxygenase for use in the enzyme composition for use in the present invention may be one or more lytic polysaccharide monooxygenase(s) encoded by a nucleic acid comprising (or consisting of, or consisting essentially of) one or more nucleotide sequence(s) selected from the group consisting of: SEQ ID No. 31 and SEQ ID No. 15.

A lytic polysaccharide monooxygenase for use in the enzyme composition for use in the present invention may be one or more lytic polysaccharide monooxygenase(s) comprising (or consisting of, or consisting essentially of) one or more of the polypeptide sequences selected from the group consisting of: SEQ ID No. 32 and SEQ ID No. 16.

The cellobiohydrolase (CBH) activity may be CBH class I (CBH I) or CBH class II (CBH II) activity or a combination of both CBH I and CBH II. Suitably the cellobiohydrolase may hydrolyse (1→4)^-D-glucosidic linkages 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-cellobiosidase activity. The

cellobiohydrolase II activity can be classified under E.C. classification EC. 3.2.1.91. The cellobiohydrolase I activity can be classified under E.C. classification EC. 3.2.1.176.

A cellobiohydrolase (CBH) for use in the enzyme composition for use in the present invention may be one or more cellobiohydrolase(s) encoded by a nucleic acid comprising (or consisting of, or consisting essentially of) one or more nucleotide sequence(s) selected from the group consisting of: SEQ ID No. 23, SEQ ID No. 21 , SEQ ID No. 19 and SEQ ID No. 17. A cellobiohydrolase (CBH) for use in the enzyme composition for use in the present invention may be one or more cellobiohydrolase(s) 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. The endoxylanase activity - may be endo-1 ,4- -xylanase activity. Preferably the endoxylanase endohydrolyses the (1→4)-p-D-xylosidic linkage in xylans. Preferably the endoxylanase is classified as E.C. 3.2.1.8.

An endoxylanase for use in the enzyme composition for use in the present invention may be one or more endoxylanase(s) encoded by a nucleic acid comprising (or consisting of, or consisting essentially of) one or more nucleotide sequence(s) selected from the group consisting of: SEQ ID No. 33, SEQ ID No. 1 1 , SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 2 and SEQ ID No. 1.

An endoxylanase for use in the enzyme composition for use in the present invention may be one or more endoxylanase(s) comprising (or consisting of, or consisting essentially of) one or more of the 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)

Pipette 1.8 ml of 1 % birchwood 4-0 methyl glucuronoxylan substrate solution into each test tube. Incubate for 10-15 minutes, allowing it to equilibrate at 50oC. Pipette 0.2 ml of enzyme dilution using positive displacement pipettes or equivalent. Vortex to mix. Incubate each sample at 50oC for exactly 5 minutes. Add 3 ml of 1 % 3,5 nitrosalicylic acid sodium salt (DNS) solution and mix. Cover the tops of the test tubes with caps to prevent evaporation. Place test tubes in a boiling bath for exactly 5 minutes. Cool test tubes for 10 minutes in ice/water bath. Incubate test tube for 10 minutes at room temperature. Transfer test tube contents to cuvettes and measure at 540 nm against deionised water. Correct the absorbance for background colour by subtracting the corresponding enzyme bank. This assay measures the release of reducing sugars by action of endoxylanase on a Birchwood xylan substrate. The rate of reducing sugar release as measured with DNS, is proportional to the enzyme activity.

One ABX unit is defined as the amount of enzyme required to generate 1 μιτιοΙ of xylose reducing sugar equivalents per minute at 50°C and pH 5.3. β-glucosidase activity as defined herein is the hydrolysis of terminal, non-reducing β-D-glucosyl residues with the release of β-D-glucose. β-glucosidase activity can be classified under E.C. classification E.C. 3.2.1.21.

A β-glucosidase for use in the enzyme composition for use in the present invention may be one or more β-glucosidase (s) encoded by a nucleic acid comprising (or consisting of, or consisting essentially of) one or more nucleotide sequence(s) selected from the group consisting of: SEQ ID No. 35 and SEQ ID No. 3.

A β-glucosidase for use in the enzyme composition for use in the present invention may be one or more p-glucosidase(s) comprising (or consisting of, or consisting essentially of) one or more of the polypeptide sequences selected from the group consisting of: SEQ ID No. 36 and SEQ ID No. 14.

"BETA-GLUCOSIDASE ACTIVITY ASSAY" (pNPG U/q)

Pipette 1 ml of 3% nitrophenyl-beta-D-glucopyranoside (pNPG) solution (prepared with 0.05M sodium acetate buffer) into duplicate test tubes for each sample and control. Place into 50°C water bath for 5 minutes. Add 200 μΙ of control or sample to their respective duplicate tubes at intervals of 15-30 seconds. To the reagent blank tube, add 200 μΙ of sodium acetate buffer. Vortex each tube after addition of sample. Let the tubes incubate for exactly 10 minutes. After the 10 minutes incubation, add 500 μΙ of 1 M sodium carbonate solution to stop the reaction. Vortex each tube after the addition and place the tube in a rack outside of the water bath. Add 10 ml of milli-Q water to each tube and vortex to mix. Using the reagent blank to zero the spectrophotometer, the concentration of the 4-nitrophenol is measured by reading each sample at 400 nm.

One pNPG unit denotes 1 μιτιοΙ of nitro-phenol liberated from para-nitrophenyl-B-D- glucopyranoside per minute at 50°C and pH 4.8.

β-xylosidase activity may hydrolyse successive xylose residues from the non-reducing termini of (1→3)^-D-xylans, e.g. the β-xylosidase may be a 1 ,3 β-D-xylosidase. 1 ,3 β-D- xylosidases may be classified under E.C. classification E.C. 3.2.1 .72 or may catalyse the hydrolysis of (1→4)^-D-xylans, to remove successive D-xylose residues from the non- reducing or reducing termini, e.g. the β-xylosidase may be a 1 ,4 β-xylosidase. 1 ,4 β- xylosidases may be classified under E.C. classification E.C. 3.2.1.37.

" B ETA-X YLOS I DAS E ACTIVITY ASSAY" The protocol for beta-xylosidase enzyme assay is similar to previously described (Ruttersmith, L.D., Daniel R.M. 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. The beta-xylosidase activity assay in accordance with the present invention is carried out as follows: substrate for beta-xylosidase is p-nitropheny^-D-xylopyranoside (ρΝβχρ) (Sigma - N2132). A dose response curve is created for each enzyme being tested. Each enzyme is tested at 7 doses (100, 50, 25, 12.5, 6.25, 3.13, 1.56 ppm) with a no-enzyme control containing only substrate and assay buffer. All solutions are made in 50 mM sodium acetate buffer. The standard reaction mixture contains 280 ί of 0.1 M sodium acetate buffer pH 5.0 (final concentration 50 mM) and 80 μΙ of substrate in 50 mM sodium acetate buffer (final concentration 1 mM) and enzyme at various dose concentrations in 50 mM sodium acetate buffer are then added to bring the final volume of the reaction mixture to 400μΙ. The reaction mixture (buffer and substrate) is placed in a 70°C water bath to prewarm prior to the enzyme addition. The enzyme is added to start the reaction. The sample mixture is incubated for 10 min, after which 0.8 ml of 0.1 MNa ₂ C0 ₃ is added to end the reaction. The concentration of p- nitrophenol released is calculated using molar extinction coefficient, ε ₄₀₀ = 18,300 M ^"1 cm ^"1 . The control is subtracted from the enzyme-containing wells, and the absorbance values (A400) are converted to concentration as described in the protocol. One unit of activity is defined as the formation of 1 pmol p-nitrophenol from ρΝβχρ per minute under assay conditions. a-L-arabinofuranosidases (E.C. 3.2.1.55) may hydrolyse arabinan to L-arabinose. "ALPHA-L-ARABINOFURANOSIDASE ACTIVITY ASSAY"

The protocol is similar to that previously described for a-L-arabinofuranosidase (Miyazaki, K. 2005). Hyperthermophilic a-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 incorporated herein by reference) with some modification. The alpha-L-arabinofuranosidase activity assay in accordance with the present invention is carried out as follows: substrate for α-L-arabinofuranosidase was p-nitrophenyl-a-L- arabinofuranoside (pNaLaf) (Sigma - N3641). A dose response curve is created for each enzyme being tested. Each enzyme is tested at 7 doses (100, 50, 25, 12.5, 6.25, 3.13, 1.56 ppm) with a no-enzyme control containing only substrate and assay buffer. All solutions are made in 50 mM sodium acetate buffer. The standard reaction mixture contains 280 μ[_ of 0.1 M sodium acetate buffer pH 5.0 (final concentration 50 mM) and 80 μΙ of substrate in 50 mM sodium acetate buffer (final concentration 1 mM) and enzyme at various dose concentrations in 50 mM sodium acetate buffer are then added to bring the final volume of the reaction mixture to 400 μΙ. The reaction mixture (buffer and substrate) is placed in 70°C water bath to prewarm prior to enzyme addition. The enzyme is added to start the reaction. The reaction mixture is incubated for 10 min, after which it is ended by adding 0.8 ml of 0.1 M Na ₂ C0 ₃ . 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). The control is subtracted from the enzyme-containing wells, and the absorbance values (A400) are converted to concentration as described in the protocol. One unit of enzyme activity is defined as the formation of 1 pmol p-nitrophenol from the substrate per minute under assay conditions.

The term "consist 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 activity or the activities characterised, but no other enzyme activity and/or no other enzyme activity which is capable of digesting lignocellulosic biomass.

By way of example only, the following enzymes may suitably be used in accordance with the present invention:

Enzymes Company Tradename

β-glucosidase Danisco Accellerase ^® BG ^{I WI}

Endoglucanase, hemi-cellulases (including

endoxylanase) & β-glucosidase Danisco Accellerase ^® Duet™

Endoglucanase, endoxylanase & β- glucosidase Danisco Accellerase ^® 1500

C1184 - Cellulase

Cellulase including endoglucanase activity from Aspergillas and endoxylanase Sigma niger

Cellulases including endoglucanase and Novozyme (Sigma- beta-glucosidase Aldrich) Celluclast® β-glucosidase Novozyme Novozyme 188

Cellulases and hemicellulases including β- glucosidase and GH61 activity Novozyme Cellic CTec3 beta-Glucanase ABVista Econase ^® BG Xylanase ABVista Econase ^® XT

Endoxylanase & beta-Glucanase Adisseo Rovabio™ Excel

Endoxylanase & beta-Glucanase BASF Natugrain® TS/L

Endoxylanase & beta-Glucanase Danisco AXTRA ^® XB

Endoxylanase & beta-Glucanase Danisco Avizyme ^® 1110

Endoxylanase Danisco Danisco Xylanase™

Endoxylanase & beta-Glucanase DSM Bio-Feed Plus™ beta-Glucanase DSM Ronozyme ^® VP

Endoxylanase & beta-Glucanase DSM Roxazyme ^® G2 beta-Glucanase Huvepharma Hostazym C ^® beta-Glucanase, Amylase, Protease &

endoxylanase Kemin Kemzyme W™ dry beta-Glucanase, Amylase & endoxylanase Kemin Kemzyme W™ liquid

Alpha-gal & beta-Glucanase Kerry Ingredients Biogalactosidase BL beta-Glucanase Le Saffre Safizyme G beta-Glucanase Lyven Feedlyve AGL endo-1 ,4- -xylanase Danisco Avizyme ^® 1100 endo-1 ,4- -xylanase Danisco Axtra ^® XB endo-1 ,4-p-xylanase Danisco Axtra ^® XAP endo-1 ,4-p-xylanase DSM Biofeed Plus™

In one embodiment the enzyme composition for use in the present invention may be a single enzyme or a combination of enzymes (e.g. an enzyme mix). In one preferred embodiment the enzyme composition according to the present invention is an enzyme mixture.

In one embodiment each of the different enzyme activities defined herein are preferably provided by a separate protein. In other words, each enzyme activity is a different enzyme protein. Preferably the defined enzyme activity is the primary (or sole) activity of the protein. In other words, preferably the defined activity is not a side activity of a protein.

BIOMASS The term "lignocellulosic" refers to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose. The term "cellulosic" refers to a composition comprising cellulose and additional components, including hemicellulose. In one embodiment the lignocellulosic biomass is any cellulosic or lignocellulosic material. The term "lignocellulosic biomass" refers to any lignocellulosic material and includes materials comprising cellulose. The lignocellulosic biomass may optionally further comprise hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein, lipid, ash, and/or extractives. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Preferably, the lignocellulosic biomass comprises at least 15% cellulose.

In one embodiment, the lignocellulosic biomass comprises at least 20% cellulose.

