BACKGROUND OF THE INVENTION

This invention relates to an enzyme food or feed additive comprising a GH10 bacterial xylanase, XT6 from Geobacillus stearothermophilus, and a GH1 1 fungal xylanase, Xyn2A from Trichoderma viride. The present invention also relates to a human food composition or non-human animal feed composition comprising the food or feed additive described and to the use of the human food composition or non-human animal feed composition in improving body weight gain and/or food or feed conversion ratio in an animal. This invention further relates to methods of increasing a food or feed conversion ratio and/or improving the nutritional value of a food or feed using the enzyme food or feed additive.

Worldwide, agricultural farming systems, such as livestock production, face an increasing challenge of maintaining future global demand for meat and dairy products because of an expected increase in population. The Food and Agriculture Organization (FAO) expects that an increase in purchasing power for food from animal sources will raise the yearly demand to 465 and 1 .043 million tonnes for meat and milk products, respectively, by 2050. Besides, the FAO estimates the growth of global population to reach 9.6 billion by the year 2050, with a doubled purchasing power for meat and dairy products.

The price of wheat increased drastically in 2007, and prices on traditional feed ingredients have in general been fluctuating since. This has encouraged meat producers to look for alternative and less expensive ingredients for feed. While most local farmers have resorted to the use of feeds developed on-site using agro-industrial wastes such as corn stover, soybean meal, canola meal, sugarcane bagasse, and wheat bagasse, these feeds contain high quantities of non-starch polysaccharides (NSPs), such as mannan, xylan, pectin and cellulose, which negatively affect the feed’s nutrient utilization.

Insoluble NSPs are associated with the encapsulation of nutrients such as starch and protein. This encapsulation causes valuable nutrients to by-pass the digestive tract of the animal, undigested. The soluble NSPs, on the other hand, increase the viscosity of the digesta and as a result, affects the digestion and assimilation of nutrients such as fatty acids by the animal. Moreover, the binding of NSPs with the intestinal brush border of animals increases the thickness of the unstirred water layer adjacent to the mucosa, leading to impaired nutrient digestion and absorption. NSPs are also associated with stimulating the growth of some pathogenic bacteria species, including Escherichia coli and Clostridium perfringens. For example, the amount of pectin (about 60 g uronic acids/kg dry matter) and the fibre matrix structure of canola most likely increases the water holding capacity of this raw material, resulting in poor nutrient availability for non-ruminants. Furthermore, the antinutritional factors (ANFs) may reduce the energetic value.

Xylanases (also called 1 ,4-6-D-xylan xylanohydrolases, EC 3.2.1.8) are enzymes which randomly cleave the b-(1 ,4)-linkages between two D-xylopyranosyl residues in xylan backbones. Xylanases have been classified into glycoside hydrolase (GH) families GH5, GH8, GH10, GH1 1 , GH30, GH43, GH62 and GH98 in the CAZy database of carbohydrate-active enzymes, with GH10 and GH1 1 being the best characterized families. GH1 1 xylanases can only hydrolyse xylosidic bonds where the two corresponding xylose moieties in subsites (-1 ) and (+1 ) are not branched, while GH10 xylanases attack the glycosidic linkage next to a single or double substituted xylose toward the non-reducing end and require two unsubstituted xylose residues between branched residues. GH1 1 fungal and bacterial xylanases often contain a catalytic domain linked to one or more non-catalytic modules such as Carbohydrate Binding Molecule Family 22 (CBM22). This appears to favour GH1 1 xylanases to display high catalytic activity on insoluble xylan substrates, while GH10 xylanases appear to be more catalytically efficient on soluble xylan substrates.

Xylanases are considered one of the industrially important microbial enzymes, which can catabolize xylan residues. Over the years, the use of xylanases at an industrial level has increased significantly. Since applications of xylanases in commercial sectors are widening, an understanding of their nature and properties for efficient and effective usage becomes crucial. Xylanases extracted from microorganisms have been used for pulp bleaching, waste paper treatment and for fabric bio-processing, such as: bio-bleaching, desizing and bio-scouring of fabrics. Xylanases have also been shown to improve rheological properties of dough, bread specific volume and crumb firmness.

Several studies have also demonstrated improvements in nutritive value, feed utilization, body weight gain, composition and activity of intestinal microbiota, and reduction in excreta volume in livestock after supplementation of agro-industrial wastes based diets with NSP-degrading enzymes such as cellulases, pectinases, xylanases and glucanases. Enzyme supplementation is a well-established method in the animal feed industry to obtain viscosity reduction and fibre degradation in situ in poultry, with xylanases being the enzymes of choice. Recently, the use of exogenous carbohydrolases in aqua feeds has also been getting more attention.

