METHOD OF PREPARING FEED ADDITIVE FIELD OF THE INVENTION

The present invention relates to methods and compositions to improve the nutritional value of lignocellulosic biomass in animal feed (particularly feed for monogastric animals). 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

Animals, e.g. 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 cereal grains in the animal's diet. Animals, particularly monogastric animals, do not have the enzymes needed to digest cellulose, and even if they did, the retention time in the intestine is a limitation for conversion of cellulose to glucose.

Direct absorption of glucose from cellulose in the small intestine of animals, particularly monogastric animals, is an efficient way to use that energy as a replacement of energy from grain. Replacement of corn and grains traditionally used in animal feed by allowing the carbohydrates from cheaper cellulosic materials to be utilized by animals as an energy source is important for reducing the cost of animal feed and releasing valuable land for growth of other non-feed crops for example. STATEMENTS OF THE INVENTION

According to 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; and

b. admixing the physically and/or chemically and/or biologically pre-treated

lignocellulosic biomass from step a. with at least one in-feed glycosyl hydrolase enzyme,

c. and optionally packaging. In another aspect there is provided a method of preparing a feed additive composition comprising: a. physically and/or chemically pre-treating lignocellulosic biomass;and b. admixing the physically and/or chemically pre-treated lignocellulosic biomass from step a. with at least one in-feed glycosyl hydrolase enzyme, c. and optionally packaging

In one aspect the method of the present invention may further comprise an optional step of treatment with at least one pre-digestive glycosyl hydrolase enzyme.

In a further aspect, the present invention provides a feed additive composition obtainable (e.g. obtained) by a method of the present invention.

In another aspect, the present invention provides a feed additive composition or a feed ingredient comprising a physically and/or chemically and/or biologically pre-treated lignocellulosic biomass in combination with at least one in-feed glycosyl hydrolase enzyme.

The present invention yet further provides a feed additive kit comprising a first container comprising a physically and/or chemically and/or biologically pre-treated lignocellulosic biomass and a second container comprising at least one in-feed glycosyl hydrolase enzyme, and instructions for co-administering same. The present invention yet further 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 the method of the present invention.

The present invention further provides a premix comprising a feed additive composition or feed ingredient according to the present invention or a feed additive composition obtainable (preferably obtained) by the method of the present invention, and at least one mineral and/or at least one vitamin.

In a yet further aspect, the present invention provides a method for improving a biophysical characteristic of an animal which method comprises administering to a subject a physically and/or chemically and/or biologically pre-treated lignocellulosic biomass in combination with an in-feed glycosyl hydrolase enzyme.

In one aspect, the present invention provides a method for improving a biophysical characteristic of an animal which method comprises administering to a subject a feed additive composition obtainable (e.g. obtained) by the method of the present invention or a feed additive composition of the present invention.

The present invention further provides the use of a physically and/or chemically and/or biologically pre-treated lignocellulosic biomass in combination with an in-feed glycosyl hydrolase enzyme for improving a biophysical characteristic of an animal.

In a yet further aspect, there is provided the use of a feed additive composition or feed ingredient according to the present invention or a feed additive composition obtainable (preferably obtained) by the method of the present invention for improving a biophysical characteristic of an animal.

In another aspect, the present invention provides a method of preparing a feedstuff comprising admixing 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 the method of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the procedure for chicken proximal gastrointestinal tract (GIT) in vitro digestions.

Figure 2 shows a nucleotide sequence (SEQ ID No. 1) encoding an endoxylanase (FoxXyn6). The signal sequence is shown in bold (upper case).

Figure 3 shows a nucleotide sequence (SEQ ID No. 2) encoding an endoxylanase (FoxXyn6).

Figure 4 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 5 shows a nucleotide sequence (SEQ ID No. 4) encoding an endoxylanase (FoxXyn4). The signal sequence is shown bold (upper case).

Figure 6 shows a nucleotide sequence (SEQ ID No. 5) encoding an endoxylanase (FoxXyn4).

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

Figure 9 shows a polypeptide sequence (SEQ ID No. 8) of an endoglucanase (EG1 ) from Aspergillus niger (CBS513.88). Figure 10 shows a nucleotide sequence (SEQ ID No. 9) encoding an endoglucanase (EG2) from Aspergillus niger CBS513.88.

Figure 11 shows a polypeptide sequence (SEQ ID No. 10) of an endoglucanase (EG2) from Aspergillus niger CBS513.88.

Figure 12 shows a nucleotide sequence (SEQ ID No. 11) encoding an endoxylanase.

Figure 13 shows a polypeptide sequence (SEQ ID No. 12) of an endoxylanase.

Figure 14 shows a nucleotide sequence (SEQ ID No. 13) encoding a β-glucosidase from Aspergillus niger CBS513.88.

Figure 15 shows a polypeptide sequence (SEQ ID No. 14) of a β-glucosidase from Aspergillus niger CBS513.88.

Figure 16 shows a nucleotide sequence (SEQ ID No. 15) encoding a lytic polysaccharide monooxygenase from Aspergillus niger CBS513.88.

Figure 17 shows a polypeptide sequence (SEQ ID No. 16) of a lytic polysaccharide monooxygenase from Aspergillus niger CBS513.88.

Figure 18 shows a nucleotide sequence (SEQ ID No. 17) encoding a CHB1A from Aspergillus niger (CBS513.88).

Figure 19 shows a polypeptide sequence (SEQ ID No. 18) of a CHB1A from Aspergillus n/ ^' ger (CBS513.88).

Figure 20 shows a nucleotide sequence (SEQ ID No. 19) encoding a CHB1 B from Aspergillus niger (CBS513.88).

Figure 21 shows a polypeptide sequence (SEQ ID No. 20) of a CHB1 B from Aspergillus n/ger (CBS513.88).

Figure 22 shows a nucleotide sequence (SEQ ID No. 21 ) encoding a CHB1 from Trichoderma reesei.

Figure 23 shows a polypeptide sequence (SEQ ID No. 22) of a CHB1 from Trichoderma reesei.

Figure 24 shows a nucleotide sequence (SEQ ID No. 23) encoding a CHB2 from Trichoderma reesei.

Figure 25 shows a polypeptide sequence (SEQ ID No. 24) of a CHB2 from Trichoderma reesei.

Figure 26 shows a nucleotide sequence (SEQ ID No. 25) encoding an endoglucanase (EG1) from Trichoderma reesei.

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

Figure 28 shows a nucleotide sequence (SEQ ID No. 27) encoding an endoglucanase (EG2) from Trichoderma reesei. Figure 29 shows a polypeptide sequence (SEQ ID No. 28) of an endoglucanase (EG2) from Trichoderma reesei.

Figure 30 shows a nucleotide sequence (SEQ ID No. 29) encoding an endoglucanase (EG3) from Trichoderma reesei.

Figure 31 shows a polypeptide sequence (SEQ ID No. 30) of an endoglucanase (EG3) from Trichoderma reesei.

Figure 32 shows a nucleotide sequence (SEQ ID No. 31) encoding a lytic polysaccharide monooxygenase from Trichoderma reesei.

Figure 33 shows a polypeptide sequence (SEQ ID No. 32) of a lytic polysaccharide monooxygenase from Trichoderma reesei.

Figure 34 shows a nucleotide sequence (SEQ ID No. 33) encoding an endoxylanase from Trichoderma reesei.

Figure 35 shows a polypeptide sequence (SEQ ID No. 34) of an endoxylanase from Trichoderma reesei.

Figure 36 shows a nucleotide sequence (SEQ ID No. 35) encoding a β-glucosidase from Trichoderma reesei.

Figure 37 shows a polypeptide sequence (SEQ ID No. 36) of a β-glucosidase from Trichoderma reesei. DETAILED DESCRIPTION

A seminal finding of the present invention is that the present inventors have surprisingly found that physically pre-treating, chemically pre-treating, physicochemically pre-treating, biologically pre-treating (or combinations thereof) lignocellulosic biomass in combination with treatment with at least one in-feed enzyme can unexpectedly and significantly improve the nutritional value of lignocellulosic biomass to animals (particularly monogastric animals).

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. Approximately 750 million tonnes of cereal grains per year are used for animal nutrition. The replacement of a proportion of these cereal grains with Iignocelluiosic biomass as taught herein provides a significant benefit to the population as a whole. The replacement of grain (e.g. corn) for cheaper cellulosic materials (e.g. Iignocelluiosic biomass) will revolutionize the animal feed markets, and/or increase the stability of the human food and energy markets for some time.

The presently claimed invention is relevant for both monogastric animals and ruminants. For example 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. Accordingly, one advantage of the present invention is to improve the nutrient value of Iignocelluiosic biomass.

Accordingly in one aspect the present invention provides a method of preparing a feed additive composition comprising: a. physically and/or chemically and/or biologically pre-treating Iignocelluiosic biomass; and

b. admixing the pre-treated Iignocelluiosic biomass from step a. with at least one in-feed glycosyl hydrolase enzyme,

c. and optionally packaging. The method of the present invention may further comprise feeding the feed additive composition to an animal.

In some embodiments the physically and/or chemically and/or biologically pre-treated Iignocelluiosic biomass in combination with the at least one in-feed glycosyl hydrolase enzyme is fed to an animal immediately after admixing the physically and/or chemically and/or biologically pre-treated Iignocelluiosic biomass with the at least one in-feed glycosyl hydrolase enzyme.

In other embodiments the in-feed glycosyl hydrolase enzyme is maintained in an inactive form on the physically and/or chemically and/or biologically pre-treated Iignocelluiosic biomass, until such time that the enzyme enters the gastrointestinal tract (GIT) of an animal. In such instances, when the enzyme enters the GIT of the animal the enzyme is activated (e.g. becomes active).

The term "in-feed" as used herein means that the enzyme is functional, preferably primarily functional, more preferably solely functional, in the GIT of the animal. In other words, the term "in-feed" as used herein means that the enzyme is substantially inactive (or is inactive) in the feed additive composition and/or on the lignocellulosic biomass prior to feeding the feed additive composition to an animal.

There term "primarily functional" means that the enzyme mainly functions on its substrate once it enters the GIT. In other words, prior to entering the GIT the level of enzyme activity defined as the amount of solubilisation of biomass material to oligosaccharides and monosaccharides is less than 20%, suitably less than 10%, preferably less than 5%, of the level of enzyme activity after it enters the GIT (particularly, after it enters the small intestine of the GIT).

The term "solely functional" as used herein means that the enzyme is inactive before entering the GIT and is activated upon entering the GIT.

The term "inactive" as used herein means that the enzyme is not active. This may mean that the enzyme's activity is somehow inhibited or that the enzyme is in an environment in which it is inactive or that the enzyme is presented to its substrate immediately prior to feeding to the animal such that there is not enough time to be active. The "inactivity" of the enzyme is in any event reversible once it enters the GIT of an animal. The term "substantially inactive" as used herein means that the enzyme has low activity compared with its activity once it has entered the GIT (e.g. in the small intestine of the animal). For instance, substantially inactive may mean that the enzyme in the feed additive composition and/or on the lignocellulosic biomass has less than 10% of its activity when compared with its activity in the GIT (particularly, in the small intestine of the GIT).

Maintaining the "in-feed" enzyme in an inactive or substantially inactive state in the feed additive composition and/or on the lignocellulosic biomass can be achieved in a number of ways known to one skilled in the art. By way of example only maintaining the water content (wt %) of the lignocellulosic biomass and/or of the in-feed enzyme and/or of the feed additive composition at less than 15%, preferably less than 10%, is sufficient to ensure that the in-feed enzyme is inactive or substantially inactive in the feed additive composition and/or on the lignocellulosic biomass.

Therefore in one embodiment the in-feed enzyme may be admixed with the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass when the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass, the in-feed enzyme or both are in a dry state or a substantially dry state.

In one embodiment the lignocellulosic biomass and/or the in-feed enzyme and/or the feed additive composition, post-admixing the lignocellulosic biomass and the in-feed enzyme, are (maintained and/or stored) in a dry state or substantially dry state.