The lignocellulosic biomass may be any cellulosic or lignocellulosic material such as agricultural residues, bioenergy crops, industrial solid waste, municipal solid waste, sludge from paper manufacture, yard waste, wood waste, forestry waste and combinations thereof.

In one embodiment the lignocellulosic biomass may be selected from the group consisting of corn cobs, crop residues such as corn husks, corn stover, grasses, beet pulp, wheat straw, wheat chaff, oat straw, wheat middlings, wheat shorts, rice bran, rice hulls, wheat bran, oat hulls, palm kernel, citrus pulp, cotton, lignin, barley straw, hay, rice straw, rice hulls, switchgrass, miscanthus, cord grass, reed canary grass, waste paper, sugar cane bagasse, sorghum bagasse, forage sorghum, sorghum stover, soybean stover, soy, components obtained from milling of trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers.

In one embodiment the lignocellulosic biomass may be selected from the group consisting wet-cake, com fibre, corn germ meal, corn bran, Hominy feed, corn gluten feed, gluten meal, wheat shorts, wheat middlings, distillers dried grain (DDG) and distillers dried grain solubles (DDGS) based e.g. on corn, wheat, sorghum, or combinations thereof. In a preferred embodiment the lignoceliulosic biomass may be selected from the group consisting of corn stover, wheat straw, corn cobs, sugarcane bagasse, switchgrass, forage sorghum and rice straw. In another embodiment the lignoceliulosic biomass may be selected from the group consisting of corn stover, wheat straw, corn cobs, sugarcane bagasse, switch grass, forage sorghum and rice straw.

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 handle.

In one embodiment the lignoceliulosic biomass may comprise less than 50% starch, preferably less than 40% starch.

In one embodiment the lignoceliulosic biomass may comprise less than 30% starch, preferably less than 10% starch.

In one embodiment the lignoceliulosic biomass may comprise less than 3% starch.

In one embodiment the lignoceliulosic biomass may comprise less than 1% starch. Suitably the lignoceliulosic biomass does not comprise starch. In one embodiment the lignoceliulosic biomass does not comprise a cereal grain. PRE-TREATMENT

In one embodiment the lignoceliulosic biomass is pre-treated by any pre-treatment process known in the art which is capable of disrupting the compact structure of lignoceliulosic biomass to expose cellulose fibers and/or which results in a decrease of the biomass crystallinity (e.g. cellulose crystallinity) and/or which increase accessible surface area for enzyme activity. A number of pre-treatment processes have been developed during the last few decades. Generally, these pre-treatments can be divided into mechanical/physical, physico-chemical, chemical, and biological pre-treatments or combinations thereof. An overview of the various pre-treatments can be found e.g. 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 incorporated herein by reference.

The pre-treatment may be any treatment known in the art which results in either low cellulose crystallinity as measured by method 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 incorporated herein by reference, or which increases accessible surface area for enzyme activity, or a combination of both.

In one embodiment the pre-treatment is a pre-treatment which reduces the crystallinity of cellulose in the lignocellulosic biomass.

In one embodiment the pre-treatment is a pre-treatment which reduces the crystallinity of cellulose in the lignocellulosic biomass by at least about 20% when measured using the Segal Method (supra). In one embodiment the pre-treatment is a pre-treatment which increases accessible surface area for enzyme activity in the lignocellulosic biomass.

In one embodiment the pre-treatment is a pre-treatment which increases accessible surface area by at least about 20%, suitably at least about 30%, suitably at least about 40% in the lignocellulosic biomass. The accessible surface area may be determined by the BET (Brunauer, Emmett and Teller) as taught in Guo et al Bioresource Technology 99 (2008) 6046-6053, which reference is incorporated herein by reference.

The available surface area of the lignocellulosic biomass may be measured using Brunauer, Emmett and Teller (BET) analysis wherein said method comprises (i) weighing 0.5g of dry sample;(ii) de-gassing with high purity nitrogen (99.999%) at room temperature (e.g. 25°C) overnight; and (iii) performing BET analysis using a surface area analyser (Quantachrome NOVA2000). In one embodiment the lignocellulosic biomass may undergo (or undergoes) a physical and/or chemical and/or biological treatment. This may be referred to herein as a physical and/or chemical and/or biological pre-treatment. In one embodiment the physical and/or chemical pre-treatment in accordance with the present invention may be a physical treatment or a chemical treatment or a physico-chemical treatment.

The term physical and chemical treatment is used herein interchangeably with physico- chemical treatment.

In one embodiment the lignocellulosic biomass may undergo (or undergoes) a physical and/or chemical pre-treatment. In one embodiment the lignocellulosic biomass may undergo (or undergoes) a physical pre- treatment.

In one embodiment the lignocellulosic biomass may undergo (or undergoes) a chemical pre- treatment.

In one embodiment the lignocellulosic biomass may undergo (or undergoes) a physicochemical pre-treatment.

The physical and/or chemical treatment and/or biological may be any physical and/or chemical and/or biological treatment (e.g. pre-treatment) known in the art.

Mechanical/physical pre-treatment

Mechanical/physical pre-treatment refers to the process of mechanical comminution by a combination of chipping, grinding, dry milling, wet milling, vibratory ball milling, rotary ball milling. The size of the feedstock is usually 10-30 mm after chipping and 0.2-2mm after milling or grinding, and the cellulose crystallinity is decreased and/or the accessible surface area is increased. Pre-pre-treatment of the biomass solid with hot water, acids or sodium bisulfate to soften feedstock can make milling less energy-intensive. In one embodiment of the invention, the biomass is pre-treated with a process for mechanical decrystallisation. The term physical pre-treatment is used herein interchangeably with mechanical pre- treatment.

Physico-chemical pre-treatments

Physico-chemical pre-treatments combine the chemical modification of the biomass compositions and physical fracture of the cell wall structure. One useful physico-chemical pre-treatment is steam explosion. In this pre-treatment biomass is treated with high-pressure saturated steam (high pressure and high temperature), optionally including addition of acids, bases or other chemicals, and then the pressure is swiftly reduced, making the materials undergo an explosive decompression.

In one embodiment the physical and/or chemical treatment (e.g. pre-treatment) may be hydrothermolysis or wet oxidation and comprises: high pressure and/or high temperature with liquid water and/or steam, optionally including addition of acids, bases or other chemicals.

In one embodiment, the pressure is in the range from 300 to 600, preferably 350 to 550, preferably 400 to 500 psi. 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.

By way of example only the pre-treatment may be a physical treatment comprising steam explosion.

In terms of cellulose crystallinity, after steam explosion the crystallinity index (Crl) of the substrate is increased. The crystallinity index (Crl) is taught in Segal (supra).

A further example of a physico-chemical pre-treatment is ammonia fiber explosion (AFEX) where the lignocellulosic material is permeated with liquid ammonia followed by increasing temperature to about 90°C. Without wishing to be bound by theory, the formed gas ammonia interacts with biomass under pressure (e.g. 17- 20 bar for 5-10 minutes) and the pressure is then rapidly released, which may result in cellulose de-crystallization, hemicellulose pre- hydrolysis and alteration of lignin structure and/or an increase in accessible surface area. The crystallinity index (Crl) might be influenced by the conditions of AFEX pre-treatment, since slight differences in the pre-treatment conditions can result in the formation of different cellulose crystal structures. Another useful physico-chemical pre-treatment process is liquid hot water pre-treatment which uses water as media to pre-treat biomass under pressure to maintain the water in the liquid state at elevated temperatures. The pre-treatment is also usually termed as hydrothermolysis or hydrothermal pre-treatment. It may solubilise approximately 40-60% of the total biomass with 4-22% of the cellulose and nearly all of the hemicelluloses to form liquid soluble oligosaccharides. As a result, an increase in accessible surface area may be seen. In addition, or alternatively, a decrease in cellulose crystallinity, a lower association of cellulose with lignin and depolymerization of cellulose may be seen which contributes to the enhancement of cellulose accessibility. In one embodiment the pre-treatment may be a physico-chemical pre-treatment comprising hydrothermolysis or a hydrothermal treatment.

In one embodiment the lignocelluosic biomass is wheat straw and the pre-treatment comprises the use of a hydrothermal treatment. The pre-treatment in accordance with the present invention may suitably include hydrothermal treatment as taught in WO2011/125056 (IBICON) the teaching of which is incorporated herein by reference.

Radiation pre-treatments involve processes to pre-treat lignocellulosic biomass with γ- irradiation, ultrasound, electron beam, or microwave. Radiation pre-treatments may also be coupled with other pre-treatments to further increase cellulose accessibility. Without wishing to be bound by theory, at a high irradiation dose the substrates become fragile owing to radiation degradation of cellulose, hemicellulose, and lignin. Cellulose crystallinity may be destroyed to a certain extent by γ-radiation. Microwave pre-treatment is usually conducted in the presence of water, an organic solvent, alkali, or a dilute acid solution.

Chemical pre-treatments

Chemical pre-treatments involve the processes using various chemicals to pre-treat biomass under various conditions. The mechanisms of these pre-treatments vary depending on the chemicals used and pre-treatment conditions. Examples of suitable chemical pre-treatment processes include but are in no way limited to: dilute acid pretreatment, alkali pre-treatment, sulfite pre-treatment, oxidative pre-treatment such as e.g. wet oxidation, cellulose solvent pre-treatment, ammonia percolation (APR), and organosolv pre-treatment.

In one embodiment the pre-treatment may be a chemical treatment comprising the use of an acid or an alkaline treatment.

By way of example the physical and/or chemical treatment may be base catalyst addition, or other methods known in the art. In one embodiment the lignocelluosic biomass is corn stover and the physical and/or chemical treatment comprises the use of an acid treatment.

In one embodiment, the pre-treatment is a dilute acid pre-treatment. For instance the lignocellulosic biomass material may be mixed with dilute acid, typically H ₂ S0 ₄ , and water to form a slurry, heated by steam to the desired temperature (usually between 160 and 220°C), and after a residence time flashed to atmospheric pressure.

In a specific embodiment, the pre-treatment may comprise the steps of: a) impregnating the ligno-cellulosic biomass (e.g. wheat straw) with 0.2 % H ₂ S0 ₄ , e.g. by soaking; b) pressing the material to a dry matter between 40-50% and c) steam pre-treating for 10 min at 190°C. Optionally, pressing the resultant slurry to filter off the liquid and optionally washing the solid residue 2-3 times and pressing to a final solids content of about 40-50%.

In a further specific embodiment, the pre-treatment may comprise the steps of a) impregnating the lignocellulosic biomass (e.g. wheat straw) with 1% acetic acid by soaking; b) pressing the material to a dry matter between 40-50% and c) steam pretreating for 10 min at 200°C. Optionally pressing the subsequent slurry filter off the liquid, and optionally washing the solid residue 2-3 times and pressing to a final solids content of about 40-50%. Alkaline pre-treatment may involve treating lignocellulosic biomass with various alkalis or bases such as NaOH, KOH, CaOH ₂ , aqueous ammonia, peroxide, and lime (using CaOH ₂ ) to pre-treat ligno-cellulosic biomass. Without wishing to be bound by theory, it is believed that during alkaline pre-treatment the intermolecular ester bonds cross-linking xylan hemicellulose and lignin are saponified, thus resulting in delignification of biomass. It has been described that alkalis are suitable agents to swell cellulose and alter cellulose crystalline polymorphs which indicates an alteration of crystalline hydrogen bond network, thus affecting the digestibility of cellulose.

In one embodiment the physical and/or chemical treatment may be a chemical treatment comprising the use of an alkaline (e.g. ammonium) treatment.

In one embodiment, the pre-treatment is anhydrous ammonia pre-treatment.

In a specific embodiment the pre-treatment may be an anhydrous ammonia pre-treatment comprising the steps of 1 ) placing lignocellulosic biomass material (e.g. corn stovers) in a vessel which is evacuated under pressure to allow better penetration of anhydrous ammonia; b) contacting of the biomass material with an aqueous solution comprising 12% ammonia is carried out at 140°C; c) pre-treating with ammonia can last up to 25 h but optimum release of glucose and xylose may be seen after a shorter time period (e.g. 15 minutes); d) removing additional ammonia solution by applying a vacuum to reach a final solids content of 50-60% dry matter.

The physico-chemical treatment may alternatively be the pre-treatment method using ammonia (e.g. dilute ammonia) described in WO 2006/1 10891 , WO 2006/1 1899, WO 2006/11900, and WO2006/1 10901 (DuPont) the teaching of these documents being incorporated herein by reference.

In one embodiment the pre-treated biomass is dilute ammonium pre-treated corn stover (DaCS).

In one embodiment, the pre-treatment is caustic delignifiction.