However, others have also reported that exogenous enzymes did not consistently enhance forage quality and utilization by ruminants. This inconsistency may be attributed to several factors such as the source of the enzyme, doses and activities of the enzyme, physical properties of the substrate, treatment duration, enzyme application method, composition of the diet to which enzyme is added and level of animal productivity.

Use of antibiotics in animal feed as growth-promoters appears to promote emergence of antibiotic resistant strains. The Guidelines for Industry issued by the Center for Veterinary Medicines of the Food and Drug Administration, USA recommend use of antibiotics only for the prevention, control and treatment of infections in animals but not for the promotion of growth, increased performance, and improved feed efficiency. Use of fibrolytic enzymes (i.e. xylanases) have been reported to lead to increased populations of probiotics such as Bifidobacteria and Lactobacilli and decreased Gram-negative bacteria overall. This shows that xylanase additives in feeds can potentially replace the use of antibiotics at subtherapeutic levels in feeds as animal growth promoters.

SUMMARY OF THE INVENTION

The present invention relates to an enzyme food or feed additive, a human food composition or non-human animal feed composition comprising the food or feed additive and to the use of the human food composition or non-human animal feed composition in improving body weight gain and/or food or feed conversion ratio in a human or a non-human animal.

According to a first aspect of the present invention there is provided for an enzyme food or feed additive comprising a GH10 xylanase derived from a Geobacillus stearothermophilus bacterium and a GH1 1 xylanase derived from a Trichoderma viride fungus.

In one embodiment of the invention, the GH10 xylanase derived from the Geobacillus stearothermophilus bacterium may be the GH10 xylanase XT6 encoded by a xt6 gene, having an amino acid sequence of at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity or substantially identical to SEQ ID NO: 1.

According to a second embodiment, the GH1 1 xylanase derived from the Trichoderma viride fungus may be the GH1 1 xylanase Xyn2A encoded by a xyn2A gene, having an amino acid sequence of at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity or substantially identical to SEQ ID NO:2.

In a third embodiment of the invention, the enzyme food or feed additive is capable of reducing non-starch polysaccharide viscosity.

According to a fourth embodiment of the present invention, the GH10 xylanase derived from the Geobacillus stearothermophilus bacterium and the GH1 1 xylanase derived from the Trichoderma viride fungus in the enzyme food or feed additive demonstrate a synergistic effect in hydrolysing non-starch polysaccharides when compared to an enzyme food or feed additive comprising a xylanase from a single source.

Further, the enzyme food or feed additive of the present invention may also comprise a physiologically acceptable carrier.

According to a second aspect of the present invention, there is provided for a human food composition or non-human animal feed composition comprising the enzyme food or feed additive as described herein. In particular, the human food composition or non-human animal feed composition may comprise a cereal, such as a cereal selected from the group consisting of barley, maize, oats, rice, rye, sorghum, triticale, wheat, and combinations thereof.

According to a third aspect of the present invention, there is provided for the use of a human food composition or non-human animal feed composition of the second aspect for improving body weight gain and/or food or feed conversion ratio in a human or a non-human animal.

According to a fourth aspect of the present invention there is provided for a method of increasing a food or feed conversion ratio and/or improving the nutritional value of a food or feed comprising:

(a) providing a cereal-based food or feed; and

(b) adding the enzyme food or feed additive of the invention as described herein to the cereal-based food or feed. It will be appreciated by one of skill in the art that the cereal-based food or feed may be selected from the group consisting of barley, maize, oats, rice, rye, sorghum, triticale, wheat, and combinations thereof.

According to a further embodiment of the present invention, the GH10 xylanase derived from the Geobacillus stearothermophilus bacterium and the GH1 1 xylanase derived from the Trichoderma viride fungus in the enzyme food or feed additive have a synergistic effect in increasing the food or feed conversion ratio and/or improving the nutritional value of the food or feed compared to a food or feed with an enzyme food or feed additive comprising a xylanase from a single source.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

Figure 1 : Amino acid sequence of GH10 xylanase XT6 from Geobacillus stearothermophilus (SEQ ID NO:1 ).

Figure 2: Amino acid sequence of GH1 1 xylanase Xyn2A from

Trichoderma viride (SEQ ID NO:2).