The term "dry state" as used herein means that the lignocellulosic biomass and/or the in-feed enzyme and/or the feed additive composition contains no or only a very low amount of water. In other words the term "dry state" as used herein may mean that the lignocellulosic biomass and/or the in-feed enzyme and/or the feed additive composition comprises less than 5%, preferably less than 1 %, water content (wt %).

The term "substantially dry state" as used herein means that the lignocellulosic biomass and/or the in-feed enzyme and/or the feed additive composition contains only a very low amount of water. In other words the term "substantially dry state" as used herein may mean that the lignocellulosic biomass and/or the in-feed enzyme and/or the feed additive composition comprises less than 15%, preferably less than 10%, water content (wt %). In one embodiment, the method according to the present invention may comprise drying the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass prior to, during or after (preferably prior to) admixing the biomass with at least one in-feed glycosyl hydrolase enzyme. In another embodiment the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass either before or after adding the at least one in-feed glycosyl hydrolase enzyme comprises less than 15 wt % moisture content.

In another embodiment the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass either before or after adding the at least one in-feed glycosyl hydrolase enzyme comprises less than 10 wt % moisture content. In another embodiment the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass either before or after adding the at least one in-feed glycosyl hydrolase enzyme comprises less than 5 wt % moisture content.

In another embodiment the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass either before or after adding the at least one in-feed glycosyl hydrolase enzyme comprises less than 1 wt % moisture content. The "in-feed" enzyme may be maintained in an inactive or substantially inactive state in the feed additive composition and/or on the lignocellulosic biomass by physically preventing the enzyme from interacting with its substrate. For example the in-feed enzyme may be encapsulated prior to admixing with the lignocellulosic biomass. When the in-feed enzyme is physically prevented from interacting with its substrate in the lignocelluosic biomass, then once in the GIT the physical barrier is removed thus allowing the interaction of the in-feed enzyme with its substrate.

By way of example only, the encapsulation may be removed by passage of the encapsulated enzyme through the stomach of an animal. The stomach of an animal is at very low (acidic) pH (e.g. pH 2-4). This acidity can be used to activate encapsulated enzymes.

In one embodiment the enzyme may be encapsulated by a polymer, such as chitin or chitosans, gelatin, gum arabic or wax for example. By way of example only the polymer may be a gelatin or gum arabic as taught in Xue et al Food Funct. 2013, Apr 25; 6 Feb (epub); 4 (4) 610-7 (which is incorporated herein by reference). Alternatively, the polymer may a chitosan-based hydrogel as taught in Zhang et al Biomacromolecules 201 1 ,12,2894-2901 (which is incorporated herein by reference). In one embodiment the at least one in-feed glycosyl hydrolase enzyme may be activated by feeding the at least one in-feed glycosyl hydrolase enzyme to an animal.

The term "inactive" as used herein may mean that the enzyme is presented to its substrate immediately prior to feeding to the animal such that there is not enough time to be active before it enters the GIT of the animal. In one embodiment the at least one in-feed glycosyl hydrolase enzyme may be admixed with the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass immediately prior to feeding the feed additive composition to an animal. In one preferred embodiment the in-feed enzyme is maintained in an inactive or substantially inactive state by maintaining the feed additive composition in a dry state or substantially dry state. This has additional benefits in that the handling, e.g. processing, packaging, storage and transport of a dry composition is easier than non-dry formulations (e.g. liquids). However, it can also be envisaged that a kit where the physically and/or chemically and/or biologically pre-treated biomass is physically separated from the in-feed enzyme (e.g. by being in separate containers) would enable the end user (e.g. farmer) to admix the physically and/or chemically and/or biologically pre-treated biomass with the in-feed enzyme immediately prior to feeding the mixture to the animal. In such a situation the physically and/or chemically and/or biologically pre-treated biomass and/or the in-feed enzyme may be in any formulation, e.g. solid, semi-solid or liquid for example.

The present inventors were the first to recognise that chemically and/or physically and/or biologically pre-treated lignocellulosic biomass can have enough nutrition value for use as a feed if one combines this biomass with in-feed enzymes. It was surprising that the relatively short period of time that the biomass is in the GIT of an animal provides sufficient time for the in-feed enzyme(s) to significantly improve the nutritional value of this sort of biomass.

Preferably the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass comprises at least 70%, preferably at least 80%, more preferably at least 90%, of the monosaccharides present in the material as oligomers or polymers.

In the present invention, the in-feed enzyme preferably generates and/or releases monosaccharides (preferably C-6 monosaccharides) from the physically and/or chemically and/or biologically treated (e.g. pre-treated) lignocellulosic biomass in the GIT of the animal. Suitably the monosaccharides produced by the in-feed enzyme are soluble monosaccharides, such as C6-sugars, such as one or more of the group consisting of glucose, galactose, fructose or mannose.

It is therefore preferred that the at least one glycosyl hydrolase in-feed enzyme(s) comprises one or more carbohydrate monomer (preferably C-6 monomer) forming activities. Glycosyl hydrolase enzymes are a widespread group of enzymes which hydrolyse the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non- carbohydrate moiety. Many of these are enzymes produced by microorganisms for the degradation of the polysaccharides in the cell walls of plants. Preferably the at least one in-feed enzyme is a glycosyl hydrolase enzyme. A glycosyl hydrolase enzyme are classified as having an E.C. classification 3.2.1.4 (in accordance with the Recommendations of the Nomenclature Committee (NC) of the International Union of Biochemistry and Molecular Biology (IUBMB) on the Nomenclature and Classification of Enzymes by the Reactions they Catalyse; as Published in Enzyme Nomenclature 1992

[Academic Press, San Diego; 0-12-227165-3] with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995), Supplement 4 (1997) and Supplement 5 (in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250; 1-6, and Eur. J. Biochem. 1999, 264, 610-650; respectively).

Suitably the at least one in-feed glycosyl hydrolase enzyme(s) may be a glycan degrading enzyme.

Preferably the at least one in-feed glycosyl hydrolase enzyme(s) hydrolyses a beta-1 ,4- glycosidic link. In one embodiment, the at least one in-feed glycosyl hydrolase enzyme(s) may comprise (or consist essentially of, or consist of) one or both of the following enzyme activities: cellulase activity and/or hemi-cellulase activity.

In one preferred embodiment, the at least one in-feed glycosyl hydrolase enzyme(s) may comprise (or consist essentially of, or consist of) at least cellulase and hemi-cellulase activity.

In a preferred embodiment, the cellulase activity may comprise (or consist essentially of, or consist of) at least the following enzyme activities: endoglucanase activity and β-glucosidase activity.

In a preferred embodiment, the hemi-cellulase activity may comprise (or consist of, or consist essentially of) at least endoxylanase enzyme activity.

Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) comprising at least one or both of cellulase activity and/or hemi-cellulase activity may further comprise one or both of the following activities: exoglucosidase activity and/or lytic polysaccharide monooxygenase activity.

In one embodiment the at least one in-feed glycosyi hydrolase enzyme(s) comprises (or consists essentially of, or consists of) one or more of the following enzyme activities: endoglucanase activity, endoxylanase activity or β-glucosidase activity.

Suitably, the at least one in-feed glycosyi hydrolase enzyme(s) comprising at least one of the following enzyme activities: endoglucanase activity, endoxylanase activity or β-glucosidase activity may further comprise one or both of the following activities: exoglucosidase activity and/or lytic polysaccharide monooxygenase activity.

In one embodiment the at least one in-feed glycosyi hydrolase enzyme(s) comprises (or consists essentially of, or consists of) two or more (e.g. suitably three) of the following enzyme activities: endoglucanase activity, endoxylanase activity or β-glucosidase activity.

Suitably the at least one in-feed glycosyi hydrolase enzyme(s) comprising two or more (e.g. suitably three) of the following enzyme activities: endoglucanase activity, endoxylanase activity or β-glucosidase activity may further comprise one or both of the following activities: exoglucosidase activity and/or lytic polysaccharide monooxygenase activity.

In one embodiment the at least one in-feed glycosyi hydrolase enzyme(s) is an enzyme composition comprising more than one enzyme. The enzyme composition comprising more than one enzyme preferably has at least one cellulase enzyme and at least one hemi- cellulase enzyme.

In another embodiment the enzyme composition comprising more than one enzyme preferably has at least one of the following: an endoglucanase, an endoxylanase or a β- glucosidase.

In one embodiment the enzyme composition comprises at least two (suitably at least three,) enzymes. The enzyme composition comprising the at least two (suitably at least three,) enzymes preferably has at least two (suitably at least three) of the following: an endoglucanase, an endoxylanase or a β-glucosidase. Suitably the enzyme composition may further comprise one or both of the following activities: exoglucosidase activity and/or lytic polysaccharide monooxygenase activity.

In a preferable embodiment the enzyme composition may comprise (or consist essentially of, or consist of) at least the following activities: endoglucanase activity, endoxylanase activity and β-glucosidase activity.

In one embodiment the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) may be characterised by its enzyme activity.

In one embodiment, the at least one in-feed glycosyl hydrolase enzyme(s) (or 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", endoxylanase activity as determined using the "Endoxylanase Activity Assay" and β-glucosidase activity as determined using the "Beta- glucosidase Activity Assay". The ranges in which these activities are present in said enzyme composition are set out in the tables below and the following paragraphs:

² 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.

³ One pNPG unit denotes 1 μπιοΙ of nitro-phenol liberated from para-nitrophenyl-B-D- glucopyranoside per minute at 50°C and pH 4.8. In one embodiment the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise about 2000-2600 CMC u/g of endoglucanase activity, more than about 3000 ABX u/g of endoxylanase activity and more than 2000 pNPG u/g of β-glucosidase activity.

Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise (or consist essentially of or consist of) at least 1000 CMC u/g endoglucanase activity (suitably at least 1500 CMC u/g activity, suitably at least 2000 CMC u/g activity) as determined using the "Endoglucanase Activity Assay".

Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise (or consist essentially of or consist of) at least 2000 ABX u/g endoxylanase activity (suitably at least 2500 ABX u/g activity, suitably at least 3000 ABX u/g activity) as determined using the "Endoxylanase Activity Assay".

Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise (or consist essentially of or consist of) at least 300 pNPG u/g β-glucosidase activity (suitably at least 500 pNPG u/g activity, suitably at least 1000 pNPG u/g activity or suitably at least 2000 pNPG u/g activity) as determined using the "Beta-glucosidase Activity Assay".

Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise (or consist essentially of or consist of) at least 1000 CMC u/g endoglucanase activity (suitably at least 1500 CMC u/g activity, suitably at least 2000 CMC u/g activity) as determined using the "Endoglucanase Activity Assay"; and at least 2000 ABX u/g endoxylanase activity (suitably at least 2500 ABX u/g activity, suitably at least 3000 ABX u/g activity) as determined using the "Endoxylanase Activity Assay"; and at least 300 pNPG u/g β-glucosidase activity (suitably at least 500 pNPG u/g activity, suitably at least 1000 pNPG u/g activity or suitably at least 2000 pNPG u/g activity) as determined using the "Beta-glucosidase Activity Assay".

Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise (or consist essentially of, or consist of) about 600 to about 4000 CMC u/g endoglucanase activity (suitably at least about 1000 to about 3000 CMC u/g activity, suitably at least about 1500 to about 2600 CMC u/g activity) as determined using the "Endoglucanase Activity Assay".

Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise (or consist essentially of, or consist of) about 2000 to about 5000 ABX u/g endoxylanase activity (suitably at least about 2500 to about 4000 ABX u/g activity, suitably at least about 3000 to about 4000 ABX u/g activity) as determined using the "Endoxylanase Activity Assay". Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise (or consist essentially of, or consist of) about 200 to about 4000 pNPG u/g β-glucosidase activity (suitably at least about 300 to about 3000 pNPG u/g activity, suitably at least about 1000 to about 3000 pNPG u/g activity or suitably at least about 2000 to about 3000 pNPG u/g activity) as determined using the "Beta- glucosidase Activity Assay".