In one embodiment the pre-treatment may be a caustic delignification pre-treatment comprising the steps of 1 ) treating the biomass with between 0.5-3% 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 between 25- 200°C at a pH in the range of 9-1 1 ; 2) recovering the pretreated biomass filtration and washing with deionised water to remove any excess alkali and dissolved byproducts.

In a specific embodiment the pre-treatment may be a caustic delignification pre-treatment comprising the steps of 1 ) treating the biomass with between 0.5-3% sodium hydroxide (NaOH) for between 0.25 and 1.5 h at about 121 °C at a pH in the range of 9-1 1 ; 2) recovering the pretreated biomass filtration and washing with deionised water to remove any excess alkali and dissolved byproducts. One way of carrying out caustic delignification is taught in Xu et al (201 ) Bioresources 6(1) 707-720 (which reference is incorporated herein by reference). In one embodiment the pretreated biomass may be a biomass treated by the caustic delignification process taught in Xu et al (201 1) supra. In one embodiment the pre-treated biomass is caustic delignified corn stover (DLcs) and/or caustic delignified switchgrass (DLswg).

In one embodiment the pre-treated biomass is caustic delignified corn stover (DLcs) and/or caustic delignified switchgrass (DLswg).

In one embodiment, sulfite pre-treatment or sulfite pulping process comprises sulfite treatment of the biomass material under acidic conditions followed by mechanical size reduction using disk refining. Sulfite pre-treatment results in removal of considerable hemicellulose, a decrease of cellulose DP, and sulfonation of lignin with increased lignin hydrophilicity.

A further chemical pre-treatment may be an oxidative pre-treatment which refers to processes with oxidants used to remove lignin and reductive substance. The oxidants usually employed for oxidative delignification comprise ozone, hydrogen peroxide, oxygen, and peracetic acids. This treatment may optionally be combined with other chemical or hydrothermal treatments. The mechanisms of the oxidative degradation of lignin vary depending on the used oxidants and reaction condition such as pH. For ozonolysis, wet- oxidation and peracids delignification, degradation of aromatic and olefinic structures involves initial electrophilic attack by oxidants, while during alkaline-H ₂ 0 ₂ pre-treatment these structures are destroyed by nucleophilic attack of hydroperoxide anions.

In one embodiment, the physical and/or chemical treatment is a wet oxidation.

In a useful embodiment, the wet oxidation is one which involves high pressure and/or high temperature with liquid water and/or steam, optionally including addition of acids, bases or other chemicals. High pressure may mean pressure in the range from 300 to 600, preferably 400 to 500, such as around 450 psi. High temperature may mean temperatures in the range 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 , the teaching of which is incorporated herein by reference). In one embodiment the high temperature may be from about 140 to 235 °C. Suitably, wet explosion is a modification of the wet oxidation pre-treatment method where the wet oxidation and steam explosion, as described above, are combined. In wet explosion, the oxidizing agent is introduced during pre-treatment after a certain residence time. The treatment is then ended by flashing to atmospheric pressure (WO 2006/032282 which is incorporated herein by reference).

By way of example only, in one embodiment the physical and/or chemical pretreatment may involve high pressure and/or high temperature with liquid water, where water exists as a mixture of liquid and vapor, optionally including addition of acids, bases or other chemicals. High pressure in this embodiment may be in the range from 50 to 300, suitably 100 to 200, for example about 150 psi. High temperature in this embodiment may be in the range from about 100 to 300 °C, suitably from about 170 to 220 °C, such as from about 170 to 200 °C.

A further useful chemical pre-treatment is organosolv pre-treatment which delignifies cellulosic biomass material by extraction using aqueous ethanol (e.g. 40-60% ethanol) at 160-200 °C for 30-60 minutes. Sulphuric acid may be added as a catalyst. In organosolv pre- treatment, the majority of hemicellulose may be removed.

Cellulose-solvent-based pre-treatment is also a chemical pre-treatment for treatment of lignocellulosic biomass based on cellulose solvents such as phosphoric acid (CPA) and ionic liquids (IL).

Biological pre-treatment

In one embodiment the lignocellulosic biomass may undergo a biological treatment (e.g. biological pre-treatment). Such a biological treatment may be any biological treatment known in the art. In biological pre-treatments microorganisms may be utilized for pre-treatment of biomass to increase the enzymatic digestibility of remaining solids. The employed microorganisms are usually capable of degrading lignin and carbohydrate polymers. For example, some types of fungi can produce lignocellulolytic enzymes, which work synergistically to degrade plant cell wall, and other types can produce hydrogen peroxide.

After biological pre-treatment, the accessible surface area may be increased resulting in the enhancement of cellulose digestibility.

The biological pre-treatment by way of example only may include treatment with a white rot fungus. Several white rot fungi such as Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Phlebia subse alis and Pleurotous ostreatus are known to efficiently metabolise lignin in a variety of lignocellulosic materials (see, for example, Singh D and Chen S. 2008. The white-rot fungus Phanerochaete chrysosporium: conditions for the production of lignin-degrading enzymes. Applied Microbiology Biotechnology 81 ,399-417, the teaching of which is incorporated herein by reference).

The cellulosic material can also be subjected to particle size reduction, pre-soaking, wetting, washing, or conditioning prior to pre-treatment (e.g. prior to physical and/or chemical and/or biological pre-treatment) using methods known in the art.

The method of the present invention may further comprise a step of contacting a feed component with the feed additive composition according to the present invention.

The present invention also relates to methods and uses for improving a biophysical characteristic of an animal.

Preferably an animal is fed a feed additive composition obtainable (e.g. 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 a feedstuff according to the present invention or a feedstuff obtainable (e.g. obtained) by a method of the present invention. The biophysical characteristic may be selected from the group consisting of one or more of the following: performance of an animal, growth performance of an animal, feed conversion ratio (FCR), ability to digest a raw material (e.g. nutrient digestibility, including starch , fat, protein, fibre digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass, feed efficiency, body fat percentage, body fat distribution, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, e.g. manure output and/or nitrogen excretion.

In the present invention, the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass is incubated with the enzyme composition.

Suitably the length of such incubation is 6-120 hours, preferably 10-60 hours, preferably 20- 50 hours, preferably 35-50 hours, preferably 40-48 hours.

In some embodiments the incubation may be from 4h to 72h. In one embodiment the incubation period is 6h to 48h. In one in embodiment the incubation period is at least about 6h. In another embodiment the incubation period is less than 72h. 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 is increased, the incubation time must be increased proportionally to achieve the same effect.

Suitably the incubation is such that about 5-20% (preferably about 10-13%) (w/w) of the pentoses (xylose and arabinose) and about 1-10% (preferably about 1-4%) (w/w) of the hexoses in the biomass are in oligo- and polymer form at the end of the incubation period. Suitably the incubation is such that about 10-29% (preferably about 16-21%) (w/w)) of glucose and about 1-10% (preferably about 4-7%) of xylose are in monomeric units at the end of the incubation period.

The length of the incubation may be dependent on the enzymes used and/or the concentration of enzyme used. The aim of the incubation period is to allow for the enzymes to degrade the biomass sufficiently. By way of example, sufficient degradation of biomass can be determined by one skilled in the art, for example by weight analysis to measure the reduction of insoluble biomass after enzyme treatment.

In one embodiment product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention are dried. The term "drying" means that the water content (wt %) of the product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention is reduced to at less than 15%, preferably less than 10%. Therefore the present invention yet further provides a dried product or a substantially-dried product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention the water content (wt %) of which is less than 30%, preferably less than 15%, preferably less than 10%, preferably less than 5%.

In some embodiments the present invention may provide a semi-liquid product or slurry product. A semi-liquid product or slurry product in accordance with the present invention is a product the water content (wt%) of which is at less than 90%, preferably less than 80%, preferably less than 70% or more preferably less than 60%.

In one embodiment the product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention, are packaged and/or stored in a dry state or substantially dry state. The terms "dried" or "dry state" as used herein means that the product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention contain no or only a very low amount of water. In other words the term "dried" or "dry state" as used herein may mean that the product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention comprises less than 5%, preferably less than 1%, water content (wt %).

The term "substantially dry state" as used herein means that the product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention contains only a very low amount of water. In other words the term "substantially dry state" as used herein may mean that the and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention comprises less than 30%, preferably less than 15%, preferably less than 10%, water content (wt %).

In one embodiment the product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention comprises less than 20 wt % moisture content.

In another embodiment the product on and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention comprises less than 15 wt % moisture content. In another embodiment the product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention comprises less than 10 wt % moisture content, In another embodiment the product and/or feed additive composition and/or premix and/or feedstuff in accordance with the present invention comprises less than 5 wt % moisture content.

Preferably the product and/or feed additive composition in accordance with the present invention comprises at least 70%, preferably at least 60%, of the xylose units present in the material as oligomers or polymers.

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, glucoronate, galacturonate (such as xylooligomers, arabinoxylan oligomers) in the product and/or feed additive composition in accordance with the present invention.

In a preferred embodiment the enzyme composition does not release or does not generate or release significant amounts of C-5 sugars (e.g. xylose) in monomer form.

Preferably less than 50% of the total C-5 sugars (e.g. xylose) in the feed additive composition and/or feed ingredient will be present in monomer form

The inventors have found that C-5 sugars (e.g. xylose and/or arabinose and/or arabinofuranose) can be maintained at a low level in the feed additive composition in accordance with the present invention by using an enzyme composition which is absent or substantially absent in β-xylosidase activity and/or a-arabinofuranosidase activity.

The method according to the present invention may further comprise feeding the feed additive composition to an animal.

In one embodiment it is envisaged that the method of the present invention further comprise admixing a feed component with the feed additive composition, e.g. thus to provide a feed or feedstuff.

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 a feed additive composition produced by a method of the present invention or a feedstuff comprising such a feed additive composition.

As used herein the term "biophysical characteristics" as used herein means one or more of the group selected from the following: performance of an animal, growth performance of an animal, feed conversion ratio (FCR), ability to digest a raw material (e.g. nutrient digestibility, including starch , fat, protein, fibre digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass, feed efficiency, body fat percentage, body fat distribution, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, e.g. manure output and/or nitrogen excretion.

The term "improving" as used herein means improved compared with feeding animal the lignocellulosic biomass which has not been treated in accordance with the present invention. The enzyme(s) may be dosed in a suitable concentration in the biomass in order to ensure efficient degradation of the lignocellulosic biomass. In some embodiments the enzyme may be in the range of approximately 2.5 g - 20 kg of active protein per tonne of pre-treated biomass, suitably approximately 5 g - 14 kg of active protein per tonne of pre-treated biomass.

By way of example only, assuming 5-60% inclusion 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.

In one embodiment the amount of enzyme in the feed may be between about 0.125 g to about 12 kg (suitably about 500 g to about 10 kg, suitably about 1 kg to about 8 kg) per MT of feed.

It is envisaged that the amount of treated biomass in the feedstuff can be varied. In one embodiment the amount of treated biomass in the feed is less than 90% w/w of the total feedstuff, preferably less than 80%, suitably less than 70%, suitably less than 60%, suitably less than 50%, suitably less than 40%. Preferably the amount of treated biomass in the feedstuff is less than 60%.

In one embodiment the amount of treated biomass in the feed is more than 30% w/w of the total feedstuff, preferably more than 5%, preferably more than 10%, preferably more than 20%, preferably more than 30%, preferably more than 40%, suitably more than 50%, suitably more than 60%. Preferably the amount of treated biomass in the feedstuff is more than 5% or 10%.

In a further embodiment the amount of treated biomass in the feed is in the range of about 5- 70%, suitably in the range of about 5-60%, suitably 5-50%, suitably 10-40% w/w of the total feedstuff.

In another embodiment the amount of treated biomass in the feed is in the range of 10-70%. In another embodiment the amount of treated biomass in the feed is in the range of 10-40%.

In one embodiment "admixing" as used herein includes any method for admixing, such as mixing, combining, spraying etc.).

In one embodiment the enzyme composition may be in a dry enzyme formulation (e.g. in the form of granules or on a carrier (such as a wheat carrier)) prior to admixing with the lignocellulosic biomass (such as the physically and/or chemically and/or biologically pre- treated lignocellulosic biomass). In another embodiment the enzyme composition may be in a liquid formulation prior to admixing with the lignocellulosic biomass (such as the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass).

When the enzyme is in a liquid formulation prior to admixing with the lignocellulosic biomass (such as the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass), the enzyme may be admixed by spraying the enzyme formulation or dipping the lignocellulosic biomass (such as the physically and/or chemically and/or biologically pre- treated lignocellulosic biomass) into the enzyme formulation for example.

