WO2020261164A1 - Additifs de xylanase pour aliments ou aliments pour animaux - Google Patents

Additifs de xylanase pour aliments ou aliments pour animaux Download PDF

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Publication number
WO2020261164A1
WO2020261164A1 PCT/IB2020/056003 IB2020056003W WO2020261164A1 WO 2020261164 A1 WO2020261164 A1 WO 2020261164A1 IB 2020056003 W IB2020056003 W IB 2020056003W WO 2020261164 A1 WO2020261164 A1 WO 2020261164A1
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Prior art keywords
food
feed
xylanase
enzyme
feed additive
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PCT/IB2020/056003
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English (en)
Inventor
Brett Ivan Pletschke
Brian Nkanyiso MATHIBE
Mpho MAFA
Samkelo MALGAS
Stones Dalitso CHINDIPHA
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Rhodes University
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Publication of WO2020261164A1 publication Critical patent/WO2020261164A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/195—Proteins from microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
    • A23L7/107—Addition or treatment with enzymes not combined with fermentation with microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry

Definitions

  • 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.
  • 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.
  • 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 increase the viscosity of the digesta and as a result, affects the digestion and assimilation of nutrients such as fatty acids by the animal.
  • 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.
  • 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.
  • the antinutritional factors (ANFs) may reduce the energetic value.
  • Xylanases also called 1 ,4-6-D-xylan xylanohydrolases, EC 3.2.1.8
  • Xylanases 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.
  • GH glycoside hydrolase
  • 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.
  • CBM22 Carbohydrate Binding Molecule Family 22
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the enzyme food or feed additive is capable of reducing non-starch polysaccharide viscosity.
  • 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.
  • the enzyme food or feed additive of the present invention may also comprise a physiologically acceptable carrier.
  • a human food composition or non-human animal feed composition comprising the enzyme food or feed additive as described herein.
  • 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.
  • 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.
  • a method of increasing a food or feed conversion ratio and/or improving the nutritional value of a food or feed comprising:
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • derived from 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • suitable dosage amounts of the xylanases, or food or feed additives, or foods or feeds containing the xylanases 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.
  • 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.
  • 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.
  • xylanases 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.
  • NDS 3,5-dinitrosalicylic acid
  • the enzyme formulation was shown to have higher hydrolytic efficiency against arabinoxylan compared to some of commercially available enzyme formulations used as feed additives.
  • 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.
  • 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.
  • 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.
  • thermophilic bacterium Clostridium stercorarium comparative characterisation and addition of new hemicellulolytic glycoside hydrolases. Biotechnol Biofuels doi: 10.1 186/s 13068-018- 1228-3

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Abstract

La présente invention concerne un additif enzymatique pour aliments ou aliments pour animaux comprenant une xylanase bactérienne Geobacillus stearothermophilus GH10 et une xylanase fongique Trichoderma viride GH11. L'invention concerne en outre une composition d'aliment destinée à la consommation humaine ou une composition d'aliment destinée à la consommation animale comprenant l'additif d'aliment pour humains ou animaux de l'invention et son utilisation pour améliorer le gain de poids corporel et/ou un rapport de conversion alimentaire chez un animal. La présente invention concerne également des procédés d'augmentation d'un rapport de conversion alimentaire pour les humains ou les animaux et/ou d'amélioration de la valeur nutritionnelle d'un aliment ou d'un aliment pour animaux à l'aide de l'additif enzymatique pour aliments ou aliments pour animaux.
PCT/IB2020/056003 2019-06-25 2020-06-25 Additifs de xylanase pour aliments ou aliments pour animaux WO2020261164A1 (fr)

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