Suitably, the at least one in-feed glycosyl hydrolase enzyme(s) (or enzyme composition) for use in the present invention may comprise (or consist essentially of, or consist of) about 600 to about 4000 CMC u/g endoglucanase activity (suitably at least about 1000 to about 3000 CMC u/g activity, suitably at least about 1500 to about 2600 CMC u/g activity) as determined using the "Endoglucanase Activity Assay"; and about 2000 to about 5000 ABX u/g endoxylanase activity (suitably at least about 2500 to about 4000 ABX u/g activity, suitably at least about 3000 to about 4000 ABX u/g activity) as determined using the "Endoxylanase Activity Assay"; and about 200 to about 4000 pNPG u/g β-glucosidase activity (suitably at least about 300 to about 3000 pNPG u/g activity, suitably at least about 1000 to about 3000 pNPG u/g activity or suitably at least about 2000 to about 3000 pNPG u/g activity) as determined using the "Beta-glucosidase Activity Assay".

In one embodiment each of the claimed enzyme activities must comprise at least 4%, preferably at least 5%, preferably at least 10%, of the total enzyme activity of the at least one in-feed glycosyl hydrolysate enzyme(s) (or enzyme composition) of the present invention.

The endoglucanase may be endo-1 ,4- -D-glucanase. 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)^-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-gluanase.

An endoglucanase for use in the at least one in-feed glycosyl hydrolase enzyme(s) 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. 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 at least one in-feed glycosyl hydrolase enzyme(s) 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/g)

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 μηιοΙ of reducing sugars (expressed as glucose equivalents) in one minute at 50°C and pH 4.8

The endoxylanase activity - may be endo-1 ,4-p-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 at least one in-feed glycosyl hydrolase enzyme(s) 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. SEQ ID No. 33, SEQ ID No. 11 , SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 2 and SEQ ID No. 1.

An endoxylanase for use in the at least one in-feed glycosyl hydrolase enzyme(s) 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. 2, 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 to equilibrate at 50°C. Pipette 0.2 ml of enzyme dilution using positive displacement pipettes or equivalent. Vortex to mix. Incubate each sample at 50°C 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 μηηοΙ 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 at least one in-feed glycosyl hydrolase enzyme(s) 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. 13.

A β-glucosidase for use in the at least one in-feed glycosyl hydrolase enzyme(s) for use in the present invention may be one or more -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/g) 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 0 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 pmol of nitro-phenol liberated from para-nitrophenyl-B-D- glucopyranoside per minute at 50°C and pH 4.8.

In one embodiment the at least on in-feed enzyme may additionally comprise one or more of the following enzyme activities: exoglucosidase activity or lytic polysaccharide monooxygenase activity. In one embodiment the at least on in-feed enzyme may additionally comprise a further enzyme which comprises (or has) one or more of the following enzyme activities: exoglucosidase activity or lytic polysaccharide monooxygenase activity.

In one embodiment a lytic polysaccharide monooxygenase for use in the at least one in-feed glycosyl hydrolase enzyme(s) 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 at least one in-feed glycosyl hydrolase enzyme(s) 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 the at least one in-feed glycosyl hydrolase enzyme(s) 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.

Exoglucosidase activity may be classified as E.C. 3.2.1.74. Exoglucosidase hydrolyses the (1->4)-linkages in (1->4)-beta-D-glucans, to remove successive glucose units.

In some embodiments the at least one in-feed glycosyl hydrolase enzyme(s) for use in the present invention may comprise (or consist essentially of, or consist of) cellobiohydrolase activity.

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 at least one in-feed glycosyl hydrolase enzyme(s) 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 at least one in-feed glycosyl hydrolase enzyme(s) 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.

In one embodiment the at least one in-feed glycosyl hydrolase enzyme for use in the present invention is able to hydrolyse lignocellulosic biomass in a crystalline state. Suitably the at least one in-feed glycosyl hydrolase enzyme for use in the present invention is able to hydrolyse cellulose in a crystalline state. By way of example only, the following enzymes may suitably be used in accordance with the present invention:

Enzymes Company Tradename

Certain cellulase activities such as

endoglucanase activity and beta-glucosidase

and certain hemi-cellulases activity such as

endoxylanase activity. Danisco Accellerase® Trio

Beta-glucosidase Danisco Accellerase® BG™

Endoglucanase, hemi-cellulases (including

endoxylanase) & beta-glucosidase Danisco Accellerase® Duet™

Endoglucanase, hemi-cellulases (including

endoxylanase) & beta-glucosidase Danisco Accellerase 1500

Cellulase including endoglucanase activity, as C1184 - Cellulase from well as endoxylanase activity Sigma Aspergillas niger

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

Beta-glucosidase Novozyme Novozyme 188

Cellulases and hemicellulases including beta- glucosidase and GH61 activity Novozyme Cellic CTec3

Hemicellulases including endoxylanase and

beta-xylosidase Novozyme Cellic HTec3 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

Danisco Xylanase™

(see EP1222256 which is incorporated herein

Endoxylanase Danisco by reference)

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-l,4" -xylanase Danisco Avizyme 1100 endo-l,4^-xylanase Danisco Axtra® XB

endo-l,4^-xylanase Danisco Axtra ^® XAP endo-l,4^-xylanase DSM Biofeed Plus™ In one embodiment the at least one in-feed glycosyl hydrolase enzyme(s) may be a single enzyme or a combination of enzymes (e.g. an enzyme mix).

In one preferred embodiment the at least one in-feed glycosyl hydrolase enzyme is an enzyme mixture.

Preferably the in-feed enzyme in accordance with the present invention is stable and active in the gastrointestinal tract (GIT) of an animal. In one embodiment the in-feed enzyme is resistant to pepsin. In one embodiment the in-feed enzyme is tolerant to bile salts. In one embodiment the in-feed enzyme is resistant to low pH. In one embodiment the in-feed enzyme can withstand pelleting temperatures (70-95°C).ln one embodiment the in-feed enzyme is active in the range of 37-40°C. In one embodiment the in-feed enzyme is active in the range of 37-40°C. The term "consist essentially of or "consists essentially of as used in the context of the activity of the at least one in-feed glycosyl hydrolase enzyme(s) means that the in-feed glycosyl hydrolase enzyme(s) has the activity or the activities characterised, but no other glycosyl hydrolase enzyme activity and/or no other enzyme activity which is capable of digesting lignocellulosic biomass.

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, corn 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 another embodiment the lignocellulosic 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 lignocellulosic biomass may comprise less than 50% starch, preferably less than 40% starch.

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

In one embodiment the lignocellulosic biomass may comprise less than 3% starch. In one embodiment the lignocellulosic biomass may comprise less than 1 % starch. Suitably the lignocellulosic biomass does not comprise starch.

In one embodiment the lignocellulosic biomass does not comprise a cereal grain. PRE-TREATMENT

In one embodiment the lignocellulosic biomass is pre-treated by any pre-treatment process known in the art which is capable of disrupting the compact structure of lignocellulosic 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 ei 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 a/ 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 2006/1 1900, 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 9-11 ; 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 (2011) 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 ) supra.

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 subserialis 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.

PRE-DIGESTIVE ENZYME(S)

In one embodiment the physically and/or chemically and/or biologically treated (e.g. pre- treated) lignocellulosic biomass may be further treated (e.g. pre-treated) with at least one pre-digestive glycosyl hydrolase enzyme (herein referred to as the pre-digestive enzyme) admixed with the physically and/or chemically and/or biologically treated (e.g. pre-treated) lignocellulosic biomass prior to admixing the lignocellulosic biomass with at least one in-feed glycosyl hydrolase enzyme, said pre-digestive enzyme utilises cellulose as a substrate (e.g. for hydrolysis).

Preferably the pre-digestive enzyme is incubated with the lignocellulosic biomass.

Preferably the pre-digestive enzyme is incubated with the lignocellulosic biomass for a minimum of 3 hours, preferably at least 4 hours, preferably at least 6 hours.

Preferably the pre-digestive enzyme is incubated with the lignocellulosic biomass for between about 3 hours to about 120 hours, preferably between about 4 hours to about 90 hours, preferably about 6 hours to about 48 hours.

As one skilled in the art will appreciate the incubation time is dependent upon factors such as enzyme activity, enzyme concentration, temperature and the like. The aim of the incubation period is to allow for the enzymes to degrade the biomass. Determining the amount of degradation of the biomass can be determined by one skilled in the art, e.g. by measuring the amount of solubilised sugars (e.g. monomeric sugar units, e.g. glucose units) produced by hydrolysis.

In the present invention, the pre-digestive enzyme(s) preferably generates oligomers in the biomass. Preferably, the lignocellulosic biomass is incubated with the pre-digestive enzyme for a period of time suitable to ensure that the biomass post incubation with the predigestive enzyme contains at least 70%, suitably at least 60%, suitably at least 50%, of the treated biomass' sugar units (e.g. glucose units) present as oligomers or polymers (preferably oligomers) which can be liberated in the gastrointestinal tract of an animal by the in-feed enzyme.

In one embodiment, preferably the pre-digestive enzyme is added to the lignocellulosic biomass before drying same. Preferably the lignocellulosic biomass treated with the pre- digestive enzyme is dried prior to admixing same with at least one in-feed glycosyl hydrolase enzyme.

In one embodiment, preferably the pre-digestive enzyme is added to the lignocellulosic biomass prior to forming a physical barrier (e.g. encapsulating) the biomass. In one embodiment, the lignocellulosic biomass treated with the pre-digestive enzyme is physically separated from the at least one in-feed enzyme.

Preferably the pre-digestive enzyme in accordance with the present invention is not active in the gastrointestinal tract of the animal and/or is inactivated immediately upon entering the gastrointestinal tract of the animal (e.g. by the pH of the stomach).

Suitably, the at least pre-digestive enzyme may be a mixture of more than one enzyme, preferably more than two enzymes. In another embodiment the digestive enzyme may comprise or consist essentially of (or consist of) a carbohydrate oligomer forming enzyme.

A pre-digestive enzyme in accordance with the present invention may comprise (or consist essentially of, or consist of) any lignocellulose depolymerising activity.

In one embodiment the pre-digestive enzyme may comprise (or consist essentially of, or consist of) at least one of the following enzyme activities: cellulase activity and hemi- cellulase activity. In a preferred embodiment, the pre-digestive enzyme may comprise (or consist essentially of, or consist of) at least the following enzyme activites: cellulase activity and hemi-cellulase activity. In another embodiment the pre-digestive enzyme may comprise (or consist essentially of, or consist of) at least one of the following enzyme activities: endoglucanase activity, endoxylanase activity or β-glucosidase activity. In a preferred embodiment, the pre-digestive enzyme may comprise (or consist essentially of, or consist of) at least the following enzyme activites: endoglucanase activity, endoxylanase activity or β-glucosidase activity.

In some embodiments the pre-digestive enzyme may further comprise (or consist essentially of, or consist of) one or both of the following enzyme activities: exoglucosidase activity and lytic polysaccharide monooxygenase activity.

In one embodiment it is envisaged that the method of the present invention further comprise a step of 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.

In one aspect, the present invention relates a method 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, wherein the biophysical characteristics of an animal is improved compared to the equivalent use of lignocellulosic biomass which has not been treated in accordance with the present invention.

In some embodiments the in-feed enzyme(s) may be dosed in the range of approximately 2.5 g - 5 kg of active protein per tonne of pre-treated biomass, suitably approximately 5 g - 1 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 - 3 kg per MT of feed.

In one embodiment the amount of in-feed enzyme(s) in the feed may be between about 0.125 g to about 3 kg (suitably about 100 g to about 2.5 kg, suitably about 500 g to about 2 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. In one embodiment the amount of treated biomass in the feed is 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% w/w. In one embodiment, the amount of treated biomass in the feedstuff is more than 5% or 0%. In one embodiment the treated biomass may be at least 60% w/w of the total feedstuff.

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 at least one in-feed glycosyl hydrolase enzyme 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 physically and/or chemically and/or biologically pre-treated biomass. In another embodiment the at least one in-feed glycosyl hydrolase enzyme may be in a liquid formulation prior to admixing with the physically and/or chemically and/or biologically pre-treated biomass.

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

Preferably at the time when the at least one in-feed enzyme(s) is admixed with the physically and/or chemically and/or biologically pre-treated biomass, the biomass is a dried and/or is solid. The physically and/or chemically and/or biologically pre-treated biomass (before or after enzyme treatment) 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, a ruminant animal, monogastric animal, 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, chickens, egg layers 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 feed additive composition and/or premix and/or feed or feedstuff according to the present invention is packaged.