The physically and/or chemically and/or biologically pre-treated biomass (before or after enzyme treatment) and/or the dried solid fraction may be milled and/or powdered and/or formed into a meal.

ANIMAL The term "animal", as used herein, means an animal that is to be or has been administered with a feed additive composition according to the present invention or a feedstuff comprising said feed additive composition according to the present invention. Preferably, the animal is a mammal, bird, fish or crustacean including for example livestock or a domesticated animal (e.g. a pet).

In one embodiment the "animal" is livestock. The term "livestock", as used herein refers to any farmed animal. Preferably, livestock is one or more of cows or bulls (including calves), pigs (including piglets, swine), poultry (including broilers, layers, chickens and turkeys), birds, fish (including freshwater fish, such as salmon, cod, trout and carp, e.g. koi carp, and marine fish, such as sea bass), crustaceans (such as shrimps, mussels and scallops), horses (including race horses), sheep (including lambs).

In another embodiment the "animal" is a domesticated animal or pet or an animal maintained in a zoological environment.

The term "domesticated animal or pet or animal maintained in a zoological environment" as used herein refers to any relevant animal including canines (e.g. dogs), felines (e.g. cats), rodents (e.g. guinea pigs, rats, mice), birds, fish (including freshwater fish and marine fish), and horses.

In one embodiment the animal is a monogastric animal. In a preferred embodiment the monogastric animal may be poultry or pig (or a combination thereof).

In another embodiment the animal is a ruminant animal.

PACKAGING

In one embodiment the enzyme composition and/or feed additive composition and/or feed ingredient and/or premix and/or feed or feedstuff according to the present invention is packaged. In one preferred embodiment the enzyme composition and/or feed additive composition and/or feed ingredient and/or premix and/or feed or feedstuff is packaged in a bag, such as a paper bag. In an alternative embodiment the enzyme composition and/or feed additive composition and/or feed ingredient and/or premix and/or feed or feedstuff may be sealed in a container. Any suitable container may be used.

FEED

The feed additive composition of the present invention may be used as - or in the preparation of - a feed.

The term "feed" is used synonymously herein with "feedstuff'.

The feed may 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.

When used as - or in the preparation of - a feed - such as functional feed - the composition of the present invention may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient.

In a preferred embodiment the feed additive composition of the present invention is admixed with a feed component to form a feedstuff.

The term "feed component" as used herein means all or part of the feedstuff. Part of the feedstuff may mean one constituent of the feedstuff or more than one constituent of the feedstuff, e.g. 2 or 3 or 4. In one embodiment the term "feed component" encompasses a premix or premix constituents.

Preferably the feed may be a fodder, or a premix thereof, a compound feed, or a premix thereof. In one embodiment the feed additive composition according to the present invention may be admixed with a compound feed, a compound feed component or to a premix of a compound feed or to a fodder, a fodder component, or a premix of a fodder. The term fodder as used herein means any food which is provided to an animal (rather than the animal having to forage for it themselves). Fodder encompasses plants that have been cut. The term fodder includes hay, straw, silage, compressed and pelleted feeds, oils and mixed rations, and also sprouted grains and legumes.

Fodder may be obtained from one or more of the plants selected from: alfalfa (Lucerne), barley, birdsfoot trefoil, brassicas, Chau moellier, kale, rapeseed (canola), rutabaga (swede), turnip, clover, alsike clover, red clover, subterranean clover, white clover, grass, false oat grass, fescue, Bermuda grass, brome, heath grass, meadow grasses (from naturally mixed grassland swards, orchard grass, rye grass, Timothy-grass, corn (maize), millet, oats, sorghum, soybeans, trees (pollard tree shoots for tree-hay), wheat, and legumes. The term "compound feed" means a commercial feed in the form of a meal, a pellet, nuts, cake or a crumble. Compound feeds may be blended from various raw materials and additives. These blends are formulated according to the specific requirements of the target animal. Compound feeds can be complete feeds that provide all the daily required nutrients, concentrates that provide a part of the ration (protein, energy) or supplements that only provide additional micronutrients, such as minerals and vitamins.

The main ingredients used in compound feed are the feed grains, which include corn, soybeans, sorghum, oats, and barley.

Suitably a premix as referred to herein may be a composition composed of microingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients. Premixes are usually compositions suitable for blending into commercial rations.

Any feedstuff of the present invention may comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from plants (e.g. cereals), such as wet-cake, distillers dried grain (DDG), and distillers dried grain solubles (DDGS), corn fibre, corn germ meal, corn bran, Hominy feed, corn gluten feed, wheat shorts, wheat middlings or combinations thereof (preferably by products of methods according to the present invention); c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried 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.

A feedstuff of the present invention may contain at least 30%, at least 40%, at least 50% or at least 60% by weight corn and soybean meal or corn and full fat soy, or wheat meal or sunflower meal.

In addition or in the alternative, a feedstuff of the present invention may comprise at least one high fibre feed material and/or at least one by-product of the at least one high fibre feed material to provide a high fibre feedstuff. Examples of high fibre feed materials include: wheat, barley, rye, oats, by products from plants (e.g. cereals), such as wet-cake, distillers dried grain (DDG), and distillers dried grain solubles (DDGS), corn fibre, corn germ meal, corn bran, Hominy feed, corn gluten feed, wheat shorts, wheat middlings or combinations thereof. Some protein sources may also be regarded as high fibre: protein obtained from sources such as sunflower, lupin, fava beans and cotton. In the present invention the feed may be one or more of the following: a compound feed and premix, including pellets, nuts or (cattle) cake; a crop or crop residue: corn, soybeans, sorghum, oats, barley, corn stover, copra, straw, chaff, sugar beet waste; fish meal; freshly cut grass and other forage plants; meat and bone meal; molasses; oil cake and press cake; oligosaccharides; conserved forage plants: hay and silage; seaweed; seeds and grains, either whole or prepared by crushing, milling etc.; sprouted grains and legumes; yeast extract.

The term "feed" in the present invention also encompasses in some embodiments pet food. A pet food is plant or animal material intended for consumption by pets, such as dog food or cat food. Pet food, such as dog and cat food, may be either in a dry form, such as kibble for dogs, or wet canned form. Cat food may contain the amino acid taurine.

The term "feed" in the present invention also encompasses in some embodiments fish food. A fish food normally contains macro nutrients, trace elements and vitamins necessary to keep captive fish in good health. Fish food may be in the form of a flake, pellet or tablet. Pelleted forms, some of which sink rapidly, are often used for larger fish or bottom feeding species. Some fish foods also contain additives, such as beta carotene or sex hormones, to artificially enhance the colour of ornamental fish.

The term "feed" in the present invention also encompasses in some embodiment bird food. Bird food includes food that is used both in birdfeeders and to feed pet birds. Typically bird food comprises of a variety of seeds, but may also encompass suet (beef or mutton fat).

As used herein the term "contacting" refers to the indirect or direct application of the composition of the present invention to the product (e.g. the feed). Examples of the application methods which may be used, include, but are not limited to, treating the product in a material comprising the feed additive composition, direct application by mixing the feed additive composition with the product, spraying the feed additive composition onto the product surface or dipping the product into a preparation of the feed additive composition. In one embodiment the feed additive composition of the present invention is preferably admixed with the product (e.g. feedstuff). Alternatively, the feed additive composition may be included in the emulsion or raw ingredients of a feedstuff.

For some applications, it is important that the composition is made available on or to the surface of a product to be affected/treated. This allows the composition to impart one or more of the following favourable characteristics: biophysical characteristics, e.g. wherein the biophysical characteristic is selected from the group consisting of one or more of the following: performance of an animal, growth performance of an animal, feed conversion ratio (FCR), ability to digest a raw material (e.g. nutrient digestibility, including starch , fat, protein, fibre digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass, feed efficiency, body fat percentage, body fat distribution, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, e.g. manure output and/or nitrogen excretion. The feed additive compositions of the present invention may be applied to intersperse, coat and/or impregnate a product (e.g. feedstuff or raw ingredients of a feedstuff) with a controlled amount of enzyme(s).

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 may be performed for up to about 1 minute; up to about 5 minutes; up to about 10 minutes; up to about 30 minutes; 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 before heating to the specified temperature are still present/active after it cools to room temperature. Preferably, at least about 80% of the enzyme components that were present and active in the additive before heating to the specified temperature are still present and active after it cools to room temperature.

In a particularly preferred embodiment the enzyme composition and/or feed additive composition is homogenized to produce a powder.

In an alternative preferred embodiment, the enzyme composition and/or feed additive composition is formulated to granules as described in WO2007/044968 (referred to as TPT granules) incorporated herein by reference. In another preferred embodiment when the enzyme composition and/or feed additive composition is formulated into granules the granules comprise a hydrated barrier salt coated over the protein core. The advantage of such salt coating is improved thermo-tolerance, improved storage stability and protection against other feed additives otherwise having adverse effect on the enzyme.

Preferably, the salt used for the salt coating has a water activity greater than 0.25 or constant humidity greater than 60% at 20°C.

Preferably, the salt coating comprises a Na ₂ S0 ₄ .

The method of preparing a feed additive composition may also comprise the further step of pelleting the powder. The powder may be mixed with other components known in the art. The powder, or mixture comprising the powder, may be forced through a die and the resulting strands are cut into suitable pellets of variable length.

Optionally, the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets. The mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection. The mixture is heated in the conditioner up to a specified temperature, such as from 60-100°C, typical temperatures would be 70°C, 80°C, 85°C, 90°C or 95°C. The residence time can be variable 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.

It will be understood that the feed additive composition of the present invention is suitable for addition to any appropriate feed material.

As used herein, the term feed material refers to the basic feed material to be consumed by an animal. It will be further understood that this may comprise, for example, at least one or more unprocessed grains, and/or processed plant and/or animal material such as soybean meal or bone meal.

As used herein, the term "feedstuff' refers to a feed material to which one or more feed additive compositions have been added. It will be understood by the skilled person that different animals require different feedstuffs, and even the same animal may require different feedstuffs, depending upon the purpose for which the animal is reared.

Preferably, the feedstuff may comprise feed materials comprising maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/ or any of the by-products, as well as protein rich components like soybean mean, rape seed meal, canola meal, cotton seed meal, sunflower seed mean, animal-by-product meals and mixtures thereof. More preferably, the feedstuff may comprise animal fats and / or vegetable oils. Optionally, the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins.

Preferably, the feedstuff is a corn soybean meal mix. In another aspect there is provided a method for producing a feedstuff. Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and enzyme may be added. Production of feedstuff may also involve an additional step that includes extrusion or expansion prior to pelleting - in particular by suitable techniques that may include at least the use of steam.

The feedstuff may be a feedstuff for a monogastric animal, such as poultry (for example, broiler, layer, broiler breeders, turkey, duck, geese, water fowl), swine (all age categories), a pet (for example dogs, cats) or fish, preferably the feedstuff is for poultry.

By way of example only a feedstuff for chickens, e.g. broiler chickens may be comprises of one or more of the ingredients listed in the table below, for example in the %ages given in the table below:

By way of example only the diet specification for chickens, such as broiler chickens, may be as set out in the Table below:

Diet specification

Crude Protein (%) 23.00 20.40

Metabolizable Energy Poultry

2950 3100

(kcal/kg)

Calcium (%) 0.85 0.85

Available Phosphorus (%) 0.38 0.38

Sodium (%) 0.18 0.19

Dig. Lysine (%) 1.21 1.07 Dig. Methionine (%) 0.62 0.57

Dig. Methionine + Cysteine (%) 0.86 0.78

Dig. Threonine (%) 0.76 0.68

By way of example only a feedstuff laying hens may be comprises of one or more of the ingredients listed in the table below, for example in the %ages given in the table below:

By way of example only the diet specification for laying hens may be as set out in the Table below:

Diet specification

Crude Protein (%) 16.10

Metabolizable Energy Poultry

2700

(kcal/kg)

Lysine (%) 0.85

Methionine (%) 0.42

Methionine + Cysteine (%) 0.71

Threonine (%) 0.60

Calcium (%) 3.85

Available Phosphorus (%) 0.42

Sodium (%) 0.16 By way of example only a feedstuff for turkeys may be comprises of one or more of the ingredients listed in the table below, for example in the %ages given in the table below:

By way of example only the diet specification for turkeys may be as set out in the Table below:

By way of example only a feedstuff for piglets may be comprises of one or more of the ingredients listed in the table below, for example in the %ages given in the table below: Ingredient Phase 1 (%) Phase 2 (%)

Maize 20.0 7.0

Wheat 25.9 46.6

Rye 4.0 10.0

Wheat middlings 4.0 4.0

Maize DDGS 6.0 8.0

Soyabean Meal 48% CP 25.7 19.9

Dried Whey 10.0 0.0

Soyabean Oil 1.0 0.7

L-Lysine HCI 0.4 0.5

DL-methionine 0.2 0.2

L-threonine 0.1 0.2

L-tryptophan 0.03 0.04

Limestone 0.6 0.7

Dicalcium Phosphate 1.6 1.6

Swine Vitamins and Micro-

0.2 0.2

minerals

Salt 0.2 0.4

By way of example only the diet specification for piglets may be as set out in the Table below:

By way of example only a feedstuff for grower/finisher pigs may be comprises of one or more of the ingredients listed in the table below, for example in the %ages given in the table below: Ingredient Grower/ Finisher (%)

Maize 27.5

Soyabean Meal 48% CP 15.4

Maize DDGS 20.0

Wheat bran 1 1.1

Rice bran 12.0

Canola seed meal 10.0

Limestone 1.6

Dicalcium phosphate 0.01

Salt 0.4

Swine Vitamins and Micro-minerals 0.3

Lysine-HCI 0.2

Vegetable oil 0.5

By way of example only the diet specification for grower/finisher pigs may be as set out in the Table below:

FORMS

The enzyme composition of the present invention and/or feed additive composition of the present invention and other components and/or the feedstuff comprising same may be used in any suitable form.

