Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K40/00—Shaping or working-up of animal feeding-stuffs
  • A23K40/25—Shaping or working-up of animal feeding-stuffs by extrusion
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
  • C12Y301/03008—3-Phytase (3.1.3.8)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
• • 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
  • A23K40/00—Shaping or working-up of animal feeding-stuffs
  • A23K40/20—Shaping or working-up of animal feeding-stuffs by moulding, e.g. making cakes or briquettes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
  • C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase
• • 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)

Definitions

• the present invention relates to the application of enzymes in industrial processes such as the manufacture of food or feed compositions.
• enzymes in animal feed for livestock has become almost common practice. These enzymes are produced by micro-organisms grown in large scale fermenters operated by industrial enzyme producers. At the end of the fermentation period, broths of micro-organisms are commonly subjected to a series of filtration steps to separate biomass from the enzyme.
• the enzyme solution is either sold as a liquid after addition of various stabilizers or processed to a dry formulation.
• Enzyme liquids and dry formulations are used on commercial scale by the feed industry. Liquids may be applied to the feed after pelleting to prevent heat inactivation of the enzymes by the pelleting process. The problem is that the amounts of enzyme in the final feed preparations are very small which makes it difficult to realize a homogenous distribution of the enzyme molecules over the feed. Liquids are notoriously more difficult to mix than dry ingredients. One needs specific equipment to add liquids to the feed after pelleting which is not currently available at most feed mills.
• Dry formulations of enzymes have the disadvantage of heat inactivation of the enzymes during pelleting.
• feed is pressed through a matrix and the formed stripes are cut into suitable pellets of variable length.
• the moisture of the mass when arriving into the pelleting equipment is generally between about 1 8% and about 1 9%.
• temperatures may rise to 60-95°C.
• the combined effect of high moisture content and high temperatures is detrimental to most enzymes.
• the method comprises the steps of: mixing a grain carrier which is physiologically acceptable and capable of absorbing an aqueous enzyme solution, with an aqueous enzyme solution for a period of time sufficient to absorb the enzyme or enzymes onto the carrier to form a carrier/enzyme complex; pelleting the carrier/enzyme complex; and drying the pelleted carrier/enzyme complex to a moisture content of between 7% and 1 5% preferably less than about 10% by weight.
• thermomechanically processed compositions comprising as active ingredient a polypeptide, which compositions are obtainable by subjecting raw materials containing transgenic plant material in which the polypeptide has been biosynthesized due to the expression of a transgene therein, to a thermomechanical process.
• the preferred thermomechanical processes are pelleting, extrusion and expansion.
• compositions of the invention discloses the use of these compositions as a feed for animals or as a premix therefor.
• the compositions of the invention are used in processes for promoting the growth of animals in which the animals are fed diets which comprises these compositions.
• the invention further discloses a process for the preparation of the thermomechanically processed compositions which comprise a polypeptide as active ingredient.
• thermomechanical processes to be applied in the present invention are frequently used in commercial feed processing, where they occur in a number of ways such as e.g. expansion, extrusion, and pelletization, or combinations thereof. All these processes are characterized by an input of thermal energy, usually in the form of steam, and mechanical energy by e.g. the drive of the screw to the feed.
• Pelleting is the classical processing method in feed production which may also be applied as thermomechanical treatment as such to any raw material.
• the raw materials are mixed and after a low pressure steam addition of several bars are conveyed into a rotating die.
• the die contains holes, usually several hundred, through which the product passes. It is pushed by rotating rolls inside the die.
• Extrusion is an alternative way for feed production which may also be applied as thermomechanical treatment as such to any raw material.
• the raw materials are put onto an endless screw consisting generally of several pressure rings of decreasing shape. This screw drives the product and leads it to the die.
• the temperature rise is caused by product friction between the screw and the jacket and by an increase in pressure.
• the pressure difference between the interior of the extruder and the exterior leads to a partial vaporisation of the water at the exit point and thus to an expansion of the product.
• Expansion is a process which is quite similar to extrusion but does not shape the material in a defined matter since it is generally not driven through a die.
• the transgenic plant materials of the invention preferably contain one or more desired polypeptides which are the expression product of transgenes contained in the plant from which the plant material is derived. This is achieved via the introduction into the plant of expression constructs comprising a DNA sequence encoding the desired polypeptides having a relevant activity such as for example the enzyme phytase (of which detailed descriptions may be found in Pen et al. EP-A-0 449 375).
• Transgenic plants and transgenic plant material are herein defined to include plants (as well as parts and cells of said plants) and their progeny, which have been genetically modified using recombinant DNA techniques to cause, enhance or alter the production of a desired polypeptide in the plant or plant organ.
• transgene is understood to refer to the specific genetic modification, i.e. nucleotide sequences, in the transgenic plant.