In one preferred embodiment the feed additive composition and/or premix and/or feed or feedstuff is packaged in a bag, such as a paper bag.

In an alternative embodiment the feed additive composition 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, wheat, rye, maize, 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, such as Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, citrus pulp, corn fibre, corn germ meal, corn bran, Hominy feed, corn gluten feed, gluten meal, wheat shorts, wheat middlings or combinations thereof; 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 Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, citrus pulp, corn fibre, corn germ meal, corn bran, Hominy feed, corn gluten feed, gluten meal, 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).

The physically and/or chemically and/or biologically treated lignocellulosic biomass and at least one in-feed glycosyl hydrolase enzyme are preferably used simultaneously (e.g. when they are in admixture together or when they are delivered by different routes). In one embodiment physically and/or chemically and/or biologically treated lignocellulosic biomass and at least one in-feed glycosyl hydrolase enzyme are applied simultaneously. Preferably the physically and/or chemically and/or biologically treated lignocellulosic biomass and at least one in-feed glycosyl hydrolase enzyme are admixed prior to being delivered to a feedstuff or to a raw ingredient of a feedstuff.

Preferably, the 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 feed additive composition is homogenized to produce a powder. In an alternative preferred embodiment, the 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 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:

Ingredients Starter (%) Finisher (%)

Maize 46.2 46.7 Wheat Middlings 6.7 10.0

Maize DDGS 7.0 7.0

Soyabean Meal 48%CP 32.8 26.2

An/Veg Fat blend 3.0 5.8

L-Lysine HCI 0.3 0.3

DL-methionine 0.3 0.3

L-threonine 0.1 0.1

Salt 0.3 0.4

Limestone 1.1 1.1

Dicalcium Phosphate 1.2 1.2

Poultry Vitamins and Micro-minerals 0.3 0.3

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

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:

By way of example only a feedstuff for turkeys may be comprises of one or more 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 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

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 feed additive composition of the present invention and other components and/or the feedstuff comprising same may be used in any suitable form. The 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)-p-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 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, hydroxypropylmethylcellu!ose (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 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 subject 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 gastrointestinal tract, e.g. the small intestine. Nutrient digestibility may be measured as the difference between what is administered to the subject and what comes out in the faeces of the subject, or between what is administered to the subject 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 gastrointestinal 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. Metabolizable 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 as subject'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 a subject, e.g. poultry or swine, comprising feeding said subject 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 also relates to the following aspects as defined in the following numbered paragraphs:

1. A method of preparing a feed additive composition comprising:

a. physically and/or chemically and/or biologically pre-treating lignocellulosic biomass;and

b. admixing the physically and/or chemically and/or biologically pre-treated

lignocellulosic biomass from step a. with at least one in-feed glycosyl hydrolase enzyme,

c. and optionally packaging.

2. A method of paragraph 1 wherein the method further comprises feeding the feed additive composition to an animal.

3. A method according to paragraph 1 or paragraph 2 wherein, when the in-feed

enzyme is added to the pre-treated biomass the pre-treated biomass contains at least 70% (suitably at least 90%) of the pre-treated biomass' sugar units (e.g. glucose units) present as oligomers or polymers which can be liberated in the gastrointestinal gut of an animal by the in-feed enzyme.

4. A method according to paragraph 3 wherein the monosaccharides are soluble

monosaccharides, such as C6-sugars, such as one or more of the group consisting of glucose, galactose, fructose or mannose.

5. A method according to any one of the preceding paragraphs wherein the in-feed

enzyme is functional, or primarily functional, in the gastrointestinal tract of the animal.

6. A method according to any one of the preceding paragraphs wherein the in-feed

enzyme is inactive (or substantially inactive) in the feed additive composition and/or on the lignocellulosic biomass prior to feeding the feed additive composition to an animal.

7. A method according to any one of the preceding paragraphs wherein the in-feed

enzyme is admixed with the physically and/or chemically and/or biologically pre- treated lignocellulosic biomass when the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass, the in-feed enzyme or both are in a dry or substantially dry state.

8. A method according to any one of the preceding paragraphs wherein the method comprising drying the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass prior to, during or after (preferably prior to) admixing the biomass with at least one in-feed glycosyl hydrolase enzyme.

A method according to paragraph 7 or paragraph 8 wherein the physically and/or chemically and/or biologically pre-treated biomass either before or after adding the at least one in-feed glycosyl hydrolase enzyme comprises less than 15 wt % moisture content.

A method according to any one of the preceding paragraphs wherein the at least one in-feed glycosyl hydrolase enzyme is encapsulated or otherwise inactivated prior to admixing with the lignocellulosic biomass.

A method according to paragraph 10 wherein the at least one in-feed glycosyl hydrolase enzyme is activated by feeding the at least one in-feed glycosyl hydrolase enzyme to an animal.

A method according to any one of the preceding paragraphs wherein the at least one in-feed glycosyl hydrolase enzyme is admixed with the physically and/or chemically and/or biologically pre-treated lignocellulosic biomass immediately prior to feeding the feed additive composition to an animal.

A method according to any one of the preceding paragraphs wherein the at least one glycosyl hydrolase in-feed enzyme(s) comprises one or more carbohydrate monomer forming activities.

A method according to any one of the preceding paragraphs wherein the at least one in-feed glycosyl hydrolase enzyme(s) is a glycan degrading enzyme.

A method according to any one of the preceding paragraphs wherein the at least one in-feed glycosyl hydrolase enzyme(s) hydrolyses a beta-1 ,4-glycosidic link.

A method according to any one of the preceding paragraphs wherein the at least one in-feed glycosyl hydrolase enzyme(s) comprises at least the following enzyme activities: cellulase activity and hemi-cellulase activity.

A method according to paragraph 16, wherein at least one in-feed glycosyl hydrolase enzyme(s) comprises at least the following enzyme activities: endoglucanase activity, endoxylanase activity and β-glucosidase activity.

A method according to paragraph 16 or paragraph 17 wherein the at least one in-feed glycosyl hydrolase enzyme(s) comprises a further enzyme comprising one or both of the following activities: exoglucosidase activity and/or lytic polysaccharide

monooxygenase activity.

A method according to any one of the preceding paragraphs wherein the

lignocellulosic biomass is any cellulosic or lignocellulosic material for example agricultural residues, bioenergy crops, industrial solid waste, municipal solid waste, sludge from paper manufacture, yard waste, wood waste, forestry waste and combinations thereof.

20. A method according to any one of the preceding paragraphs wherein the

lignocellulosic biomass is 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.

21. A method according to any one of the preceding paragraphs wherein the

lignocellulosic biomass is selected from the group consisting of corn stover, wheat straw, corn cobs, sugarcane bagasse, switch grass, forage sorghum and rice straw. 22. A method according to any one of the preceding paragraphs wherein the physical and/or chemical and/or biological pre-treatment is a treatment comprising steam explosion.

23. A method according to any one of the preceding paragraphs wherein the physical and/or chemical and/or biological pre-treatment is a treatment comprising the use of an acid or an alkaline treatment.

24. A feed additive composition obtainable (e.g. obtained) by the method of any one of paragraphs 1-23.

25. A feed additive composition or a feed ingredient comprising a physically and/or

chemically and/or biologically pre-treated lignocellulosic biomass in combination with at least one in-feed glycosyl hydrolase enzyme.

26. A feed additive kit comprising a first container comprising a physically and/or

chemically and/or biologically pre-treated lignocellulosic biomass and a second container comprising at least one in-feed glycosyl hydrolase enzyme, and instructions for co-administering same.

27. A feed additive composition or feed additive kit according to paragraph 25 or

paragraph 26 wherein the at least one in-feed glycosyl hydrolase enzyme(s) comprises one or more carbohydrate monomer forming activities.

28. A feed additive composition or feed additive kit according to any one of paragraphs 25 to 27 wherein the at least one in-feed glycosyl hydrolase enzyme(s) is a glycan degrading enzyme. 29. A feed additive composition or feed additive kit according to any one of paragraphs 25-28 wherein the at least one in-feed glycosyl hydrolase enzyme(s) hydrolyses a beta-1 ,4-glycosidic link.

30. A feed additive composition or feed additive kit according to any one of paragraphs 25-29 wherein the at least one in-feed glycosyl hydrolase enzyme(s) comprises at least the following enzyme activities: cellulase activity and hemi-cellulase activity.

31. A feed additive composition or feed additive kit according to any one of paragraphs 25-30 wherein the at least one in-feed glycosyl hydrolase enzyme(s) comprises at least the following enzyme activities: endoglucanase activity, endoxylanase activity and β-glucosidase activity.

32. A feed additive composition or feed additive kit according paragraph 30 or paragraph 31 wherein the at least one in-feed glycosyl hydrolase enzyme(s) further comprises an enzyme comprising one or both of the following activities: exoglucosidase activity and/or lytic polysaccharide monooxygenase activity.

33. A feed or feedstuff comprising a feed additive composition or feed ingredient

according to any one of paragraphs 24-32 or a feed additive composition obtainable (preferably obtained) by the method of any one of paragraphs 1-23.

34. A premix comprising a feed additive composition or feed ingredient according to any one of paragraphs 24-32 or a feed additive composition obtainable (preferably obtained) by the method of any one of paragraphs 1-23, and at least one mineral and/or at least one vitamin.

35. A method for improving a biophysical characteristic of an animal which method

comprises administering to a subject a physically and/or chemically and/or biologically pre-treated lignocellulosic biomass in combination with an in-feed glycosyl hydrolase enzyme, and optionally at least one feed component and/or at least one mineral and/or at least one vitamin.

36. A method for improving a biophysical characteristic of an animal which method

comprises administering to a subject a feed additive composition obtainable (e.g. obtained) by the method of any one of paragraphs 1-23 or a feed additive

composition according to any one of paragraphs 24-32 or a feed according to paragraph 32 or a premix according to paragraph 34.

37. A method according to paragraph 35 or paragraph 36, wherein the biophysical

characteristic is selected from the group consisting of one or more of the following: 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.

38. Use of a physically and/or chemically and/or biologically pre-treated lignocellulosic biomass in combination with an in-feed glycosyl hydrolase enzyme for improving a biophysical characteristic of an animal.

39. Use of a feed additive composition or feed ingredient according to any one of

paragraphs 24-32 or a feed additive composition obtainable (preferably obtained) by the method of any one of paragraphs 1-23 or a feed according to paragraph 33 or a premix according to paragraph 34 for improving a biophysical characteristic of an animal.

40. A use according to paragraph 38 or paragraph 39, 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.

41. A method according to any one of paragraphs 1-23, wherein the method further

comprise a step of contacting a feed component with the feed additive composition.

42. A method of preparing a feedstuff comprising contacting a feed component with a feed additive composition or feed ingredient according to any one of paragraphs 24- 32 or a feed additive composition obtainable (preferably obtained) by the method of any one of paragraphs 1-23.

43. A method, or feed additive composition, or use, or kit, or feed, or feedstuff, or premix a generally taught herein with reference to the Examples and Figures.

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

EXAMPLES

Example 1 : Effects of Accel lerase ^® BG and Accel lerase ^® Trio™ on the digestibility of the fibre found in dilute ammonia p retreated corn stover using an in-vitro model simulating upper tract digestion in chickens Materials and Methods

In vitro digestions of the simulated feed containing corn stover biomass material

Composition of the in vitro digestion treatments is shown in Table 1. To mimic chicken feed, 70% of the biomass material was mixed with 30% soybean meal (SBM). For biomass samples, two types of upper gastrointestinal (GIT) digestions were done. First, samples were prepared, that were not treated either with Accellerase ^® Trio™ or Accellerase ^® BG (samples referred as 'None'). Secondly, samples that were treated with Accellerase ^® Trio™ and Accellerase ^® BG during the uGIT in vitro digestions (samples referred as Ίη-feed samples') were prepared. Furthermore, two control samples of pure corn and SBM based feed without biomass material were prepared: either digested with Accellerase ^® Trio™and Accellerase ^® BG referred as 'Control In-feed' or digested without Accellerase ^® Trio™ and Accellerase ^® BG referred as 'Control'. Accellerase ^® Trio™ is a commercial enzyme available from Danisco (now part of DuPont) and contains certain cellulase activities such as endoglucanase activity and beta-glucosidase activity and certain hemi-cellulases activity such as endoxylanase activity.