The enzyme composition of the present invention and/or feed additive composition of the present invention may be used in the form of solid or liquid preparations or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, pellets, tablets, dusts, and granules which may 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.

In some applications, feed additive composition of the present invention may be mixed with feed or administered in the drinking water.

Suitable examples of forms include one or more of: powders, pastes, boluses, pellets, tablets, pills, capsules, ovules, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled- release applications.

By way of example, if the composition of the present invention is used in a solid, e.g. pelleted form, 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 glycollate, 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.

Examples of nutritionally acceptable carriers for use in preparing the forms include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

Preferred excipients for the forms include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions and/or elixirs, the composition of the present invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, propylene glycol and glycerin, and combinations thereof.

COMBINATION WITH OTHER COMPONENTS The feed additive composition, or feed ingredient, or feed or feedstuff or premix of the present invention may be used in combination with other components. The combination of the present invention feed additive composition, or feed ingredient, or feed or feedstuff or premix of the present invention and another component which is suitable for animal consumption and is capable of providing a medical or physiological benefit to the consumer. In one embodiment the "another component" may be one or more enzymes.

Suitable additional enzymes 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); celliobiohydrolases (E.C. 3.2.1.91), β-glucosidases (E.C. 3.2.1.21), cellulases (E.C. 3.2.1.74), lichenases (E.C. 3.1.1.73), lipases (E.C. 3.1.1.3), lipid acyltransferases (generally classified as E.C. 2.3.1.x), phospholipases (E.C. 3.1.1.4, E.C. 3.1.1.32 or E.C. 3.1.1.5), phytases (e.g. 6-phytase (E.C. 3.1.3.26) or a 3-phytase (E.C. 3.1.3.8), alpha-amylases (E.C. 3.2.1.1), xylanases (E.C. 3.2.1.8, E.C. 3.2.1.32, E.C. 3.2.1.37, E.C. 3.1.1.72, E.C. 3.1.1.73), glucoamylases (E.C. 3.2.1.3), proteases (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)) and/or mannanases (e.g. a β-mannanase (E.C. 3.2.1.78)).

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 (E.C. 3.2.1.8, E.C. 3.2.1.32, E.C. 3.2.1.37, E.C. 3.1.1.72, E.C. 3.1.1.73), an amylase (including a-amylases (E.C. 3.2.1.1), G4-forming amylases (E.C. 3.2.1.60), β-amylases (E.C. 3.2.1.2) and γ- amylases (E.C. 3.2.1.3); and/or a protease (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)). In one embodiment (particularly for feed applications) the other component may be a combination of an amylase (e.g. a-amylases (E.C. 3.2.1.1)) and a protease (e.g. subtilisin (E.C. 3.4.21.62)).

In one embodiment (particularly for feed applications) the other component may be a β- glucanase, e.g. an endo-1 ,3(4)^-glucanases (E.C. 3.2.1.6). In one embodiment (particularly for feed applications) the other component may be a mannanases (e.g. a β-mannanase (E.C. 3.2.1.78)).

In one embodiment (particularly for feed applications) the other component may be a lipase (E.C. 3.1.1.3), a lipid acyltransferase (generally classified as E.C. 2.3.1.x), or a phospholipase (E.C. 3.1.1.4, E.C. 3.1.1.32 or E.C. 3.1.1.5), suitably a lipase (E.C. 3.1.1.3).

In one embodiment (particularly for feed applications) the other component may be a protease (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)).

In one embodiment the additional component may be a stabiliser or an emulsifier or a binder or carrier or an excipient or a diluent or a disintegrant. The term "stabiliser" as used here is defined as an ingredient or combination of ingredients that keeps a product (e.g. a feed product) from changing over time.

The term "emulsifier" as used herein refers to an ingredient (e.g. a feed ingredient) that prevents the separation of emulsions. Emulsions are two immiscible substances, one present in droplet form, contained within the other. Emulsions can consist of oil-in-water, where the droplet or dispersed phase is oil and the continuous phase is water; or water-in-oil, where the water becomes the dispersed phase and the continuous phase is oil. Foams, which are gas-in-liquid, and suspensions, which are solid-in-liquid, can also be stabilised through the use of emulsifiers.

As used herein the term "binder" refers to an ingredient (e.g. a feed ingredient) that binds the product together through a physical or chemical reaction. During "gelation" for instance, water is absorbed, providing a binding effect. However, binders can absorb other liquids, such as oils, holding them within the product. In the context of the present invention binders would typically be used in solid or low-moisture products for instance baking products: pastries, doughnuts, bread and others. Examples of granulation binders include one or more of: polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia. "Carriers" mean materials suitable for administration of the enzyme and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner.

The present invention provides a method for preparing a composition (e.g. a feed additive composition) comprising admixing 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 wheat component, sucrose, starch, Na ₂ S0 ₄ , Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose, propylene glycol, 1 ,3-propane diol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate and mixtures thereof.

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.

Examples of "disintegrants" include one or more of: starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates.

Examples of "diluents" include one or more of: water, ethanol, propylene glycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are in admixture together or even when they are delivered by different routes) or sequentially (e.g. they may be delivered by different routes) to the feed additive of the present invention.

In one embodiment preferably the feed additive composition, or feed ingredient, or feed or feedstuff or premix according to the present invention does not comprise chromium or organic chromium. In one embodiment preferably the feed additive composition, or feed ingredient, or feed or feedstuff or premix according to the present invention does not contain sorbic acid.

BIOPHYSICAL CHARACTERISTIC

As used herein, "biophysical characteristic" means any biophysical property of an animal which improves its health and/or performance and/or output. By way of example, the biophysical characteristic of interest may be one or more of the group selected from: performance of the animal, growth performance of an animal, feed conversion ratio (FCR), ability to digest a raw material (e.g. nutrient digestibility, including starch , fat, protein, fibre digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass, feed efficiency, body fat percentage, body fat distribution, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, e.g. manure output and/or nitrogen excretion.

In a preferred embodiment the biophysical characteristic arising from a method or use in accordance with the present invention may be selected from the group consisting of one or more of the following: performance of an animal, growth performance of an animal, feed conversion ratio (FCR), ability to digest a raw material (e.g. nutrient digestibility, including starch , fat, protein, fibre digestibility), nitrogen retention, carcass yield, growth rate, weight gain, body weight, mass, feed efficiency, body fat percentage, body fat distribution, growth, egg size, egg weight, egg mass, egg laying rate and environmental impact, e.g. manure output and/or nitrogen excretion.

In one embodiment the biophysical characteristic of the animal means the performance of the animal. PERFORMANCE

As used herein, "performance of the animal" may be determined by the feed efficiency and/or weight gain of the animal and/or by the feed conversion ratio and/or by the digestibility of a nutrient in a feed (e.g. amino acid digestibility) and/or digestible energy or metabolizable energy in a feed and/or by nitrogen retention.

Preferably "performance of the animal" is determined by feed efficiency and/or weight gain of the animal and/or by the feed conversion ratio. By "improved performance of the animal" it is meant that there is increased feed efficiency, and/or increased weight gain and/or reduced feed conversion ratio and/or improved digestibility of nutrients or energy in a feed and/or by improved nitrogen retention in the animal resulting from the use of feed additive composition of the present invention compared with feeding the animal the lignocellulosic biomass which has not been treated in accordance with the present invention. Preferably, by "improved animal performance" it is meant that there is increased feed efficiency and/or increased weight gain and/or reduced feed conversion ratio.

As used herein, the term "feed efficiency" refers to the amount of weight gain in an animal that occurs when the animal is fed ad-libitum or a specified amount of food during a period of time.

By "increased feed efficiency" it is meant that the use of a feed additive composition according the present invention in feed results in an increased weight gain per unit of feed intake compared with an animal fed with the lignocellulosic biomass which has not been treated in accordance with the present invention.

FEED CONVERSION RATIO (FCR) As used herein, the term "feed conversion ratio" refers to the amount of feed fed to an animal to increase the weight of the animal by a specified amount.

An improved feed conversion ratio means a lower feed conversion ratio. By "lower feed conversion ratio" or "improved feed conversion ratio" it is meant that the use of a feed additive composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the lignocellulosic biomass which has not been treated in accordance with the present invention is used in or as the feed.

NUTRIENT DIGESTIBILITY

Nutrient digestibility as used herein means the fraction of a nutrient that disappears from the gastro-intestinal tract or a specified segment of the gastro-intestinal tract, e.g. the small intestine. Nutrient digestibility may be measured as the difference between what is administered to the animal and what comes out in the faeces of the animal, or between what is administered to the animal and what remains in the digesta on a specified segment of the gastro intestinal tract, e.g. the ileum. Nutrient digestibility as used herein may be measured by the difference between the intake of a nutrient and the excreted nutrient by means of the total collection of excreta during a period of time; or with the use of an inert marker that is not absorbed by the animal, and allows the researcher calculating the amount of nutrient that disappeared in the entire gastro- intestinal tract or a segment of the gastro-intestinal tract. Such an inert marker may be titanium dioxide, chromic oxide or acid insoluble ash. Digestibility may be expressed as a percentage of the nutrient in the feed, or as mass units of digestible nutrient per mass units of nutrient in the feed. Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.

Energy digestibility as used herein means the gross energy of the feed consumed minus the gross energy of the faeces or the gross energy of the feed consumed minus the gross energy of the remaining digesta on a specified segment of the gastro-intestinal tract of the animal, e.g. the ileum. etabolizable energy as used herein refers to apparent metabolizable energy and means the gross energy of the feed consumed minus the gross energy contained in the faeces, urine, and gaseous products of digestion. Energy digestibility and metabolizable energy may be measured as the difference between the intake of gross energy and the gross energy excreted in the faeces or the digesta present in specified segment of the gastro-intestinal tract using the same methods to measure the digestibility of nutrients, with appropriate corrections for nitrogen excretion to calculate metabolizable energy of feed. NITROGEN RETENTION

Nitrogen retention as used herein means an animal's ability to retain nitrogen from the diet as body mass. A negative nitrogen balance occurs when the excretion of nitrogen exceeds the daily intake and is often seen when the muscle is being lost. A positive nitrogen balance is often associated with muscle growth, particularly in growing animals.

Nitrogen retention may be measured as the difference between the intake of nitrogen and the excreted nitrogen by means of the total collection of excreta and urine during a period of time. It is understood that excreted nitrogen includes undigested protein from the feed, endogenous proteinaceous secretions, microbial protein, and urinary nitrogen. CARCASS YIELD AND MEAT YIELD

The term carcass yield as used herein means the amount of carcass as a proportion of the live body weight, after a commercial or experimental process of slaughter. The term carcass means the body of an animal that has been slaughtered for food, with the head, entrails, part of the limbs, and feathers or skin removed. The term meat yield as used herein means the amount of edible meat as a proportion of the live body weight, or the amount of a specified meat cut as a proportion of the live body weight. WEIGHT GAIN

The present invention further provides a method of increasing weight gain in an animal, e.g. poultry or swine, comprising feeding said animal a feedstuff comprising a feed additive composition according to the present invention.

An "increased weight gain" refers to an animal having increased body weight on being fed feed comprising a feed additive composition compared with an animal being fed a feed comprising or consisting of lignocellulosic biomass which has not been treated in accordance with the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning 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, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.

The term "protein", as used herein, includes proteins, polypeptides, and peptides.