• polypeptides are applied as active ingredients, whereby an active ingredient is defined as being encoded by a transgene.
• the polypeptides to be used in the invention may be produced constitutively in the transgenic plants in all tissues, during all stages of development. In some instances, e.g. in the case of plant cell wall degrading enzymes, it may be required to limit the expression of the transgene to specific parts of the plant in which the expressed desired polypeptide do not interfere with the development of the host plant.
• the genes encoding the desired polypeptides can therefore also be expressed in a stage- and/or tissue-specific manner, or, alternatively, these genes can be put under control of an inducible promoter.
• seeds are used, either intact or after milling or grinding, in which the desired polypeptide has been specifically expressed using a seed-specific promoter. Nevertheless, it will be evident to those skilled in the art that expression of polypeptides in other plant organs is equally feasible and may lead to similar advantages during application as disclosed in the present invention.
• plants to be selected include, but are not Iimited to crops producing edible flowers such as cauliflower (Brassica oleracea), artichoke (Cynara scolymus), fruits such as apple (Ma/us, e.g. domesticus), banana (Musa, e.g. ac ⁇ minata), berries (such as the currant, Ribes, e.g. rubrum), cherries (such as the sweet cherry, Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus), grape ( Vitis, e.g.
• Brassica oleracea Brassica oleracea), endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce (Lactuca, e.g. sativa), spinach (Spinacia e.g. oleraceae), tobacco (Nicotiana, e.g. tabacum), roots, such as arrowroot (Maranta, e.g. arundinacea) , beet (Beta, e.g. vulgaris), carrot (Daucus, e.g. carota), cassava (Manihot, e.g. esculenta), turnip (Brassica, e.g.
• rapa radish
• Raphanus e.g. sativus
• yam Dioscorea , e.g. esculenta
• sweet potato Ipomoea batatas
• seeds such as bean (Phaseolus, e.g. vulgaris), pea (Pisum, e.g. sativum), soybean (Glycin, e.g. max), wheat ( Triticum, e.g. aestivum), barley (Hordeum, e.g. vulgare), corn (Zea, e.g. mays), rice (Oryza, e.g.
• sativa sativa
• rapeseed Brasset
• Millet Pulp L.
• sunflower Helianthus annus
• oats A vena sativa
• tubers such as kohlrabi (Brassica, e.g. oleraceae), potato (Solanum, e.g. tuberosum) and the like.
• polypeptides when added to a composition as active ingredient and in the form of a transgenic plant material are more resistant to inactivation during the thermomechanical process steps such as pelleting, as compared to conventional forms in which polypeptides were added to such compositions. It will be apparent that these advantages are not necessarily Iimited to the specific embodiments of the specific examples of the present application, i.e. enzymes such as phytase or xylanase which are added to compositions to be pelleted and used as feed for animals. In contrast, the advantages of the present invention should apply to any composition which is to be subjected to a thermomechanical treatment and which contains one or more polypeptides as active ingredients.
• the advantages of the invention will be most pronounced in the case of polypeptides with a sensitive biological activity, e.g. sensitive to thermal inactivation.
• Thermosensitive polypeptides are herein defined as polypeptides of which a substantial amount of biological activity is lost upon being subjected to the thermomechanical processes applied in the invention.
• polypeptides and/or the DNA sequences encoding them are heterologous with respect to the transgenic host plant in which the DNA sequences are expressed.
• Heterologous is herein understood to mean that the DNA sequences and/or polypeptides are not native to the host organism, i.e. which are derived from other plant species, mammals, or microorganisms, such as fungi or bacteria.
• the present invention is particularly advantageous when glycosylated proteins are expressed in the transgenic plants, whereby their glycosylation may differ from the naturally occurring counterparts of these proteins.
• Preferred enzymes to be included in the present invention are enzymes to be applied in animal feed. Animal feed is herein understood to also include pet food.
• feed enzymes such as e.g. phytase
• function of these enzymes is to improve the feed conversion rate, e.g. by reducing the viscosity or by reducing the antinutritional effect of certain feed compounds.
• feed enzymes such as e.g. phytase
• Preferred enzymes for these purposes are: phosphatases, of which preferably phytases and/or acid phosphatases; carbohydrases, such as amylolytic enzymes and plant cell wall degrading enzymes of which preferably cellulases, hemicellulases, or galactanases; proteases of which preferably proteases with a neutral and/or acidic pH optimum; and lipases of which preferably phospholipases such as the mammalian pancreatic phospholipases A2.
• the polypeptides to be included in the invention could also comprise non-enzymatic biological activities such as antigenic determinants to be used as vaccines and/or polypeptides engineered to have an increased content of essential amino acids.
• the pelleting-stability of Aspergillus niger phytase has been tested when the enzyme is contained in milled seeds of oilseed rape (Brassica nap ⁇ s) in which the A.niger phytase gene has been expressed. Cloning of the A.niger phytase gene has been described elsewhere (van Gorcom et al. EP-A-0 420 358).