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

Table 1. Composition of the in-vitro digestion treatments.

ccellerase Trio : Combination of cellulase activities including endoglucanase activity and beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity ² Accellerase ^® BG: beta-glucosidase

The chicken upper GIT in vitro digestion was conducted using the procedure depicted in Figure 1. A total of one, two or three grams of dry matter (DM) was weighed per simulation unit, depending on the target dry matter content (DM% End) during small intestinal digestion step. After mixing the feed with water and adjusting the pH with HCI, feed enzymes, Accellerase ^® Trio™ and Accellerase ^® BG were dosed at 0.2 imL/1 g DM and 0.05 mL/1 g DM, respectively. In the end of in vitro digestion, liquid phase was separated by centrifugation at 30 000 g for 30 minutes (10°C), and stored at -20°C until analysed for soluble carbohydrate composition.

The solid residues were oven-dried at 75°C for 24 to 48 hours depending on the amount of DM in the simulation unit. The weighed dry pellets (indigestible dry matter) were homogenised by crushing for the analysis of insoluble non-starch polysaccharide (NSP) sugars, neutral detergent fibre (NDF) and acid detergent fibre (ADF).

Assessment of Fiber Digestion and Release of Soluble Sugars

Determination of soluble sugars after in vitro digestion

The centrifuged supernatant samples were purified on a C18 solid phase extraction column and the free monosaccharides were analysed with HPLC as described below. Total soluble sugars were determined after acid hydrolysis of the supernatant in 1 M H ₂ S0 ₄ in an oven at 100°C for three hours. After dilution with water and filtration, the monosaccharides were analysed by HPLC as described below. The amount of soluble polymeric sugars (oligo- and polysaccharides) in the sample solution was calculated as follows: oligo- and polysaccharides = total soluble sugars - free soluble monosaccharides

Determination of insoluble NSP (non starch polysaccharide) sugars after in vitro digestion

An aliquot of the dry pellet was dispersed in 0.05 M phosphate buffer solution (pH 6) and starch was degraded with heat stable alpha-amylase (Sigma-Aldrich) by incubating the sample in a water bath at 95-100°C for 30 min. Next, the pH was adjusted to 4.5, amyloglucosidase added, and the sample was incubated in a water bath at 60°C for 60 min. Then ethanol was added and the sample centrifuged. The supernatant was discarded and the pellet dried in an oven at 50°C.

To hydrolyse the polymeric sugars, the dried residue was dispersed in 12 M H ₂ S0 ₄ and incubated in a water bath at 35°C for 60 min. Then, water was added to dilute the H ₂ S0 ₄ from 12 M to concentration of 1 M and the sample was incubated in an oven at 100°C for two hours. The sample was diluted with water, filtrated and the monosaccharides were determined by HPLC as described below. Determination of monosaccharides by HPLC

The monosaccharides (rhamnose, arabinose, galactose, glucose, xylose, mannose) were separated and detected using high pH anion exchange chromatography with pulsed electrochemical detection. The pre-column was CarboPac PA1 (4 x 40 mm) and the analytical column CarboPac PA1 (4 x 250 mm). The flow rate was 1 mL/min and the mobile phase consisted of A: water and B: 0.2 M NaOH as following gradient:

Time (min) A (%) B (%)

0 90 10

1.1 90 10

5 100 0

29 100 0

30 0 100

40 0 100

41 90 107

58 90 10

Analysis of neutral detergent (NDF) and acid detergent (ADF) fibre after in vitro digestion

For the feed and in vitro digestion residues, dry matter values were determined by oven- drying at 105°C for 18 hours. The applied gravimetric NDF and ADF methods were performed by MTT Agrifood Research Finland, using Fibertec TM System M (FOSS

Analytical, Denmark) extraction system. For determining the amount of NDF, extraction with the neutral solution of sodium lauryl sulfate and EDTA (Van Soest et al., 1991 ) was applied.

Acid detergent fibre (cellulose, lignin) was recovered by extraction with 1 N sulphuric acid containing CTAB (cetyl trimethylammonium bromide) (AOAC 973.18, 1990). After over-night drying at 100°C, the remaining of the sample was weighed and the amount of fibre calculated as percent of dry matter content.

Statistical analyses

The statistical analyses were carried out using One-way ANOVA (Graphpad Prism version 6.01) with Turkey's post-hoc test. The P-value P<0.05 was considered statistically significant. Mean + SD is shown for all the figures. RESULTS

Digestion of fibre and release of soluble sugars from biomass materials

For studying the effect of physicochemical pretreatment alone (none) or in combination with in feed enzymes (In-feed Accellerase ^® Trio™ & Accellerase®BG) on hydrolysis of carbohydrates during simulated upper GIT digestion, several analyses were performed. The results for indigestible dry matter residue, released soluble sugars, insoluble NSP sugar composition, and the amount of NDF and ADF are shown for the control feed and corn stover treatment. The data for control diet (com 70% - 30% SBM) is shown in Tables 2-4 and for the dilute ammonia pretreated corn stover containing treatments (CS 70% - SBM 30%) in Tables 5-7.

Control diet (corn 70% - SBM 30%)

There was no difference in the amount of indigestible residue (%) between Control and Control In-feed Accellerase®Trio™ & Accellerase ^® BG after in vitro digestions (Table 2).

Table 2. Indigestible dry matter residue of Control diet (corn 70% - SBM 30%)

after in vitro digestions (n=4) with 3 g (DM) of test material.

Treatment Indigestible residue

(%) SD

Control 77 1 .3

Control In-feed Accellerase®Trio™ &

76 2.1

Accellerase®BG ²

1 -_TM.

beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity ² Accellerase®BG: β-glucosidase

The amount of total soluble sugars, free monosaccharides, soluble oligo- and polymeric sugars, and insoluble NSP sugars after in vitro digestions of Control treatments, as well as approximate total NSP sugar composition before digestions, are presented in Table 3.

Table 3. Control diet (corn 70% - SBM 30%): Sugar analysis after in vitro digestions with 3 g

(DM) of test material and different treatments. Approximate NSP sugar composition before the digestions.

Glucose Xylose Arabinose Galactose Mannose

Total soluble sugars mg/g SD mg/g SD mg/g SD mg/g SD mg/g SD after digestions

Control 115 ^B 14 0 ^b 0 1.2° 0 15 1.6 1.2° 0 Control In-feed 138 ^a 8.8 2.3 ^a 0.2 3.2 ^a 0.4 16 ^a 0.5 6.4 ^a 0.8 Accellerase ^® Trio™ ¹ &

Accellerase ^® BG ²

Free soluble

monosaccharides

after digestions

Control 12° 3 0 0 0 ^b 0 0.2 ^D 0.1 0 ^b 0

Control In-feed 35 ^a 8.9 0 0 0.6 ^a 0.2 0.4 ^a 0.1 0.2 ^a 0.2 Accellerase ^® Trio™ &

Accellerase ^® BG

Soluble oligo- and

polymeric sugars

after digestions

Control 104 13 0 ^b 0 1.2 ^β 0.1 15 1.4 1.2° 0.2

Control In-feed 102 15 2.3 ^a 0.2 2.7 ^a 0.2 16 0.4 6.2 ^a 0.8 Accellerase ^® Trio™ &

Accellerase ^® BG

Insoluble NSP

sugars after

digestions

Control 42 ^a 4 19 3 15 2 12 2 13 1

Control In-feed 34° 3 17 2 15 1 12 1 12 1 Accellerase ^® Trio™ &

Accellerase ^® BG

Approx. total NSP

sugars before

digestions

Control 46 18 19 16 8

Control In-feed 46 18 19 16 8

Accellerase ^® Trio™ &

Accellerase ^® BG

Accellerase'nYio Combination o ^: cellulase ac tivities including endoglucanase ac tivity and beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity ² Accellerase®BG: β-glucosidase

a Mean values within a column with unlike superscript letters were significantly different (P<0.05; ANOVA followed by Turkey's post hoc test).

Table 4. Amount of neutral detergent and acid detergent fibre (% of dry matter) after in vitro digestions with Control diet. NDF ADF

Treatment % DM SD % DM SD

Control 22.46 3.60 5.81 0.70

Control In-feed Accellerase ^® Trio™ ¹ &

Accellerase®BG ² 22.50 3.23 6.04 0.62 beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity ² Accellerase®BG: β-glucosidase

Table 5. Indigestible dry matter residue of Corn stover - SBM (70%-30%)

after in vitro digestions (n=4) with 3 g (DM) of test material and different

treatments.

Treatment Indigestible residue

(%) SD

CS None 87 08

CS In-feed Accellerase®Trio™ ¹ &

n „ 72 ^a 1.6

Accellerase®BG ²

^T Accellerase ^® Trio™: Combination of cellulase activities including endoglucanase activity and beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity ² Accellerase®BG: β-glucosidase

a Mean values within a column with unlike superscript letters were significantly different (P<0.05; ANOVA followed by Turkey's post hoc test).

Table 6. Corn stover - SBM (70%-30%): Sugar analysis after in vitro digestions with 3 g (DM) of test material and different treatments. Approximate NSP sugar composition before the digestions.

Glucose Xylose Arabinose Galactose Mannose

Total soluble mg/g SD mg/g SD mg/g SD mg/g SD mg/g SD sugars after

digestions

CS None 22 ^b 4.2 14 ^b 2.2 7 ^b 1.3 16 ^aD 2.7 1.5 ^b 0.2

CS In-feed 87 ^a 1.8 69 ^a 1.2 16 ^a 0.5 19 ^a 0.5 6.0 ^a 0.2 Accellerase ^® Trio™ ¹

& Accellerase ^® BG ²

Free soluble

monosaccharides

after digestions

CS None 0.9 ^D 0.4 0 ^b 0 0 ^b 0 0 0 0 0 CS In-feed 60 ^a 1.4 44 ^a 1.2 5.6 ^a 0.2 0 0 0 0 Accellerase ^® Trio™ &

Accellerase ^® BG

Soluble oligo- and

polymeric sugars

after digest.

₇ a

CS None 21 4 14 ^b 2.2 1.2 16 2.8 1.5 ^b 0.2

CS In-feed 27 2.4 25 ^a 2.2 1 1 ° 0.4 19 0.7 6.0 ^a 0.2 Accellerase ^® Trio™ &

Accellerase ^® BG

Insoluble NSP

sugars after

digestions

CS None 239 ^a 7.9 127 ^a 3.1 22 ^a 0.5 15 ^a 0.7 12 ^a 1.4

CS In-feed 171° 9.6 67° 3.8 11 ° 0.5 12° 0.9 12 ^a 1 Accellerase ^® Trio™ &

Accellerase ^® BG

Approx. total NSP

sugars before

digestions

CS None 229 127 27 19 13

CS In-feed 229 127 27 19 13

Accellerase ^® Trio™ &

Accellerase ^® BG

¹ Accellerase ^® Trio ^{, M} : Combination of cellulase activities including endoglucanase activity and beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity ² Accellerase®BG; β-glucosidase

a ^b Mean values within a column with unlike superscript letters were significantly different (P<0.05; ANOVA followed by Turkey's post hoc test).

Table 7. Amount of neutral detergent and acid detergent fibre (% of dry matter) after digestions with com stover containing treatments.

NDF ADF

Treatment % DM SD % DM SD

CS None 49.78 ^a 1 .18 39.88 ^a 1 .08

CS In-feed Accellerase®Trio™ ¹

& Accellerase®BG ² 40.55 ^b 1.66 38.90 ^a 1 .31 ¹ Accellerase ^® Trio™: Combination of cellulase activities including endoglucanase activity and beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity ^z Accellerase ^® BG: β-glucosidase

a ^b Mean values within a column with unlike superscript letters were significantly different (PO.05).