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing are shown using standard three letter abbreviations for amino acids. In the accompanying sequence listing:

SEQ ID NO:1 - amino acid sequence of XT6 from Geobacillus s tearothermophilus

SEQ ID NO:2 - amino acid sequence of Xyn2A from Trichoderma viride

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As used throughout this specification and in the claims that follow, the singular forms“a”,“an” and“the” include the plural form , unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms“comprising”,“containing”, “having” and“including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

The present invention relates to the use of a synergistic combination of xylanases, specifically a combination of a GH10 bacterial xylanase and a GH1 1 fungal xylanase, as an additive to a foodstuff or an animal feed, and to foodstuffs or feeds that contains the synergistic combination of xylanases. The invention also relates to a method for improving the food or feed conversion ratio and/or the apparent metabolizable energy of foodstuffs or feedstuffs using the synergistic combination of xylanases. Combining xylanases of different GH families has previously been shown to result in varied substrate specificities (Christakopoulos et al. 2003; Beaugrand et al. 2004; Broeker et al. 2018) Therefore, combining different GH family xylanases generally leads to a diversity and higher production of XOS (Goncalves et al. 2015; Malgas and Pletschke 2019). However, based on a review of the prior art, the degree of synergism between the xylanases varies depending on the sources of the enzymes (Goncalves et al. 2015), with some combinations showing no synergism and anti synergism , whereby combining enzymes leads to lower hydrolysis compared to that achieved by single enzymes (Beaugrand et al. 2004). Thus, it is surprising that the particular combination of the present invention shows a synergistic effect, particularly in improving the feed conversion ratio and/or the apparent metabolizable energy of feedstuffs. It is submitted that the synergistic effect observed is dependent on the species/organism the enzymes are derived from and the enzyme structure and/or sequence, which affect the biochemical properties of the enzymes.

As used herein a“xylanase” means a protein, or a polypeptide, having xylanase activity. Xylanase activity can be measured using any assay, in which a substrate is employed, that includes 1 ,4-8-D-xylosidic endo-linkages in xylans. Different types of substrates are available for the determination of xylanase activity e.g. Xylazyme cross- linked arabinoxylan tablets, or insoluble powder dispersions and solutions of azo-dyed arabinoxylan.

The xylanases of the present invention may be formulated as a food or feed additive by methods known to those skilled in the art. Physiologically acceptable ingredients may be used. The term "physiologically acceptable" refers to properties and/or substances which are acceptable for administration to an animal from a toxicological point of view. Further“physiologically acceptable” refers to factors such as formulation, stability, patient acceptance and bioavailability, which will be known to a person skilled in the art.

As used herein the term“derived from” with reference to a xylanase refers to any wild-type xylanase isolated from the organism in question, synthetic versions thereof, as well as variants or fragments thereof which retain xylanase activity.

The xylanases of the invention may be“purified”, in that they may be comprised in a protein-enriched preparation, in which a substantial amount of low molecular components, typical residual nutrients and minerals have been removed. Such purification can e.g. be by conventional chromatographic methods such as ion- exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

As used herein a“substantially identical” amino acid sequence is an amino acid sequence that differs from a reference sequence only by one or more non -conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the activity of the expressed protein or polypeptide. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein. This definition also refers to, or may be applied to, the compliment of a given sequence. The term“animal” refers to all non-human animals, as well as human beings. Examples of non-human animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goat, and cattle, e.g. cow such as beef cattle and dairy cows. In a particular embodiment, the animal is a non-ruminant animal. Non-ruminant animals include mono-gastric animals, e.g. pig or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chickens (including but not limited to broiler chicks, layers) ; fish (including but not limited to salmon, trout, tilapia, catfish and carp); and crustaceans (including but not limited to shrimp and prawn). The xylanase compositions of the invention can be fed to the non-human animal or the human before, after, or simultaneously with the diet.

The term“food”,“food composition”,“feed”,“feed composition”, or“diet” means any compound, preparation, mixture, or composition suitable for, or intended for intake by a non-human animal or by a human.

The “suitable forms” of the food or feed additive may be combined with “physiologically acceptable carriers” and other elements known in the art to produce additives for foods or feeds. The food or feed additive may further be combined with other ingredients which promote absorption by the digestive tract. By“physiologically acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the xylanases and/or food or feed additives to a non-human animal or a human. The physiologically acceptable carrier may be a cereal or derived from a cereal. Such cereals include milled wheat, maize, soya, sugars, starches or a by-product of any of these. Such carriers are conventional in this technical art, and so are not described in any further detail.