• a pelleting-stable formulation of the Aspergillus endoxylanase is provided by expression of the fungal gene encoding this enzyme in tobacco seeds. The cloning of the Aspergillus endoxylanase gene has been described in detail by de Graaff et al. EP-A-0 463 706.
• the seed-specific transcription of the phytase cDNA and termination thereof are controlled by the 5'- and 3'-regulatory sequences derived from gene encoding the Brassica napus 12S storage protein cruciferin (the cruA gene; Ryan et al., 1 989, Nucl. Acids Res. _V7: 3584), respectively.
• the Alfalfa Mosaic Virus RNA4 leader sequence is used to stabilize the mRNA (Bredero et al., 1 980, Nucl. Acids Res.
• Agrobacterium strain LBA4404 Hoekema et al., 1 983, Nature 303: 1 79
• This strain was used to transform rapeseed (Brassica napus cv. Westar).
• rapeseed Brainssica napus cv. Westar.
• surface-sterilized stem segments taken from 5 to 6 week-old plants, just before flowering, are preconditioned for 24 h on MS medium (Fry et al. ( 1 987) Plant Cell Reports 6, 321 ) and then co-cultivated for 48 h with Agrobacterium on fresh plates with the same medium.
• Transgenic plantlets were regenerated from shoots that grow on selection medium (500 mg/l carbenicilline, 40 mg/l paromomycin) and further analyzed as described in by Pen et al. in EP-A-0 449 375.
• selection medium 500 mg/l carbenicilline, 40 mg/l paromomycin
• expression levels of phytase in oilseed rape reached levels up to 10% of soluble protein corresponding to about 500 FTU/g.
• FTU One unit of phytase activity
• FTU is defined as that amount of enzyme which liberates inorganic phosphorus from 1 .5 mM sodium phytate at the rate of 1 ⁇ mol/min at 37°C and at pH 5.5.
• Example 2 Stability of phytase in oilseed rape during pelleting Pelleting conditions were such that the pellets obtained a temperature of 55 °C after conditioning and 75 °C after pelleting.
• Two independent trials have been conducted, one on labscale and one on industrial scale.
• the Aspergillus niger phytase produced in oilseed rape is substantially more resistant to pelleting conditions than the current commercial Aspergillus niger phytase formulations. Since milled seeds were used, the stability of the enzyme is apparently not dependent on the integrity of the seeds.
• Natuphos ® is a commercial product containing the Aspergillus niger phytase, which may be obtained from BASF, Ludwigshafen, Germany.
• the Aspergillus endoxylanase gene has been cloned as described in detail by de Graaff et al. EP-A-0 463 706.
• the E.coli plasmid plMI OO containing the Aspergillus endoxylanase gene has been deposited at the Centraal Bureau voor Schimmelcultures on July 19, 1990 and was assigned the designation CBS 322.90.
• a fusion PCR with plMI OO as template the single intron in the xylanase gene was removed.
• This artificial cDNA was expressed in tobacco seeds following procedures similar to those described in detail for the expression of phytase in tobacco (Pen et al., 1 993, Bio/technology JJ.: 81 1 ).
• the cDNA fragment encoding the mature enzyme was fused in ⁇ frame to the sequence encoding the signal peptide of the tobacco PR-S protein (Cornelissen et al., 1 986, Nature 321 : 531 ) in the binary expression vector pMOG29 (Pen et al., 1 992, Bio/technology 10: 292).
• the expression cassette in pMOG29 consists of the following elements: the constitutive cauliflower mosaic virus (CaMV) 35S promoter with double enhancer (Guilley et al., 1982, Cell 30: 763); the synthetic Alfalfa Mosaic Virus RNA4 leader sequence to stabilize the mRNA (Bredero et al., 1980, Nucl. Acids Res.
• This binary vector containing the chimeric endoxylanase gene was mobilized, in a triparental mating with the E. coli K-1 2 strain RK201 3 (containing plasmid pRK201 3) (Ditta et al., 1 980, Proc. Natl. Acad. Sci.
• Agrobacterium strain LBA4404 Hoekema et al., 1 983, Nature 303: 1 79
• This strain was used to transform Tobacco (Nicotiana tabacum cv Petit Havana SR1 ) leaf discs according to the procedure of Horsch et al. (1 985, Science 227: 1229) .
• Transgenic plants were selected on media containing 100 mg/l kanamycin.
• EXU endoxylanase activity
• Milled seeds and the microbial enzyme product sold under the brandname Lyxasan Forte ® (obtainable from BASF, Ludwigshafen, Germany) were added to the meal to a final concentration of 6500 EXU/kg of feed
• endoxylanase produced in seeds is substantially more resistant to pelleting than the current formulation containing the microbial enzyme.

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