DISCUSSION

Assessment of fibre digestibility

Due to addition of in-feed enzymes (In-feed Accellerase ^® Trio™ & Accellerase ^® BG) on Control diet containing 70% corn, there was no significant difference in fibre digestion parameters of indigestible DM residue, NDF or ADF. In contrast, the amount of insoluble NSP glucose was significantly lower (-19%), being consistent with detected higher release of free glucose with Ίη-feed Accellerase ^® Trio™ & Accellerase ^® BG'. With xylose, arabinose, and mannose containing CHO structures, mainly solubilisation of oligo- and polymeric sugars during in vitro digestion was observed.

Addition of in-feed enzymes (In-feed Accellerase ^® Trio™ & Accellerase ^® BG) to dilute ammonia pretreated corn stovers resulted in a significant (P<0.05) increase in the solubilisation of glucose, xylose, arabinose and mannose compared to dilute ammonia pretreatment of corn stovers alone (CS None). In addition, the majority of the soluble sugar release as a result of in-feed enzymes (In-feed Accellerase ^® Trio™ & Accellerase ^® BG) was detected as monosaccharides with the exception of mannose where the release of polymeric mannose accounted for the increase in mannose solubilisation. Residual amount of neutral detergent fibre was lower also with Ίη-feed Accellerase ^® Trio™ & Accellerase ^® BG' compared to OS None' however there was no change in acid detergent fibre between treatments. Consistent with the NDF results, the amount of indigestible DM residue and insoluble NSP sugars was decreased by the addition of in-feed enzymes (In-feed Accel lerase ^® Trio™ & Accellerase ^® BG). CONCLUSION

In-vitro digestion of dilute ammonia pretreated corn stover resulted in 10 and 11 % conversion of the total NSP sugars fraction to soluble glucose and xylose, respectively. However, the addition of Accellerase ^® Trio™and Accellerase ^® BG to pretreated corn stovers during in-vitro digestion further increased glucose and xylose conversion to 38 and 54% of the total NSP sugar fraction. A corresponding decrease in the NDF fraction while the ADF fraction remained the same indicates that in-feed enzyme addition to dilute ammonia pretreated corn stovers solublised the hemicellulose fraction of the total NSPs. Example 2: Effects of endoxylanase and Accellerase ^® Trio™ on the total tract digestibility of nutrients in pigs fed diets containing 30% dilute ammonia pretreated corn stover as a partial replacement for corn

Materials and Methods

The use of animals and experimental protocol is approved by the Animal Experiment Committee. The basal diet, as fed, is formulated to be balanced for energy and protein, and to exceed the nutrient requirements for growing pigs of this age by 15% (Table 1 ). A common digestibility marker (chromic oxide) is included at 6 g/kg to allow determination of digestibility of dietary components. Low levels of a synthetic antimicrobial (Virginamycin) is included in the diet to balance for any traces of antimicrobials that may be present in ammonia pretreated corn stover and the diet is supplied as a mash. For treatments 2 and 3, 30% of the basal diet is replaced with ground corn stovers or dilute ammonia pretreated corn stovers (on a dry matter basis) according to the treatments detailed in Table 2.

The basal diet is divided into portions which are then treated with the enzymes identified in Table 2. During feed mixing, the mixer is flushed to prevent cross contamination of diet. Samples are collected from each treatment diet from the beginning, middle, and end of each batch and blended together to confirm enzyme activities in feed. Samples from each treatment diet are retained during mixing and stored at -20°C until required.

Table 1 : Examples of basal diet composition for pigs 25 to 60 kg body weight (%, as

Table 2: Experimental diets identification

beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity The experiment is planned and conducted to correspond to growing phase (<25 to ~60 kg body weight).

Growing barrows (initial body weight of 30 kg) are housed in individual pens (1.2 χ 1.5 m) in an environmentally controlled room. Each pen was equipped with a feeder and a nipple drinker and had fully slatted concrete floors. The experiment is designed and conducted to give a minimum of 12 replicates per treatment. All pigs are fed at a level of 3 times their maintenance energy requirement (197 kcal ME per kg MW ⁶⁰ ; NRC, 2012), and provided at two equal portions at 0800 and 1700 h. Animals are allowed free access to water through a bowl-type drinker. Pig weights are recorded at the beginning and at the end of each period and the amount of feed supplied each day are recorded. Experimental period lasts for 14 d. The initial 10 days of each period are considered an adaptation period to the diet. Fresh grab fecal samples are collected on d 1 1 and 12 using standard operating procedures. Following collection fecal samples are immediately frozen at -20°C for subsequent analysis Fecal samples are thawed, mixed within animal and diet, and a sub-sample collected for chemical analysis. A sample of basal diet is also collected and analyzed. Fecal samples are dried in an oven and finely ground for analysis. All samples are analyzed for dry matter, digestibility marker, gross energy, crude protein, fat and neutral detergent fibre according to standard procedures (AOAC, 2005).

The values for total digestibility of energy and nutrients are calculated as described previously (Stein et al., 2007. J. Anim. Sci, 85: 172-180). The pen is the experimental unit. Data are subjected the MIXED procedures of SAS. Significance is claimed at P<0.05. RESULTS

The apparent total tract digestibility (ATTD) of dry matter, organic matter, crude protein, gross energy and fiber in pigs fed the control corn soybean diet is higher than all other treatments as expected as this diet does not contain any high fiber biomass material (Table 3: P < 0.01). Treated groups of pigs fed the whole combination of endoxylanase plus Accellerase ^® Trio™ together with 30% dilute ammonia pretreated corn stovers have higher apparent total tract digestibility of dry matter, organic matter, crude protein, gross energy and fiber compared to pigs fed diets containing 30% ground corn stover that has not undergone any pretreatment process or the addition of in-feed enzymes.

Table 3. Apparent total digestibility (ATTD) of energy and nutrients in growing pigs from Trial BMN1204 fed high fibre diets with or without exogenous enzymes.

Diets

Ground DAcs ¹ +

Control

corn Enzymes ²

Item, % SEM P-value

stover

Dry matter 87.53 ^a 72.04° 77.68 0.65 <0.01

Organic matter 89.36 ^a 73.84° 79.47 ^b 0.64 <0.01

Crude protein 87.58 ^a 75.79° 78.74 0.93 <0.01

Gross energy 86.64 ^a 72.36° 76.47 0.74 <0.01

Crude fiber 68.93 ^a 48.04° 57.64 ^b 2.14 <0.01

ADF 61.16 ^a 43.12° 51.60 2.82 <0.01

NDF 60.08 ^a 1.69° 33.25 ^b 2.31 <0.01

DAcs - Dilute ammonia pretreated corn stovers

2Enzymes - endoxylanase included at 4400 U/kg and Accellerase®Trio™ - a cellulase, hemicellulase enzyme combination developed by DuPont included at 12,500 BGU/kg ^ab °Within a row, means with different superscripts are significantly different (P < 0.05)

DISCUSSION

Generally, physico-chemical pre-treatment technologies maximise the activity of the enzymes by reducing the crystallinity of the cellulose and maximising the cellulase accessibility to cellulose (CAC). Dilute ammonia pretreatment successfully opens up the cellulose structure thus increasing the surface area for enzymes to function. This was very evident in this study, where the dry matter and nutrient digestibility of corn stover was improved with the combination of dilute ammonia pretreatment and in-feed enzymes compared to ground corn stover alone when evaluated in pigs.

Example 3: Effects of endoxylanase and Accellerase ^® Trio™ on the true metabolisable energy of physicochemically pretreated biomass materials when fed to conventional roosters using a precision-fed model

The use of animals and experimental protocol is approved by the Animal Experiment Committee. Treatments include biomass materials fed with or without the enzymes (outlined in Table 4). Roosters are housed in individual wire mesh cages fitted with excreta collection trays. Feed is withheld 24 hours before intubation (30 g) of each treatment and for 48 hours after intubation of each treatment. The experiment is designed and conducted to give a minimum of 8 replicates per treatment. Total excreta is collected for 48 hours, weighed, freeze dried and ground with mortar and pestle. The excreta is analysed for gross energy and nitrogen. Excreta from fasted roosters is used to calculate endogenous nitrogen losses. The nitrogen value attributed to endogenous losses is used as a correction factor allowing for the calculation of nitrogen corrected true metabolisable energy (T E _n ).

Table 4: Experimental diets identification

¹ Accellerase ^® Trio : Combination of cellulase activities including endoglucanase activity and beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity RESULTS

Results suggest that treated groups fed physicochemically pretreated biomass materials in combination with enzymes (endoxylanase plus Accellerase ^® Trio™) have higher digestibility of energy (T E _n ) than roosters fed physicochemically pretreated biomass or untreated biomass without enzymes (milled corn stovers - treatment 1).

Table 5. True metabolism energy values for pretreated biomass materials with or with exogenous enzymes from trial conducted using a rooster model ¹ .

Biomass Material TME _n (kcal/kg DM) ²

Milled corn stover 980

DLcs ³ 917

DLcs + enzymes ⁴ 1056

AII test materials were fed as a mixture of 30% test material and 70% corn to facilitate ease of feeding. The GE value of corn was used to factor out its energy contribution in the mixture. 2TME _n means are an average of 8 observations

3DLcs - caustic delignified corn stover

Enzymes - Endoxylanase was included at 2500 U/kg and Accellerase®Trio™ was included at 12,500 BGU/kg Example 4: Effects of Danisco Xylanase™ and Accellerase ^® Trio™ on the digestibility of the fibre found in physicochemically pretreated biomass materials using an in-vitro ruminant digestibility model Materials & Methods

The fermentation of test ingredients alone (Table 1) or as part of a complete ruminant diet (Table 2) with or without the addition of enzymes is carried out using a novel, wireless, automated system for measuring fermentation gas production kinetics of feed ingredient described previously by Adesogan et al. (2005). The experiments are designed and conducted to give 5 replicates per treatment and the incubation lasts 24 hours. Following fermentation, the quantity of total gases produced is quantified. The residuals remaining following fermentation are characterised according to their acid detergent fibre (ADF) andneutral detergent fibre (NDF) composition.

Table 1. Treatments for experiment 1

Accellerase Trio : Combination of cellulase activities including endoglucanase and beta- glucosidase activities, and hemi-cellulase activities including endoxylanase activity and Danisco xylanase™ which contains endoxylanase activity.

Table 2. Treatments for experiment 2

Accellerase Trio : Combination of cellulase activities including endoglucanase and beta- glucosidase activities, and hemi-cellulase activities including endoxylanase activity and Danisco xylanase™ which contains endoxylanase activity.

RESULTS Results from experiment 1 evaluating biomass materials alone indicate that the addition of enzymes (Accel lerase^rio™ and Danisco Xylanase™) to dilute ammonia pretreated corn stovers and caustic delignified corn stover and switchgrass results in higher dry and organic matter digestibility along with increased fiber digestibility as measured by acid and neutral detergent fiber digestibility (Table 3). Gas production from the pretreated biomass materials was also enhanced by the addition of in-feed enzymes.

Table 3. In vitro apparent digestibility of dry matter (DMD), organic matter (OMD), acid

(ADFD) and neutral (NDFD) detergent fiber of pretreated biomass materials.

Treatment DMD (%) OMD (%) NDFD (%) ADFD Gas

(%) production

(ml)

Corn stover (untreated - 19.8 20.4 0.7 -4.3 262

CS)

Dilute ammonia 35.7 35.8 19.7 14.4 422

and Danisco xylanase™ which contains endoxylanase activity.

Results from experiment 2 where pretreated biomass constitutes 60% of a complete ruminant diet, dry matter digestibility along with fiber digestibility as assessed by neutral and acid detergent fiber digestibility are increased when in-feed enzymes are added. Likewise, gas production is higher when pretreated biomass included as part of a complete ruminant diet is combined with in-feed enzymes compared pretreated biomass alone.

Table 4. In vitro apparent digestibility of dry matter (DMD) and acid (ADFD) and neutral (NDFD) detergent fiber and fermentation gas production from the experimental diets ¹ .