The food or feed additive of present invention may be combined with other food or feed components to produce a cereal-based food or feed. Such other food or feed components include one or more other enzyme supplements, vitamin food or feed additives, mineral food or feed additives and amino acid food or feed additives. The resulting food or feed additive may then be mixed in an appropriate amount with the other food or feed components such as cereal and protein supplements to form an animal feed or human food. Although the cereal component of a cereal-based feed constitutes a source of protein, it may be necessary to include sources of supplementary protein in the food or feed such as those derived from fish-meal, meat- meal or vegetables. Sources of vegetable proteins include at least one of full fat soybeans, rapeseeds, canola, soybean-meal, rapeseed-meal and canola-meal. The food or feed provided by the present invention may also include other enzyme supplements such as one or more of b-glucanase, glucoamylase, mannanase, a-galactosidase, phytase, lipase, a-arabinofuranosidase, protease, a-amylase and pectinase.

Processing of these components into an animal feed can be performed using any of the currently used processing methods and apparatuses such as a double- pelleting machine, a steam pelleter, an expander or an extruder.

The use of the xylanases, or food or feed additives, or foods or feeds containing the xylanases, entails administration of an effective amount of the xylanases, or food or feed additives, or foods or feeds containing the xylanases, to an animal or a human in order to promote digestion and improve the nutritional value of an animal feed or human food.“Improving the nutritional value of a food or feed” means improving the availability of nutrients, whereby the growth rate, weight gain, and/or food or feed conversion (i.e. the weight of ingested food or feed relative to weight gain) of the animal or human is/are improved.

Although some indications have been given as to suitable dosage amounts of the xylanases, or food or feed additives, or foods or feeds containing the xylanases, the exact amount and frequency of administration will be dependent on several factors and can be optimised. These factors include the individual components used, the formulation of the food or feed additives, or foods or feeds containing the xylanases, the age, weight and general physical condition of the animal, and other factors as are known to those skilled in the art.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Formulation of an Enzyme Cocktail for Use as an Animal Feed Additive

Using commercial xylanases supplied by Megazyme (Ireland, Wicklow) and Sigma Aldrich (USA, St. Louis), namely the GH1 1 xylanase Xyn2A from the fungus Trichoderma viride and the GH10 xylanase XT6 from the bacterium Geobacillus stearothermophilus, an enzyme cocktail relevant for use as an animal feed additive was formulated. The enzyme formulation comprises 75:25 % of Xyn2A to XT6 at protein mass dosage/loading. EXAMPLE 2

Comparison of the Enzyme Formulation Activity to Other Xylanase Animal Feed Products

The activity of the enzyme formulation from Example 1 was compared to that of commercially available xylanases; XynA from Thermomyces lanuginosus, XT6 from Geobacillus stearothermophilus, Xyn2A from Trichoderma viride, and Xynl OD from Cellvibrio japonicus, during the degradation of wheat-flour arabinoxylan, a predominant NSP in agricultural grains and feedstocks. The enzyme loading was kept at 0.25 mg protein/g biomass in all the reactions. Activity of the xylanases was determined by incubating them with 1 % (w/v) wheat-flour arabinoxylan (in 50 mM sodium citrate buffer, pH 5.0) at 50 ^° C for 12 hours and estimating the reducing sugar release using the 3,5-dinitrosalicylic acid (DNS) method with absorbance readings at 540 nm. Table 1 below shows the sugar release in mg/ml of the xylanases.

Table 1. Evaluation of wheat-flour arabinoxylan hydrolysis by the enzyme formulation and other xylanases from different sources and/or GH families. ANOVA analysis for differences in hydrolysis with respect to reducing sugar release by the different enzymes (p value < 0.05). Different superscript letters denote statistical significant difference between values. Values are represented as mean values ± SD (n=4).

As can be seen from Table 1 above, the enzyme formulation was shown to have higher hydrolytic efficiency against arabinoxylan compared to some of commercially available enzyme formulations used as feed additives.

EXAMPLE 3

Arabinoxylan Viscosity Reduction by the Enzyme Formulation

The NSP (wheat-flour arabinoxylan) viscosity reduction ability of the enzyme formulation of Example 1 was determined using a Cannon-Manning Semi-Micro viscometer (size 50) at room temperature upon hydrolysis of 0.5 % (w/v) wheat flour arabinoxylan by 0.25 mg protein/g biomass of the enzyme formulation at pH 5.0 and 50 ^° C after 12 hours and compared to an undigested/un-hydrolysed sample (negative control). Table 2 below shows the viscosity reduction of the enzyme formulation relative to the negative control.