As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in conformity with the lUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" 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 "the feed" includes reference to one or more feeds and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

The invention will now be described, by way of example only, with reference to the following Figures and Examples. EXAMPLES

SUMMARY Due to population growth and improving living standards in BRIC countries, food and feed prices have been soaring. The present invention seeks to provide alternative feed ingredients for animal production.

The present inventors have surprisingly found that the elimination of C5-monomer sugar production and promotion of C5-oligomer production together with cellulase activities (e.g. to produce C6-oligomer and C-6 monomer sugars) is advantageous in the production of plant materials (e.g. lignocellulosic biomass) for use in or as feedstuffs.

MATERIALS & METHODS

The enzymes tested were:

• Cellulase SC is an enzyme composition comprising the following enzyme activities: endoglucanase activity, β-glucosidase activity and endoxylanase activity and which has no or substantially no β-xylosidase activity and a-L-arabinofuranosidase activity. Cellulase SC was kept at -20°C and thawed before use.

• Endoxylanase (endo-1 ,4-p-xylanase) - Danisco Xylanase™ 40000L (synonym:

Xylanase Y5 or Y5) available from Danisco Animal Nutrition, DuPont A/S - this was diluted to 9293U/g.

• Endoxylanase (endo-1 ,4- -xylanase) - Econase XT™ available from ABVista. This was in purified form at 0.51 mg protein/ml. Econase XT™ is a xylanase variant from the actinomycete Nonomuraea flexuosa (Leskinen et al. Appl Microbiol Biotechnol (2005) 67: 495-505).

• Endoxylanase (endo-1 , 4-p-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.

• Endoxylanase (endo-1 ,4-p-xylanase) - (synonym: FveABC5, FoxXyn4, FveXyn4) as taught in PCT/CN2012/079650 (which is incorporated herein by reference) which is encoded by the nucleotide sequence SEQ ID No. 4 or SEQ ID No. 5 and has the polypeptide sequence SEQ ID No. 6. • AcceIlerase ^® Trio™ is a comparative enzyme composition comprising an endoglucanase, β-glucosidase and endoxylanase activity as well as C-5 monomer forming enzyme activities such as β-xylosidase activity and a-L-arabinofuranosidase activity. The recommended dosage for Accellerase ^® Trio™ is 0.05 - 0.3 ml_ per gram cellulose or roughly 0.03 -0.16 ml_ per gram of biomass (depending on biomass composition). Accellerase^rio™ was kept at -20°C before use.

P re-treated biomasses: Dilute ammonium pre-treated corn stover (DaCS) was from DuPont (Lot no. HP90-HP93). It has -65% solids and contains some low level of acetamide. It was produced according to DuPont Patent application (WO 2006/110901 A2 - which is incorporated herein by reference).

Analysis of sugar using Aminex HPX-87P column (300 x 7.8 mm) from Biorad (Hercules, CA USA). Release of sugars and oligomers is an indication of biomass hydrolysis. According to the manufacturer, the following sugars can be separated well: 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: column oven temperature set at 80°C, flow rate set at 0.5ml milliQ water/min. HPLC was Dionex Summit with P580 pump and Shodex Rl detector. Glucose, xylose and arabinose standards (1 % (w/v)) were used. Injection volumes were 10 or 20ul.

Analysis of glucose, xylose, arabinose and oligosaccharides was performed using by CarboPac column from Dionex on a Dionex HPLC. Sugars, oligomers and polymers released were also analyzed on a CarboPac column on a Dionex HPLC.

For free monosaccharides determination: the sample was centrifuged for 5 min at 16000 x g and the supernatant saved. A strong anion exchanger solid phase extraction column (SAX SPE) (available from Sigma Fine Chemicals) (500 mg of sorbent) was conditioned with 2 ml of methanol and 2 ml of water. 0.1 ml of sample solution was applied to the column and the sample was sucked via vacuum into a test tube. The column was washed with 2 ml of water into the same test tube. Each sample was diluted to 10 ml with water (or other suitable volume) depending on the concentration of monosaccharides. For total sugars determination after acid hydrolysis: 0.100 ml of the sample, 0.100 ml of water and 0.200 ml of 2 M H ₂ S0 ₄ was added into a 35 ml test tube. The tube was closed with a stopper and allowed to stand at 100°C for 3 h. The tube was allowed to cool and 29.6 ml of water added. The sample was then centrifuged for 5 min at 13000 x g and filtered through a 0.45 μιη filter paper. It was further diluted with water depending on sugar concentration. The amount of polymeric sugars was calculated as follows:

The process samples were analyzed on Dionex HPLC using CarboPacTM PA1 (4 x 250mm) with CarboPacTM AI (4 x 40mm) as pr-ecolumn, (available from Dionex) Column oven: 30°C. Mobile phase: A: HPLC-grade water, B: 0.2 M NaOH , Injection volume: 25□!. flow rate, 1 ml/min. The gradient program used was: Omin, 10% B, 1.1 min, 10% B, 5.0min, 0% B, 33min, 0% B, 34min, 100% B, 41 min, 100% B, 42min, 10% B, 56min, 10% B. 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. Cellulase SC hydrolysis of dilute ammonium treated corn stover (DaCS).

Dilute ammonium treated corn stover (DaCS): to 50ml Falcon tube, add 15g DaCS, tap water, adjust the pH to 5.0 using 2N HCI. 1.33 ml of Cellulase SC (product lot no. 1 18, (protein concentration 76.6 g/L) referred to herein as Cellulase SC118) was added to start the reaction. The final concentration of biomass DaCS was 18.4% (w/v). Cellulase SC118: Biomass ratio (w/w) was 1.08:100.

The reaction was performed at 50°C with shaking at 200rpm for 70h. Liquid samples were taken at 21.5h, 45.5h, 62.5h and at the end of the reaction 70.5h after short spin of the biomass in the Falcon tube. Sampling size at the time intervals was 60μΙ, which was then mixed 60μΙ water used to rinse the pipette tips used to suck the samples. The samples collected were kept at 5°C before HPLC analysis. Each of the samples was then further mixed with 120ul milliQ water, heated at 100°C for 5min and filtered. The filtrate 10μΙ was injected on HPLC for analysis of released sugars. To each of the 3 Falcon tubes 14ml milliQ water was added to wash the residual biomass obtained after centrifugation. The washed residual biomass was dried at 80°C for 20h and weighed. Their weights were used to compare the weights of the biomass treated in the absence of the enzyme composition.

Results of HPLC analysis of Example 1

From Figure 1 shows the glucose and xylose yield obtained from treating DaCS with Cellulase SC. Figure 1 is based on the data detailed in the table (Table 1) below. As can be seen from the table, xylose released is less than 1/3 of glucose released from DaCS at 50°C pH5.0 for up to 70.5 hours.

TABLE 1

Results of weight analysis of Example 1 From Table 2, under the in vitro conditions, it can be seen that 56% of DaCS has been converted. Improvement can be achieved by further optimizing the mixing.

Table 2:. Biomass hydrolysis of corn stover by Cellulase SC cellulase at 50oC and 70.5h

The results indicate that the most efficient cellulase for biomass hydrolysis is Accellerase ^® Trio™. The products are glucose, xylose, and a minor amount of arabinose as seen from HPLC chromatograms. Cellulase SC cellulase mixture hydrolysed DaCS, but different from comparative enzyme composition Accellerase ^® Trio™ (Figure 2) Cellulase SC was less efficient overall and also produced fewer C5 sugars of xylose and arabinose. The formation of fewer C5 sugars is desirable as monogastric animals are inefficient in using C5 sugars. This result indicates that Cellulase SC has low (or no) β-xylosidase activity and a-L-arabinofuranosidase activity.

Cellulase SC plus an endoxylanase (Danisco Xylanase™) was found to promote the release of glucose and xylooligosaccharides. This was found to be highly beneficial for use in feedstuffs.

EXAMPLE 2. Cellulase SC hydrolysis of dilute ammonium treated corn stover (DaCS) and the effect of adding an endoxylanase. To each of the 9 plastic tubes with a volume of 12ml, 1g of DaCS was added, 5ml MilliQ water and mixed and the tubes were let stand at 5°C overnight to ensure the DaCS was well hydrated. The following day 150ul 1 N HCI was added to adjust the pH to 4.6-5.1. Enzymes (listed in Table 3 below) were added to start the reaction. The reaction was performed at 50°C with a shaking speed of 180 rpm for 40h. Liquid samples (120μΙ) were collected at a reaction time of 6h, 19h, and 40h, diluted with 120μΙ MilliQ water and saved at 5°C waiting for analysis. At the end of the enzyme reaction, 240μΙ milliQ water was further added to each of the collected samples, which were then heated at 100°C water bath for 5min, followed by centrifugation at 10000g for 10min. 250μΙ of supernatant was then filtered and the filtrate 10μΙ were injected for HPLC analysis. Glucose and xylose standards at 1 % (w/v) were filtered and analyzed on HPLC at the same conditions with the same injection volume as the hydrolyzate samples collected.

Biomass weight reduction analysis: For the residual biomass in the 9 tubes obtained after centrifugation, after removal of the supernatant, 4ml water was added and shaken to wash out anything soluble from the precipitate. The suspension was centrifuged again to get the washed biomass residue, which was then dried at 60°C for 48h.

Table 3

Tube no. Dilute ammonium Cellulases added

treated corn stover

(DaCS)

Results of HPLC analysis Example 2 Cellulase SC cellulase (Cellulase SC 1 18 and Cellulase SC 151) produced much lower level of xylose than Accellerase ^® Trio™ (See Figure 2 and Figure 3). The glucose produced by Cellulase SC cellulase preparations was also lower than Accellerase ^® Trio™. This is also true in the whole reaction process from 6, 19 and 40h (Figure 2 below). Cellulase SC together with an endoxylanase (FoxXyn4) promoted the glucose release and also promoted the xylose release in the whole reaction time course. The xylose (e.g. C5 sugar) release by Cellulase SC together with the xylanase was much lower than that released by Accellerase^Trio™ The hydrolyzates generated in Table 2 were further analyzed using CarboPac column on a Dionex HPLC. The results were given in Figure 3. From Figure 3 one can see that the results are similar to Figure 2 with regard to the total amount of monosugars produced. In addition, Figure 3 shows that for the total soluble polymeric sugars, the Cellulase SC or Cellulase SC plus endoxylanase treatment had resulted in a higher amount than that of Acceilerase^rio™ treatment. From Figure 3 one can see that the total soluble sugar in polymeric form released by Cellulase SC with or without the addition of xylanase is 4 times higher than Accellerase^rio™, which had beta-xylosidase and alpha-L-arabinofuranosidase activities. Sugars were analysed using CarboPac column from Dionex.

EXAMPLE 3. A further study of Cellulase SC hydrolysis of dilute ammonium treated corn stover (DaCS) and the effect of adding various xylanases. Take 1g DaCS to each of the tubes listed in Table 3 with a volume of 12ml, add 4.6ml water and 0.15ml 1 N HCI, mix. The pH measured was pH 5.03-5.17. Then enzymes of Accellerase ^® Trio™, Cellulase SC 118 or Cellulase SC 118 plus 10-120ul of the 5 different xylanases were added to start the reaction at 50°C, shaking at 200rpm for 45h. Tubes 22-24 were controls as no enzymes were added. Tube 3k, 6k, 12k, 15k, 18k, and 21 k are blanks.