Treatment DMD NDFD ADFD Gas production,

(%) (%) (%) psi_

Basal diet 59.3 32.7 32.7 549

Basal + 70% DAcs 45.9 25.9 19.9 372

Basal + 70% DAcs + Enzymes ¹ 55.6 38.6 39.8 448

Basal + 70% DLcs 51.7 48.2 43.5 479

Basal + 70% DLcs + Enzymes 51.9 50.3 43.7 485 Basal + 70% DLswg 41.6 39.7 38.5 156

Basal + 70% DLswg + Enzymes 44.1 44.2 41.2 500

¹ Enzymes: Accellerase ^® Trio™: Combination of cellulase activities including endoglucanase and beta-glucosidase activities, and hemi-cellulase activities including endoxylanase activity and Danisco xylanase™ which contains endoxylanase activity.

DAcs - dilute ammonia pretreated com stover; DLcs - caustic delignified corn stover; DLswg - caustic delignified switchgrass

DISCUSSION

As was observed in experiment 1 , physico-chemical pretreatment alone increases the fiber digestibility and gas production from biomass material however; these responses are enhanced further when in-feed enzymes in the form of Accellerase ^® Trio™ and Danisco xylanase™ are included in the digestion.

Example 5: Effects of Accellerase 1500 and Econase XT on the digestibility of the fibre found in dilute ammonia pretreated corn stover using an in-vitro model simulating upper tract digestion in chickens

Materials and Methods

In vitro digestions of the simulated feed containing corn stover biomass material

Composition of the in vitro digestion treatments is shown in Table 1. To mimic chicken feed, 70% of the biomass material is mixed with 30% soybean meal (SBM). For biomass samples, two types of upper gastrointestinal (GIT) digestions are conducted. First, samples are prepared, that are not treated either with Accellerase ^® 1500 or Econase ^® XT (samples referred as 'None'). Secondly, samples that are treated with Accellerase ^® 1500 and Econase ^® XT during the uGIT in vitro digestions (samples referred as Ίη-feed samples') are prepared. Furthermore, two control samples of pure corn and SBM based feed without biomass material are prepared: either digested with Accellerase ^® 1500 and Econase ^® XT referred as 'Control In-feed' or digested without Accellerase ^® 1500 and Econase ^® XT referred as 'Control'. 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 is a commercial enzyme available from AB Vista and has endo1-4 Beta xylanase activity. Table 1. Composition of the in-vitro digestion treatments.

Accellerase 1500: Combination of cellulase activities including endoglucanase activity, cellobiohydrolase activities, beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity

2 Econase ^® XT: endoxylanase

The chicken upper GIT in vitro digestion is done with the procedure depicted in Figure 1. A total of one, two or three grams of dry matter (DM) is weighed per simulation unit, depending on the target dry matter content (DM% End) during small intestinal digestion step. After mixing the feed with water and adjusting the pH with HCI, feed enzymes, Accellerase ^® 1500 and Econase ^® XT are dosed at 0.3 mL/1 g DM and 4.6 μΙ/g DM, respectively. In the end of in vitro digestion, liquid phase is separated by centrifugation at 30 000 xg for 30 minutes (10°C), and stored at -20°C until analysed for soluble carbohydrate composition.

Release of soluble sugars, determination of insoluble non-starch polysaccharide sugars, analysis of neutral and acid detergent fibre, following in-vitro digestion are assessed as described by the methods in example 1.

RESULTS

Digestion of fibre and release of soluble sugars from biomass materials

Preliminary results indicate that the addition of Accellerase ^® 1500 & Econase ^® XT to dilute ammonia pretreated corn stovers increases the release of soluble sugars and decreases the amount of residual neutral detergent fibre, indigestible DM residue and insoluble NSP sugars compared to dilute ammonia pretreated corn stovers alone. Example 6: Effects of Celluclast®, Danisco Xylanase™ and Accel lerase®BG™ on the digestibility of the fibre found in dilute ammonia pretreated corn stover using an in- vitro model simulating upper tract digestion in chickens

Materials and Methods

In vitro digestions of the simulated feed containing corn stover biomass material Composition of the in vitro digestion treatments is shown in Table 1. To mimic chicken feed, 70% of the biomass material is mixed with 30% soybean meal (SBM). For biomass samples, two types of upper gastrointestinal (GIT) digestions are done. First, samples are prepared, that are not treated either with Celluclast®, Danisco Xylanase™ and Accellerase®BG™ (samples referred as 'None'). Secondly, samples that are treated with Celluclast®, Danisco Xylanase™ and Accellerase®BG™ during the uGIT in vitro digestions (samples referred as Ίη-feed samples') are prepared. Furthermore, two control samples of pure corn and SBM based feed without biomass material are prepared: either digested with Celluclast®, Danisco Xylanase™ and Accellerase®BG™ referred as 'Control In-feed' or digested without Celluclast®, Danisco Xylanase™ and Accellerase®BG™ referred as 'Control'.

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

Danisco Xylanase™ is a commercial enzyme available from Danisco (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.

Table 1. Composition of the in-vitro digestion treatments.

Celluclast ^® : Combination of cellulase activities including endoglucanase activity, cellobiohydrolase activities and beta-glucosidase activity.

² Danisco Xylanase™: endoxylanase

³ Accellerase®BG™ : beta-glucosidase The chicken upper GIT in vitro digestion is conducted using the procedure depicted in Figure 1. A total of one, two or three grams of dry matter (DM) is weighed per simulation unit, depending on the target dry matter content (DM% End) during small intestinal digestion step. After mixing the feed with water and adjusting the pH with HCI, feed enzymes, Celluclast ^® , Danisco Xylanase™ and Accellerase®BG™ are dosed at .0.1 mL/g DM, 2.3 μΙ/g DM (40,000 U/g), and 0.15 mL/g DM, respectively. In the end of in vitro digestion, liquid phase is separated by centrifugation at 30 000 xg for 30 minutes (10°C), and stored at -20°C prior to soluble carbohydrate composition analysis.

Release of soluble sugars, determination of insoluble non-starch polysaccharide sugars, analysis of neutral and acid detergent fibre, following in-vitro digestion are assessed as described by the methods in example 1.

RESULTS

Digestion of fibre and release of soluble sugars from biomass materials

Preliminary results indicate that the addition of Celluclast ^® , Danisco Xylanase™ and Accellerase®BG™ to dilute ammonia pretreated corn stovers increases the release of soluble sugars and decreases the amount of residual neutral detergent fibre, indigestible DM residue and insoluble NSP sugars compared to dilute ammonia pretreated corn stovers alone.

Example 7: Effects of Cellulase from Aspergillus niger (C1184 - Sigma) and Accellerase®BG™ on the digestibility of the fibre found in dilute ammonia pretreated corn stover using an in-vitro model simulating upper tract digestion in chickens Materials and Methods

In vitro digestions of the simulated feed containing corn stover biomass material

Composition of the in vitro digestion treatments is shown in Table 1. To mimic chicken feed, 70% of the biomass material is mixed with 30% soybean meal (SBM). For biomass samples, two types of upper gastrointestinal (GIT) digestions is done. First, samples are prepared, that are not treated either with Cellulase or Accellerase®BG™ (samples referred as 'None'). Secondly, samples that are treated with Cellulase or Accellerase®BG™ during the uGIT in vitro digestions (samples referred as Ίη-feed samples') are prepared. Furthermore, two control samples of pure corn and SBM based feed without biomass material are prepared: either digested with Cellulase or Accel lerase®BG™ referred as 'Control In-feed' or digested without Cellulase or Accellerase®BG™ referred as 'Control'.

Cellulase from Aspergillus niger is a commercial enzyme available from Sigma-Aldrich (C1184) and contains certain cellulase activities including 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. Table 1. Composition of the in-vitro digestion treatments.

Cellulase from Aspergillus niger: Combination of cellulase activities including endoglucanase activity and endoxylanase activity.

2 Accellerase®BG™ : beta-glucosidase

The chicken upper GIT in vitro digestion is done with the procedure depicted in Figure 1. A total of one, two or three grams of dry matter (DM) is weighed per simulation unit, depending on the target dry matter content (DM% End) during small intestinal digestion step. After mixing the feed with water and adjusting the pH with HCI, feed enzymes, Cellulase (C1 184) or Accellerase®BG™ were dosed at 0.2 ml_/1 g DM and 0.15 mL/1 g DM, respectively. In the end of in vitro digestion, liquid phase is separated by centrifugation at 30 000 xg for 30 minutes (10°C), and stored at -20°C prior to soluble carbohydrate composition analysis.

Release of soluble sugars, determination of insoluble non-starch polysaccharide sugars, analysis of neutral and acid detergent fibre, following in-vitro digestion are assessed as described by the methods in example 1.

RESULTS

Digestion of fibre and release of soluble sugars from biomass materials

Preliminary results indicate that the addition of Cellulase (from Aspergillus niger) and Accellerase®BG™ to dilute ammonia pretreated corn stovers increases the release of soluble sugars and decreases the amount of residual neutral detergent fibre, indigestible DM residue and insoluble NSP sugars compared to dilute ammonia pretreated corn stovers alone.

Example 8: Effects of Accellerase ^® 1500 and Econase ^® XT on the digestibility of the fibre found in physico-chemically pretreated biomass material using an in-vitro model simulating upper tract digestion in pigs Materials and Methods In vitro digestions of the simulated feed containing physico-chemically pretreated biomass material

Composition of the in vitro digestion treatments is shown in Table 1. Three types of physicochemically pretreated biomass (DAcs: dilute ammonia pretreated corn stover, DLcs: caustic delignified corn stover, DLswg: caustic delignified switchgrass) and corn are digested under simulated conditions of the upper digestive tract of a pig in the presence or absence of carbohydrase enzymes Accellerase ^® 1500 and Econase ^® XT.

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 is a commercial enzyme available from AB Vista and has endo1 -4 Beta xylanase activity. Table 1. Composition of the in-vitro digestion treatments.

Accellerase 1500: Comb nation of cellulase activities including endoglucanase activity, cellobiohydrolase activities, beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity

2 Econase ^® XT: endoxylanase

3 DAcs - dilute ammonia pretreated corn stover

⁴ DLswg - caustic delignified switchgrass

⁵ DLcs - caustic delignified corn stover To assess the effect of different enzymes on the fermentation characteristics of feed ingredients in the upper part of the digestive system, gastric and small intestinal conditions are simulated in batch tests. Gastric juice (28 mL) containing mucin and pepsin (pH 3.5) are added to 3 g (DM) of test material and enzymes, and incubated for 2 h at 39°C in a shaking incubator set at 90 rpm. Accellerase ^® 1500 and Econase ^® XT 5P are dosed at 0.3 mL/g DM and 1.4 mg/g DM, respectively. During stomach incubation, pH is measured at regular time intervals and adjusted to pH 3.5. After stomach incubation, 12 mL of pancreatic juice is added, containing oxgall (6 g/L), pancreatin (6 g/L), sodium bicarbonate (12.5 g/L) and phosphate buffer (pH 6.8) and the digest is incubated for 6 h at 39°C while shaking. During the small intestine incubation, the pH is measured regularly and adjusted to pH 6.8 if needed. Soluble sugar release (Total: glucose, xylose, arabinose, galactose, mannose) are measured by means of HPAE-PAD (High Performance Anion Exchange Chromatography- Pulsed Amperometric Detection) (Dionex Technical Note 20). Determination of soluble and insoluble non-starch polysaccharides are assessed as described by Englyst et al. (1992).

RESULTS

Digestion of NSPs and release of soluble sugars from physico-chemically pretreated biomass materials

Results indicate that the addition of Accellerase ^® 1500 & Econase ^® XT to physicochemically pretreated biomass (dilute ammonia pretreated corn stovers, caustic deiignified corn stover and switchgrass) increases the release of total soluble arabinose, galactose, glucose, xylose and mannose compared to physicochemical pretreatment alone (Table 2),

Table 2. Soluble sugar analysis after in vitro digestions under simulated conditions of the upper digestive tract of a pig with 3 g (DM) of t material with or without in-feed Accellerase ^® ! 500 & Econase ^® XT.