Table 2. Viscosity reduction of 0.5 % wheat flour arabinoxylan by the enzyme formulation. ANOVA analysis for differences in hydrolysis with respect to reducing sugar release by the different enzymes (p value < 0.01 ). Different superscript letters denote statistical significant difference between values. Values are represented as mean values ± SD (n=4).

As can be seen from Table 2 above, significant reduction in NSP viscosity was proven using the enzyme formulation.

EXAMPLE 4

Comparison of the Enzyme Activities of Formulations Prepared Using Available GH10 and GH11 Xylanases

Two GH10 xylanases (XT6 from Geobacillus stearothermophilus and Xyn10D from Cellvibrio japonicus) and three GH1 1 xylanases (XynA from Thermomyces lanuginosus, Xyn2A from Trichoderma viride and Xyn1 1A from Neocallimastix patriciarum) were used to formulate various GH10 to GH1 1 xylanase formulations in order to ascertain whether all GH10 to GH1 1 combinations behave synergistically or not. Six formulations of the various GH10 to GH1 1 xylanases at varying ratios were assessed and were as follows: XT6 to XynA, XT6 to Xyn2A, XT6 to Xyn1 1 A, Xyn10D to XynA, Xyn10D to Xyn2A and Xyn10D to Xyn1 1 A. The activities of the GH1 0 to GH1 1 xylanase formulations were compared during the degradation of wheat-flour arabinoxylan (Table 3). The enzyme loading was kept at 0.25 mg protein/g biomass in all the reactions. Activity of the xylanases was determined by incubating them with 1 % (w/v) wheat-flour arabinoxylan (in 50 mM sodium citrate buffer, pH 5.0) at 50 ^° C for 12 hours and estimating the reducing sugar release using the 3, 5-Dinitrosalicylic acid (DNS) method with absorbance readings at 540 nm . Table 3. Comparison of wheat-flour arabinoxylan hydrolysis by the six combinations of GH10 (Xynl OD and XT6) to GH1 1 xylanases (XynA, Xyn2A and Xyn1 1 A). ANOVA analysis for differences in hydrolysis with respect to reducing sugar release by the different enzymes (p value < 0.05). Different superscript letters denote statistical significant difference between values. Values are represented as mean values ± SD (n=4).

Only two out of the six considered GH10 to GH1 1 xylanase formulations yielded results which were statistically improved (p > 0.05) compared to 100% of the most active enzyme in the combination, and these were 50:50% of Xyn10D to XynA and 25:75% of XT6 to Xyn2A (Table 4).

Table 4. Comparison of wheat-flour arabinoxylan hydrolysis by the synergistic enzyme formulations and the most active xylanase contained in the formulations. ANOVA analysis for differences in hydrolysis with respect to reducing sugar release by the different enzymes (p value < 0.05). Different superscript letters denote statistical significant difference between values. Values are represented as mean values ± SD (n=4).

As can be seen from Table 4 above, the enzyme formulation (25:75% of XT6 to Xyn2A) was also shown to have higher hydrolytic efficiency against arabinoxylan compared to another GH10 to GH1 1 xylanase enzyme formulation (50:50% of Xyn10D to XynA) developed in-house.

REFERENCES

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Broeker J, Mechelke M, Baudrexl M, et al (2018) The hemicellulose-degrading enzyme system of the thermophilic bacterium Clostridium stercorarium : comparative characterisation and addition of new hemicellulolytic glycoside hydrolases. Biotechnol Biofuels doi: 10.1 186/s 13068-018- 1228-3

Christakopoulos P, Katapodis P, Kalogeris E, et al (2003) Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and 1 1 endoxylanases. Int J Biol Macromol 31 :171— 175.

Goncalves GAL, Takasugi Y, Jia L, et al (2015) Synergistic effect and application of xylanases as accessory enzymes to enhance the hydrolysis of pretreated bagasse. Enzyme Microb Technol 72:16-24. doi: 10.1016/j.enzmictec.2015.01.007

Malgas S, Pletschke Bl (2019) The effect of an oligosaccharide reducing-end xylanase, Bh Rex8A, on the synergistic degradation of xylan backbones by an optimised xylanolytic enzyme cocktail. Enzyme Microb Technol 122:74-81 .