Table 3. "-" means no addition of the relevant component

Tube DaCS, Additional Enzyme composition Xylanase

no. g MilliQ (cellulases) μΙ

water

added, μΙ

1 1 220 Accellerase ^® Trio ^{I M} , 30 -

2 1 220 Accellerase ^® Trio ^{I M} , 30 -

3 1 220 Accellerase ^® Trio ^{I M} , 30 -

3k - 220 Accellerase ^® Trio ^{I M} , 30 -

4 1 200 Cellulase SC1 18, 50 -

5 1 200 Cellulase SC1 18, 50 -

6 1 200 Cellulase SC1 18, 50 -

6k - 200 Cellulase SC118, 50 -

7 1 240 Cellulase SC1 18, 50 10μΙ Danisco Xylanase™

product, 9293U/g

8 1 240 Cellulase SC1 18, 50 10μΙ Danisco Xylanase™

product, 9293U/g

9 1 240 Cellulase SC118, 50 10μΙ Danisco Xylanase™

product, 9293U/g

9k 240 Cellulase SC1 18, 50 10μΙ Danisco Xylanase™

product, 9293U/g 1 150 Cellulase SC118, 50 100μΙ Danisco Xylanase™ pure, 0.63mg protein/ml

1 150 Cellulase SC118, 50 100μΙ Danisco Xylanase™ pure, 0.63mg protein/ml

1 150 Cellulase SC118, 50 100μΙ Danisco Xylanase™ pure, 0.63mg protein/mlk 150 Cellulase SC118, 50 100μΙ Danisco Xylanase™ pure, 0.63mg protein/ml

1 150 Cellulase SC118, 50 100μΙ FoxXyn4,

0.6mgprotein/ml

1 150 Cellulase SC118, 50 100μΙ FoxXyn4,

0.6mgprotein/ml

1 150 Cellulase SC118, 50 100μΙ FoxXyn4,

0.6mgprotein/ml

k 150 Cellulase SC118, 50 100μΙ FoxXyn4,

0.6mgprotein/ml

1 150 Cellulase SC118, 50 100μΙ FoxXyn6, 0.71 mg protein/ml

1 150 Cellulase SC118, 50 100μΙ FoxXyn6, 0.71 mg protein/ml

1 150 Cellulase SC118, 50 100μΙ FoxXyn6, 0.71 mg protein/ml

k 150 Cellulase SC118, 50 100μΙ FoxXyn6, 0.71 mg protein/ml

1 150 Cellulase SC118, 50 120μΙ Pure Econase ^* XT™,

0.51 mg protein/ml

1 150 Cellulase SC118, 50 120μΙ Pure Econase* XT™,

0.51 mg protein/ml

1 150 Cellulase SC118, 50 120μΙ Pure Econase* XT™,

0.51 mg protein/ml

k 150 Cellulase SC118, 50 120μΙ Pure Econase* XT™,

0.51 mg protein/ml

1 250 - -

1 250 - -

1 250 - - * Pure Econase XT is a more purified version of Econase XT which displays only 1 band on SDS-PAGE gel corresponding to the xylanase

At the end of the reaction, all the tubes were centrifuged at 3500 rpm for 10min and the supernatant was collected. The precipitate was washed two times with 4ml and 3,5 milliQ water, respectively and the centrifugation was repeated. All the supernatants were pooled and adjusted to a final volume of 12ml. 0.3ml of it were heated at 100oC for 7min, filtered, and injected 10ul for HPLC sugar analysis. Standards of arabinose, xylose and glucose (w/v) were also filtered and injected 10ul. Two HPLC runs for each sample were performed. Biomass weight analysis: The final residual biomass precipitates were placed at 80°C for 48h drying and weighed to get the net weight.

Results of HPLC analysis of Example 3 As can be seen from Figure 4, a combination of Cellulase SC with all different xylanases all increased the production of glucose by some 30-40%, which is in line with the glucose yield produced by Accellerase ^® Trio™. The production of xylose also increased but its level is much lower than that achieved by Accellerase^Trio™.

A typical HPLC chromatogram of DaCS hydrolyzate produced by Accellerase^Trio™ shows the monosaccharide peak of glucose, xylose and arabinose (see Figure 5) with quite flat peaks in the polymer area (Peak 2 with retention of 9.22min).

A typical HPLC chromatogram of DaCS hydrolyzate produced by Cellulase SC (lower trace) and Cellulase SC in combination with Danisco Xylanase™ (upper trace) shows glucose, xylose and arabinose peaks (see Figure 6), compared to the much higher and wider peaks of oligomers and polymers of Figure 5 with Accellerase ^® Trio™.

Figure 5 and Figure 6 show that Cellulase SC and Cellulase SC in combination with a xylanase generated a much reduced xylose peak and also a reduced glucose peak. On the other hand it has high contents of olio- or polymers in the region between retention time of 6.5 - 11.5min.

The inventors have surprisingly found that reduced amount of xylose (and even glucose) and increased amount of oligomers and polymers are advantageous when the hydrolyzate is used as a feed ingredient, particularly for monogastric animals. The inventors have found that xylose is not an efficient source of energy for monogastric animals as approximately 50% of consumed xylose is excreted in the urine. Oligomers and polymers on the other hand can be fermented by microbiota occupying the large intestine to short chain fatty acids which can be absorbed into the blood stream and be used by the animals as an energy source.

Results of weight analysis Example 3

From Figure 7 below, it can be seen that in the presence of Cellulase SC, the addition of an endoxylanase (e.g. Danisco Xylanase™, Econase XT™, FoxXyn4 or FoxXyn6 or the more highly purified forms of Danisco Xylanase™, Econase XT™) all increased hydrolysis based on the reduced weight of the residual biomass obtained after the enzyme reaction. The hydrolysis represented in Figure 7 was conducted using non-industrial processing conditions and for shorter periods of time to accommodate bench-scale testing constraints. Therefore, the extent of overall hydrolysis was suboptimal but is still reflective of the improved hydrolysis that occurs when Cellulase SC and an endoxylanase are used in combination.

EXAMPLE 4. A further study of Cellulase SC hydrolysis of dilute ammonium treated corn stover (DaCS) and the dose effect of Cellulase SC and endoxylanase (Danisco Xylanase™).

1g of DaCS was placed in each of the tubes listed in Table 4, water was added to a volume of 12 ml along with 0.15ml 1 N HCI and mixed. Then enzymes of Cellulase SC 1 18 50-200 μΙ or Cellulase SC 1 18 50-200 μΙ plus 10ul Danisco Xylanase™ were added to start the reaction at 50°C, with shaking at 250rpm for 40h. After the reaction the tubes were centrifuged and the supernatant collected. The precipitate was washed twice with 4ml and 3.5ml water, respectively by re-suspension and centrifugation. The supernatants were pooled and the volume adjusted to 12ml by adding additional water. The pooled supernatants (2ml) were placed at 100°C for 10min to inactivate the enzymes, then centrifuged and filtered. The filtrate was analyzed via HPLC with an injection volume of 10μΙ. The washed precipitates were dried at 80°C for 48h.

Table 4

Tube no. Danisco Cellulase DaCS, g 50°C 1 N HCI, Water, ml

Xylanase™, SC, μΙ Ml μΙ

1-3 - - ¹ - 150 4.85 -6 10 - ¹ Yes 150 4.84

7-9 10 50 ¹ Yes 150 4.79

10-12 10 100 Yes 150 4.74

13-15 10 150 ¹ Yes 150 4.69

16-18 10 200 ¹ Yes 150 4.64

19-21 - 50 ¹ Yes 150 4.80

22-24 - 100 ¹ Yes 150 4.75

25-27 - 150 ¹ Yes 150 4.70

28-30 - 200 ¹ yes 150 4.65

Results of weight analysis of Example 4

From Figure 8 it can be seen that DaCS had a dry matter content of 63.3% (Column 1 from left). Incubation of the DaCS at 50°C for 40h in water with pH adjusted to pH5 using HCI, 16.0% of the dry matter became soluble (Column 2 from left of Figure 9). Addition of the endoxylanase at 10μΙ (93 units) further increased the solubility of DaCS to 26.9%. Addition of Cellulase SC1 18 cellulase alone increased the solubility to 41.7%. With a combination of 10μΙ of endoxylanase and 50μΙ of Cellulase SC118, the solubility reached around 51.7%, indicating that xylanase and Cellulase SC have an additive effect. To reach the level of solubility by 10μΙ xylanase and 50μΙ Cellulase SC one needs to add 3 times or more Cellulase SC alone (150μΙ and 200ul). The conclusion is that for the solubilization of DaCS, a combination of 50μΙ Cellulase SC with 0μΙ endoxylanase is superior to Cellulase SC alone at 50, 100 and 150μΙ. Cellulase SC 50μΙ plus endoxylanase 10μΙ is the optimal with a DaCS dry matter load of 10% (w/v) because 100, 150 and 200μΙ Cellulase SC plus endoxylanase at 10μΙ did not further increase the solubility. It is generally agreed that the higher the solubility the higher the in vivo digestibility.

Results of HPLC analysis of Example 4

From Figure 0 it can be seen that there is no free glucose in the soluble fraction of DaCS without added enzyme or with Danisco Xylanase™ at 10ul (93units). This indicates that the 26.9% soluble matter released by the addition of Danisco Xylanase™ alone is in principle oligomers and polymers (Figure 9). With the addition of Cellulase SC at a dose from 50, 100, 150 to 200ul in the reaction, there is an increased release of glucose and xylose but the increase is not linear in relation to the Cellulase SC dose (Figure 10). From Figure 10 it can also be seen that the amount of released glucose is higher for 50μΙ Cellulase SC plus 10μΙ Danisco Xylanase™ than 100μΙ Cellulase SC alone; 100μΙ Cellulase SC plus 10μΙ Danisco Xylanase™ is roughly equal to 150μΙ Cellulase SC alone, and 150μΙ Cellulase SC plus 10μΙ Danisco Xylanase™ is roughly equal to 200μΙ Cellulase SC alone.

To conclude, it is clear that Danisco Xylanase™ and Cellulase SC have a synergy in release of glucose and xylose as the glucose released by a combination of Cellulase SC at 50μΙ and Danisco Xylanase™ at 10μΙ is greater than the sum of glucose released by Danisco Xylanase™ alone (which is basically none) and Cellulase SC at 50μΙ (Figure 10).

Figure 1 1 shows the total soluble sugar concentration in the 12 ml supernatant obtained after the enzyme hydrolysis of DaCS followed by centrifugation and washing. It can see seen that the major sugars are glucose, xylose, arabinose; the minor components are galactose and mannose. CONCLUSIONS

Various cellulase preparations for sugar release were studied. For animal nutrition commercial enzyme combinations particularly those used for breakdown of lignocellulosic biomass in the bioethanol industry are not ideal for use in animal nutrition, mainly as such compositions produce a lot of xylose. We have found that xylose is a sugar which monogastric animals cannot metabolize and utilize efficiently.

An enzyme composition comprising at least the following activities: endoglucanase activity, β-glucosidase activity 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 a-L-arabinofuranosidase activity was found to be the most effective composition for degrading lignocellulosic biomass whilst producing a product that was most suited for use as an animal feed composition or as a feedstuff.

EXAMPLE 5: Xylose as an energy source for broilers

A total of 240 male broiler chicks (1 day old) of the Ross 308 strain were used in this study. The chicks were individually weighed at hatch and divided into 12 groups of 5 birds balanced for body weight. They were then group weighed and each group was housed in a cage in electrically heated Petersime battery brooders. The brooder and room temperatures were set at 32 and 29°C, respectively, during the first week. Thereafter, heat supply in the brooder was switched off and room temperature was maintained at 29°C throughout the experiment. The study duration was 21 days during which time birds were fed one of the following four treatments: 1 : basal diet (corn soybean meal based diet) + corn starch 25%, D-xylose 0%, 2: basal diet + corn starch 20%, D-xylose 5%, 3: basal diet + corn starch 10%, D-xylose 15%, and 4: basal diet + corn starch 0%, D-xylose 25%. Fresh water and feed were made available to all chicks for ad libitum intake throughout the study. During the study period, the feed consumption and body weight of the birds was monitored weekly for calculation of body weight gain, voluntary feed intake, and feed conversion efficiency.

The study duration was 21 days, however, following 14 days of treatment birds on treatment 4 were removed due to high mortality rates and signs of illness. The basal diet was a corn soybean meal diet. 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). At 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.

Following 21 days of feeding treatment diets, birds fed 10% corn starch and 15% xylose in the diet had significantly lower body weight gain and poorer feed efficiency than birds fed 25% starch & 0% xylose (P<0.05) (see Figure 13 and Figure 14). Birds fed 5% xylose and 20% corn starch demonstrated weight gain and FCR intermediate to the two other treatments but was not significantly different from either.

In conclusion, xylose replacing 25% of the energy source in the diet is detrimental to the health of broiler chickens. Broilers appear to tolerate up to 5% xylose in the diet before demonstrating significant adverse effects on growth performance however, even at 5% xylose inclusion in the diet, there is a numerical decrease in growth performance compared to the control diet.

EXAMPLE 6. Accellerase®1500™ and Econase®XT™ hydrolysis of dilute ammonium treated corn stover (DaCS) and caustic deli nified switch grass (DLswg) To each of 8 plastic tubes with a volume of 12ml, 1g of DaCS and 5ml MilliQ water are added and mixed. Likewise, 1g of DLswg and 5ml MilliQ water are added to another 8 plastic tubes with a volume of 12ml, and mixed. All tubes are let stand at 5°C overnight to ensure the DaCS and DLswg are well hydrated. The following day 150ul 1 HCI is added to adjust the pH to 4.6-5.1. Enzymes (listed in Table 5 below) are added to start the reaction. The reaction is performed at 50°C with a shaking speed of 180 rpm for 40h. Liquid samples (120μΙ) are collected at a reaction time of 6h, diluted with 120μΙ MilliQ water and maintained at 5°C until analysis is performed. At the end of the enzyme reaction, 240μΙ milliQ water is further added to each of the collected samples, which are then heated at 100°C in a water bath for 5min, followed by centrifugation at 10000 xg for 10min. 250μΙ of supernatant is then filtered and the filtrate 10μΙ is injected for HPLC analysis. Glucose and xylose standards at 1 % (w/v) are filtered and analyzed by HPLC at the same conditions with the same injection volume as described in the materials and methods section. Table 5.