ND - below the limit of detection

¹ Accellerase ^® 1500: Combination of cellulase activities including endoglucanase activity, cellobiohydrolase activities, beta-glucosidase activity, a hemi-cellulase activities including endoxylanase activity

² Econase ^® XT: endoxylanase

³ DAcs - dilute ammonia pretreated corn stover

⁴ DLcs - caustic delignified corn stover

⁵ DLswg - caustic delignified switchgrass

Following analysis of NSP sugars after in-vitro digestion under simulated conditions of the upper digestive tract of a pig, the addition of in-feed Accellerase®1500 and Econase® XT was found to decrease the release of total NSP sugars from dilute ammonia pretreated and caustic delignified corn stover and caustic delignified switch grass compared to physicochemical pretreatment alone (Table 3). The majority of this reduction was comprised of reduced release of insoluble glucose, xylose and arabinose. Table 3. Non-starch polysaccharide (NSP) sugar analysis after in vitro digestions under simulated conditions of the upper digestive tract of a pig with 3 g (DM) of test material with or without in-feed Accellerase 1500 and Econase XT.

cellobiohydrolase activities, beta-glucosidase activity, and hemi-cellulase activities including endoxylanase activity

² Econase ^® XT: endoxylanase

³ DAcs - dilute ammonia pretreated corn stover

DLcs - caustic delignified corn stover

⁵ DLswg - caustic delignified switchgrass

CONCLUSION

The addition of in-feed enzymes with at least endoglucanase, endoxylanase and β- glucosidase activity to physicochemically pretreated biomass under simulated pig digestive conditions (upper GIT), resulted in increased release of soluble sugars and decreased release of total non-starch polysaccharides in particular the insoluble fraction compared to physicochemical pretreatment alone. The reduction in total non-starch polysaccharides (NSPs) is desirable as animals are unable to digest these polysaccharides in vivo. Therefore the addition of the in-feed enzymes capable of breaking NSPs (particularly the insoluble fraction) has great significance in the release of nutrients to an animal.

Example 9: Effects of Celluclast®, Danisco Xylanase™ and Accellerase®BG™ on the digestibility of the fibre found in physico-chemically pretreated biomass material using an in-vitro model simulating upper tract digestion in pigs

Materials and Methods In vitro digestions of the simulated feed containing physicochemically pretreated biomass materials

Composition of the in vitro digestion treatments is shown in Table 1. Three types of physicochemically pretreated biomass (DAcs: dilute ammonia pretreated corn stover, DLcs: caustic delignified corn stover, DLswg: caustic delignified switchgrass) and corn are digested under simulated conditions of the upper digestive tract of a pig in the presence or absence of carbohydrase enzymes Celluclast ^® _j Danisco Xylanase™ and Accellerase®BG™.

Celluclast is a commercial enzyme available from Sigma-Aldrich (C2730) and contains certain cellulase activities including endoglucanase, and beta-glucosidase.

Danisco Xylanase™ is a commercial enzyme available from Danisco (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.

Table 1. Composition of the in-vitro digestion treatments.

cellobiohydrolase activities and beta-glucosidase activity.

2 Danisco Xylanase™: endoxylanase

3 Accellerase®BG™: beta-glucosidase

⁴ DAcs - dilute ammonia pretreated corn stover

⁵ DLswg - caustic delignified switchgrass

⁶ DLcs - caustic delignified corn stover To assess the effect of different enzymes on the fermentation characteristics of feed ingredients in the upper part of the digestive system, gastric and small intestinal conditions are simulated in batch tests as described in Example 8. Celluclast ^® , Danisco Xylanase™ and Accel lerase®BG are dosed at 0.1 mlJg DM, 3.5 uL/g DM and 0.15 mL/g DM, respectively. Soluble sugar release (Total: glucose, xylose, arabinose, galactose, mannose) and soluble and insoluble non-starch polysaccharides are assessed as described in Example 8. RESULTS

Digestion of NSPs and release of soluble sugars from physicochemically pretreated biomass materials

Results indicate that the addition of Celluclast ^® , Danisco Xylanase™ and AccelleraseOBG to physicochemically pretreated biomass (dilute ammonia pretreated corn stovers, caustic delignified corn stover and switchgrass) increases the release of total soluble arabinose, galactose, glucose, xylose and mannose compared to physicochemical pretreatment alone (Table 2).

Table 2. Soluble sugar analysis after in vitro digestions under simulated conditions of the upper digestive tract of a pig with 3 g (DM) of t material with or without in-feed enzymes.

ND - below the limit of detection

Celluclasf ^® : Combination of cellulase activities including endoglucanase activity, cellobiohydroiase activities and beta-glucosidase activity.

² Danisco Xylanase™: endoxylanase

³ Accellerase®BG™: beta-glucosidase

DAcs - dilute ammonia pretreated corn stover

⁵ DLcs - caustic delignified corn stover

⁶ DLswg - caustic delignified switchgrass

Following analysis of NSP sugars after in-vitro digestion under simulated conditions of the upper digestive tract of a pig, the addition of in-feed Celluclast ^® , Danisco Xylanase™ and Accellerase®BG was found to decrease the release of total NSP sugars from dilute ammonia pretreated and caustic delignified corn stover and caustic delignified switch grass compared to physicochemical pretreatment alone (Table 3). The majority of this reduction was comprised of reduced release of insoluble glucose, xylose and arabinose.

Table 3. Non-starch polysaccharide (NSP) sugar analysis after in vitro digestions under simulated conditions of the upper digestive tract of a pig with 3 g (DM) of test material with or without in-feed enzymes.

Corn + Celluclas ¹ , Danisco Xylanase™

and Accellerase®BG™ ³ 2.8 2.9 1.7 0.8 8.9

DAcs 25.3 13.6 2.4 1.1 43.6

DAcs + Celluclasf ¹ _, Danisco Xylanase™

and Accellerase®BG™ ³ 18.3 6.4 1.1 1.0 28.1

DLcs 45.9 20.1 2.7 0.7 70.3

DLcs + Celluclasf ¹ _, Danisco Xylanase™

and Accel lerase®BG™ ³ 30.5 10.9 1.3 0.6 44.0

DLswg 37.8 18.0 2.4 0.8 59.6

DLswg + Celluclasf ¹ , Danisco

Xylanase™ ² and Accellerase®BG™ ³ 28.7 8.0 1.0 0.6 39.2 cellobiohydrolase activities and beta-glucosidase activity.

² Danisco Xylanase™: endoxylanase

³ Accel lerase®BG™: beta-glucosidase

⁴ DAcs - dilute ammonia pretreated corn stover

⁵ DLcs - caustic delignified corn stover

⁶ DLswg - caustic delignified switchgrass

CONCLUSION

The addition of in-feed enzymes with at least endoglucanase, endoxylanase and β- glucosidase activity to physicochemically pretreated biomass under simulated pig digestive conditions (upper GIT), resulted in increased release of soluble sugars and decreased release of total non-starch polysaccharides in particular the insoluble fraction compared to physicochemical pretreatment alone.

Example 10: Effects of Cellulase from Aspergillus niger (C1184 - Sigma) and Accellerase®BG™ on the digestibility of the fibre found in physico-chemically pretreated biomass material using an in-vitro model simulating upper tract digestion in pigs

Materials and Methods

In vitro digestions of the simulated feed containing physicochemically pretreated biomass material

Composition of the in vitro digestion treatments is shown in Table 1. Three types of physicochemically pretreated biomass (DAcs: dilute ammonia pretreated corn stover, DLcs: caustic delignified corn stover, DLswg: caustic delignified switchgrass) and corn are digested under simulated conditions of the upper digestive tract of a pig in the presence or absence of carbohydrase enzymes Cellulase from Aspergillus niger (C1184 - Sigma) and Accellerase®BG™.

Cellulase from Aspergillus niger is a commercial enzyme available from Sigma-Aldrich (C1184) and contains certain cellulase activities including endoglucanase activity as well as endoxylanase activity.

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

Table 1. Composition of the in-vitro digestion treatments.

¹ Cellulase from Aspergillus niger: Combination of cellulase activities including endoglucanase activity and endoxylanase activity.

² Accellerase®BG™ : beta-glucosidase

³ DAcs - dilute ammonia pretreated corn stover

4 DLswg - caustic delignified switchgrass

5 DLcs - caustic delignified corn stover

To assess the effect of different enzymes on the fermentation characteristics of feed ingredients in the upper part of the digestive system, gastric and small intestinal conditions are simulated in batch tests as described in Example 8. Cellulase from Aspergillus niger (C1 184 - Sigma) and Accel lerase®BG™ are dosed at 0.2 g/g DM and 0.15 mL/g DM, respectively. Soluble sugar release (Total: glucose, xylose, arabinose, galactose, mannose) and soluble and insoluble non-starch polysaccharides are assessed as described in Example 8.

RESULTS

Digestion of fibre and release of soluble sugars from physicochemically p retreated biomass materials

Results indicate that the addition of Celluiase from Aspergillus niger (C1184 - Sigma) and Accellerase®BG™ to physicochemically pretreated biomass (dilute ammonia pretreated corn stovers, caustic delignified corn stover and switchgrass) increases the release of total soluble arabinose, galactose, glucose, xylose and mannose compared to physicochemical pretreatment alone (Table 2).

Table 2. Soluble sugar analysis after in vitro digestions under simulated conditions of the upper digestive tract of a pig with 3 g (DM) of t material with or without in-feed enzymes.

ND - below the limit of detection

¹ Cellulase from Aspergillus niger: Combination of cellulase activities including endoglucanase activity and endoxylanase activity.

² Accellerase®BG™: beta-glucosidase

³ DAcs - dilute ammonia pretreated corn stover

⁴ DLcs - caustic delignified corn stover

⁵ DLswg - caustic delignified switchgrass

Following analysis of NSP sugars after in-vitro digestion under simulated conditions of the upper digestive tract of a pig, the addition of in-feed Cellulase from Aspergillus niger (C1 184 - Sigma) and Accellerase®BG™ was found to decrease the release of total NSP sugars from dilute ammonia pretreated and caustic delignified corn stover and caustic delignified switch grass compared to physicochemical pretreatment alone (Table 3). The majority of this reduction was comprised of reduced release of insoluble glucose, xylose and arabinose.

Table 3. Non-starch polysaccharide (NSP) sugar analysis after in vitro digestions under simulated conditions of the upper digestive tract of a pig with 3 g (DM) of test material with or without in-feed enzymes.

Aspergillus niger ¹ and

Accellerase®BG™ ²

Total NSP sugars after digestion

Corn 2.3 2.1 1.4 0.6 7.1

Corn + Cellulase from Aspergillus

niger ¹ and Accellerase®BG™ ² 2.6 2.5 1.4 0.6 7.7

DAcs 25.3 13.6 2.4 1.1 43.6

DAcs + Cellulase from Aspergillus

niger ¹ and Accellerase®BG™ ² 20.6 6.4 1.0 0.8 29.5

DLcs 45.9 20.1 2.7 0.7 70.3

DLcs + Cellulase from Aspergillus

niger ¹ and Accellerase®BG™ ² 38.5 9.3 1.1 0.6 50.1

DLswg 37.8 18.0 2.4 0.8 59.6

DLswg + Cellulase from

Aspergillus niger ¹ and

Accellerase®BG™ ² 36.9 1 1.7 1.4 0.6 51.3

¹ Cellulase from Aspergillus niger: Combination of cellulase activities including endoglucanase activity and endoxylanase activity.

² Accellerase®BG™: beta-glucosidase

³ DAcs - dilute ammonia pretreated corn stover

⁴ DLcs - caustic delignified corn stover

⁵ DLswg - caustic delignified switchgrass

CONCLUSION

The addition of in-feed enzymes with at least endoglucanase, endoxylanase and β- glucosidase activity to physicochemically pretreated biomass under simulated pig digestive conditions (upper GIT), resulted in increased release of soluble sugars and decreased release of total non-starch polysaccharides in particular the insoluble fraction compared to physicochemical pretreatment alone. 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. REFERENCES

Adesogan AT, Krueger NK, Kim SC (2005). A novel wireless automated system for measuring fermentation gas production kinetics of feeds and its application to feed characterization. Anim. Feed Sci. Technol., (123-124): 211-223.

Dionex Technical Note 20. Analysis of carbohydrates by high-perfromance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD): http://www.dionex.com/en-us/webdocs/5023-TN20 LPN032857-04.pdf. Englyst, HN, Quigley ME, Hudson GJ, Cummings JH (1992). Determination of dietary fibre as non-starch polysaccharides by gas chromatography. Analyst. 117(1 1 ): 1707-1714.