Tube no. Dilute Caustic delignified switch Cellulases added

ammonium grass (DLswg)

treated corn

stover (DaCS)

Tube 1 i g Accellerase ^® 1500 ^, - 0.3 ml/g

DM

Tube 2 ig Accellerase ^® 1500 ^{I M} - 0.3 ml/g

DM

Tube 3 —

g Accellerase ^® Trio™ - 0.2 ml/g DM

Tube 4 i g — Accellerase ^® Trio™ - 0.2 ml/g DM

Tube 5 —

i g Econase®XT 5P (1.4 mg/g DM)

Tube 6 —

i g Econase®XT 5P (1.4 mg/g DM)

Tube 7 i g Accellerase ^w 1500 ^{I M} (0.3 ml/g) +

Econase®XT 5P (1.4 mg/g DM)

Tube 8 i g Accellerase ^® 1500 ^I (0.3 ml/g) +

Econase®XT 5P (1.4 mg/g DM)

Tube 9 i g Accellerase ^® 1500 ^{I M} - 0.3 ml/g

DM

Tube 10 i g Accellerase ^® 1500™ - 0.3 ml/g

DM

Tube 1 1 — i g Accellerase ^® Trio ^{I M} - 0.2 ml/g DM

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 hemi-cellulases activity such as endoxylanase activity.

Econase ^® XT 5P is a commercial enzyme available from AB Vista and has endo1-4 Beta xylanase activity.

RESULTS

Results indicate that the addition of Accellerase® 1500™ and Econase®XT 5P to dilute ammonia pre-treated corn stover and caustic delignified switchgrass have a synergy in release of glucose as the 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 glucose released by each enzyme alone (Table 6). The combination of endoglucanase, endoxylanase and β- glucosidase activities as is provided in this example by the addition of Accellerase®1500™ and Econase®XT 5P resulted in substantial release of glucose similar to Accellerase®Trio™. The combination of Accellerase®1500™ and Econase®XT 5P resulted in significantly less xylose release due to the absence of significant amounts of a-L-arabinofuranosidase and β- xylosidase activity present in this enzyme combination compared to Accellerase®Trio™. Table 6. Release of xylose and glucose from dilute ammonia pretreated corn stover (DAcs) and caustic delignified switch grass (DLswg) using Accellerase ^® Trio™, Accellerase ^® 1500™

¹ DAcs - dilute ammonia pretreated corn stovers

2DLswg - caustic delignified switch grass

n.d. - not detected

Example 7. Celluclast®, Danisco Xylanase™ and Accellerase®BG™ hydrolysis of dilute ammonium treated corn stover (DaCS) and caustic delignified switchgrass (DLswg)

To each of 12 plastic tubes with a volume of 12ml, 1g of DaCS and 5ml MilliQ water are added and mixed. Likewise, 1g of DLswg and 5ml MilliQ water are added to another 12 plastic tubes with a volume of 12ml, and mixed. All tubes are let stand at 5°C overnight to ensure the DaCS and DLswg are well hydrated. The following day 150ul 1 N HCI is added to adjust the pH to 4.6-5.1. Enzymes (listed in Table 7 below) are added to start the reaction. The reaction is performed at 50°C with a shaking speed of 180 rpm for 40h. Liquid samples (120μΙ) are collected at a reaction time of 6h, , diluted with 120μΙ MilliQ water and maintained at 5°C until analysis is performed. At the end of the enzyme reaction, 240μΙ milliQ water is further added to each of the collected samples, which are then heated at 100°C in a water bath for 5min, followed by centrifugation at 10000 xg for 10min. 250μΙ of supernatant is then filtered and the filtrate 10μΙ is injected for HPLC analysis. Glucose and xylose standards at 1% (w/v) are filtered and analyzed by HPLC at the same conditions with the same injection volume as described in the materials and methods section.

Table 7.

Tube no. Dilute Caustic Cellulases added ammonium delignified

treated corn switchgrass

stover (DaCS) (DLswg)

Tube 1 ig — Celluclast® (0.1 ml/g DM)

Tube 2 ig — Celluclast® (0.1 ml/g DM)

Tube 3 ig — Danisco Xylanase™ (3.5 ul/g DM)

Tube 4 ig — Danisco Xylanase™ (3.5 ul/g DM)

Tube 5 ig — Accellerase®BG™ (0.15 ml/g DM)

Tube 6 ig — Accellerase®BG™ (0.15 ml/g DM)

Tube 7 i g Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)

Tube 8 i g Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)

Tube 9 ig Celluclast® (0.1 ml/g

DM)+Accellerase®BG™ (0.15 ml/g DM)

Tube 10 ig Celluclast® (0.1 ml/g

DM)+Accellerase®BG™ (0.15 ml/g DM)

Tube 11 ig Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)+Accellerase®BG™ (0.15 ml/g DM)

Tube 12 ig Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)+Accellerase®BG™ (0.15 ml/g DM)

Tube 13 — i g Celluclast® (0.1 ml/g DM)

Tube 14 — ig Celluclast® (0.1 ml/g DM)

Tube 15 — i g Danisco Xylanase™ (3.5 l/g DM)

Tube 16 — ig Danisco Xylanase™ (3.5 ul/g DM)

Tube 17 — ig Accellerase®BG™ (0.15 ml/g DM)

Tube 18 — ig Accellerase®BG™ (0. 5 ml/g DM)

Tube 19 ig Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)

Tube 20 ig Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)

Tube 21 ig Celluclast® (0.1 ml/g

DM)+Accellerase®BG™ (0.15 ml/g DM) Tube 22 ig Celluclast® (0.1 ml/g

DM)+Accellerase®BG™ (0.15 ml/g DM)

Tube 23 i g Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)+Accellerase®BG™ (0.15 ml/g DM)

Tube 24 i g Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)+Accellerase®BG™ (0.15 ml/g DM)

Tube 25k ig — No enzyme added

Tube 26k — i g No enzyme added

Tube 27k No biomass Celluclast ^® , (0.1 ml/g DM)

Tube 28k No biomass Danisco Xylanase™, (3.5 ul/g DM)

Tube 29k No biomass Accellerase®BG™ (0.15 ml/g DM)

Tube 30k No biomass Celluclast® (0.1 ml/g DM)+Danisco

Xylanase™ (3.5 ul/g DM)+Accellerase®BG™ (0.15 ml/g DM)

Celluclast ^® is a commercial enzyme available from Sigma-Aldrich (C2730) and contains certain cellulase activities such as endoglucanase activity, and beta-glucosidase.

Danisco Xylanase™ is a commercial enzyme available from Dansico (now part of DuPont) and has endoxylanase activity.

Accellerase®BG™ is a commercial enzyme available from Danisco (now part of DuPont) and has β-glucosidase activity.

RESULTS

Results indicate that the addition of Celluclast®, Danisco Xylanase™ and Accellerase®BG™ to dilute ammonia pre-treated corn stover and caustic delignified switchgrass have a synergy in release of glucose as the glucose released by the combination of Celluclast® (0.1 ml/g DM), Danisco Xylanase™ (3.5 ul /g DM) and AcceIlerase®BG™ (0.15 ml/g DM) is greater than the glucose released by each enzyme alone or a combination of only 2 enzymes (Table 8). The combination of endoglucanase, endoxylanase and b-glucosidase activities as is provided in this example by the addition of Celluclast®, Danisco Xylanase™ and Accellerase®BG™ resulted in similar glucose release to Accellerase®Trio™. The combination of Celluclast®, Danisco Xylanase™ and Accellerase®BG™ resulted in significantly less xylose release due to the absence of significant amounts of a-L- arabinofuranosidase and β-xylosidase activity present in this enzyme combination compared to Accellerase®Trio™.

Table 8. Release of xylose and glucose from dilute ammonia pretreated corn stover (DAcs) and caustic delignified switch grass (DLswg) using Accellerase ^® Trio™, Celluclast®, Danisco X lanase™ and Accellerase®BG™

¹ DAcs - dilute ammonia pretreated corn stovers

2DLswg - caustic delignified switch grass

n.d. - not detected Example 8. Cellulase from Aspergillus niger (Sigma C1184) and Accellerase®BG™ hydrolysis of dilute ammonium treated corn stover (DaCS) and caustic delignified switch grass (DLswg)

To each of 6 plastic tubes with a volume of 12ml, 1g of DaCS and 5ml MilliQ water are added and mixed. Likewise, 1 g of DLswg and 5ml MilliQ water are added to another 8 plastic tubes with a volume of 12ml, and mixed. All tubes are let stand at 5°C overnight to ensure the DaCS and DLswg are well hydrated. The following day 150ul 1 N HCI is added to adjust the pH to 4.6-5.1. Enzymes (listed in Table 9 below) are added to start the reaction. The reaction is performed at 50°C with a shaking speed of 80 rpm for 40h. Liquid samples (120μΙ) are collected at a reaction time of 6h, diluted with 120μΙ MilliQ water and maintained at 5°C until analysis is performed. At the end of the enzyme reaction, 240μΙ milliQ water is further added to each of the collected samples, which are then heated at 100°C in a water bath for 5min, followed by centrifugation at 10000 xg for 10min. 250μΙ of supernatant is then filtered and the filtrate 10μΙ is injected for HPLC analysis. Glucose and xylose standards at 1 % (w/v) are filtered and analyzed by HPLC at the same conditions with the same injection volume as described in the materials and methods section.

Table 9.

Tube no. Dilute Caustic delignified Cellulases added

ammonium switchgrass (DLswg)

treated corn

stover (DaCS)

Tube 1 i g Cellulase from A. Niger (0.2 mg/g

DM)

Tube 2 i g Cellulase from A. Niger (0.2 mg/g

DM)

Tube 3 i g — Accellerase ^® BG ^{I M} (0.15 ml/g)

Tube 4 i g — Accellerase ^® BG ^{I M} (0.15 ml/g)

Tube 5 ig Cellulase from A. Niger (0.2 mg/g

DM)+Accellerase ^® BG™ (0.15 ml/g)

Tube 6 ig Cellulase from A. Niger (0.2 mg/g

DM)+Accellerase ^® BG™ (0.15 ml/g)

Tube 7 ig Cellulase from A. Niger (0.2 mg/g

DM)

Tube 8 ig Cellulase from A. Niger (0.2 mg/g

DM)

Tube 9 — ig Accellerase ^® BG ^{I M} (0.15 ml/g)

Tube 10 — ig Accellerase ^® BG™ (0.15 ml/g)

Tube 11 i g Cellulase from A. Niger (0.2 mg/g

DM)+Accellerase ^® BG™ (0.15 ml/g)

Tube 12 i g Cellulase from A. Niger (0.2 mg/g

DM)+Accellerase ^® BG™ (0.15 ml/g)

Tube 13k —

i g No enzyme added

Tube 14k — ig No enzyme added

Tube 15k No biomass Cellulase from A. niger, (0.2 mg/g

DM)

Cellulase from Aspergillus niger is a commercial enzyme available from Sigma-Aldrich (C1 184) and contains certain cellulase activities such as endoglucanase activity as well as endoxylanase activity.

Accellerase®BG™ is a commercial enzyme available from Danisco (now part of DuPont) and has β-glucosidase activity.

RESULTS

Results indicate that the addition of Cellulase from A. niger and Accellerase®BG™ to dilute ammonia pretreated corn stover and caustic delignified switchgrass have a synergy in release of glucose as the glucose released by the combination of Cellulase from A. niger and Accellerase®BG™ is greater than the glucose released by each enzyme alone (Table 10). The combination of endoglucanase, endoxylanase and b-glucosidase activities as is provided in this example by the addition of cellulase from A. niger and Accellerase®BG™ resulted in similar glucose release to Accellerase®Trio™. The combination of cellulase from A. niger and Accellerase®BG™ resulted in significantly less xylose release due to the absence of significant amounts of a-L-arabinofuranosidase and β-xylosidase activity present in this enzyme combination compared to Accellerase®Trio™. Table 10. Release of xylose and glucose from dilute ammonia pretreated corn stover (DAcs) and caustic delignified switch grass (DLswg) using Accellerase^Trio™, Cellulase from A. ni er and Accellerase®BG™

DAcs - dilute ammonia pretreated corn stovers

2DLswg - caustic delignified switch grass All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should 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 those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.