WO1991015966A1 - Fourrage traite par voie enzymatique pour la conservation en silo - Google Patents

Fourrage traite par voie enzymatique pour la conservation en silo Download PDF

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Publication number
WO1991015966A1
WO1991015966A1 PCT/FI1991/000118 FI9100118W WO9115966A1 WO 1991015966 A1 WO1991015966 A1 WO 1991015966A1 FI 9100118 W FI9100118 W FI 9100118W WO 9115966 A1 WO9115966 A1 WO 9115966A1
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Prior art keywords
enzyme composition
xylanase
pectinase
cellulase
forage
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PCT/FI1991/000118
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English (en)
Inventor
Robert Charlick
Kari Hissa
Pirjo Hissa
Markku Virkki
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Ssv-Development Oy
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Publication of WO1991015966A1 publication Critical patent/WO1991015966A1/fr

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    • 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)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
    • A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
    • A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
    • 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)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • 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)
    • C12Y302/01002—Beta-amylase (3.2.1.2)
    • 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)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • 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)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)
    • 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)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
    • 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)
    • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)

Definitions

  • This invention relates to use of enzymes with and without bacteria to preserve and enhance the nutritive value, in particular energy and protein utilisation, of forage for silage and to improve the palatability, digestibility and rate of digestion of treated forage by ruminants.
  • compositions have usually either been administered directly to livestock for their consumption or have been applied to assorted plant materials prior to human or farm animal consumption to improve their nutritive value, digestibility, or taste.
  • the technique of administering certain enzyme has usually either been administered directly to livestock for their consumption or have been applied to assorted plant materials prior to human or farm animal consumption to improve their nutritive value, digestibility, or taste.
  • compositions to single-stomach animals such as pigs, hens, horses, etc. to improve the feed efficiency and promote digestion has been widely adopted in the livestock industry.
  • U.S. Patent No. 4,144,354 discloses a method for promoting the secretion of milk of livestock and improving the quality of the milk by
  • This enzyme corposition may include cellulase and pectinase as well as other enzymes.
  • glucose oxidase in ensiling forage material has been described in U.S. Patent No. 4,751,089. This patent discusses the depletion of oxygen and concommitant production of pH-decreasing gluconic acid.
  • Certain enzyme compositions have also been used in treating selected plant materials in order to improve their value as food materials.
  • U.S. Patent No. 3,757,582 discloses a process for treating tea extracts with a pectinase enzyme preparation to prepare high bulk density tea powders.
  • U.S. Patent No. 3,615,721 discloses a process for preparing an edible food product having improved nutritive value, digestibility and storage stability which comprises treating food materials and food by-products with a mixture of enzymes exhibiting catalytic cellulase, hemicellulase and pectinase activity.
  • U.S. Patent No. 4,617,383 discloses a method for treating decorticated plant bast fiber with an aqueous acidic solution of fungal pectinase to remove the pectin from the plant fiber.
  • silage is more palatable allowing maximum dry matter intake of feed.
  • orchardgrass bluegrass, bermudagrass
  • corn silage Other crops such as sorghum, clover, potatoes, sunflowers, cabbages, root tops, fruit residues, brewers/distillers grains, or cereals can be ensiled for winter feeding.
  • the nutritive value and microbiological quality of the forage continually and progressively deteriorates because of the growth of harmful bacteria and the concommitant degradation of the forage material resulting in unpalatable silage with low dry matter intake potential.
  • the ensiled plant material like protein and sugars, undergoes a sequential fermentation during which the plant tissue first dies and the supply of oxygen is rapidly depleted.
  • the growth of harmful microorganisms in the ensiled forage is initially slowed due to the presence of lactic acid bacteria.
  • the lactic acid bacteria, as well as the harmful species of bacteria, such as clostridia, are normally present on the surface of the forage plants at the time they are harvested and ensiled.
  • lactic acid bacteria convert glucose to lactic acid which reduces the pH of the ensiled forage to a level sufficient to prevent or inhibit the growth of other microorganisms.
  • the level at which a certain pH will inhibit growth of harmful microorganisms such as clostridia depends on several variables, including water activity or buffering capacity.
  • Lactic acid bacteria will start to produce lactic acid from sugars as soon as anaerobic conditions are achieved, i.e., when crop pH is about 6.0 - 7.0. Lactic acid will continue to be produced as long as monomer sugars are
  • clostridia can metabolise both sugars and lactic acid producing butyric acid, carbon dioxide and hydrogen. Such byproducts will cause pH to rise, proteins will be degraded and the resulting silage will be odorous, unpalatable and of low quality.
  • the original forage composition is better preserved since the harmful microorganisms are net allowed to develop.
  • the lactic acid bacteria are able to survive at the low pH and high osmotic pressure levels present in the ensiled forage.
  • lactic acid production is often constrained by the lack of water soluble carbohydrates (sugars) available in the ensiled forage, the production of lactic acid eventually drops, the pH rises, as a result of breakdown of plant and fermentation acids, and the harmful bacteria once again can begin to grow and degrade the forage resulting in unpalatable silage with low nutrient value.
  • additives which have been used or experimented with to a limited extent include salts, antibiotics, urea, ammonia, ammonium salts and formaldehyde.
  • the water soluble carbohydrates required by the lactic acid bacteria for the lactic acid fermentation process may be added to the forage at the time of ensiling in an attempt to prolong the production of lactic acid by lactic acid bacteria.
  • These water soluble carbohydrate additives may take the form of molasses, starch, and sugars such as lactose and sucrose. These additives provide a source of fermentable carbohydrate which aids lactic fermentation.
  • molasses starch
  • sugars such as lactose and sucrose.
  • These additives provide a source of fermentable carbohydrate which aids lactic fermentation.
  • a large amount of sugar addition is needed, i.e. 10 - 20 kilograms ⁇ ugar/tn. Molasses requires a usage rate of between 80 and 100.
  • the lactic acid bacteria themselves may be introduced into the ensiled forage to lower the pH of the ensiled forarje down to the desired level of between 4.0 and 5.0, depending on crop moisture content.
  • this technique also suffers frcm the lack of water soluble
  • feed supplements containing concentrated, easily digested nutrients.
  • feed concentrates may supplement dietary energy and may contain corn and other cereals, or protein supplements containing soybean or other high protein sources.
  • these food concentrates are used but they are a more expensive source of energy.
  • these types of food concentrates are also well suited for human consumption, as well as for non-ruminant animal consumption. Therefore, their use as feed for ruminants is not very economical, particularly since on many farms such feed concentrates cannot be produced from home-produced crops and have to be purchased from outside sources. Consequently, new methods for improving the
  • the application of the enzyme composition of the present invention to forage also improves the digestibility of forage and thereby increases the net energy and protein intake of ruminant animals.
  • the enzyme composition of the present invention may preferably be used in combination with the addition of an effective amount of homolactic bacteria.
  • the enzyme composition of the present invention preferably contains at least one enzyme selected from the group consisting essentially of pectinase, cellulase, xylanase, amylase, arabinosidase, cutinase, lipase, and esterase.
  • the enzyme compesitier of the present invention contains the enzymes cellulase and amylase.
  • the enzyme composition of the present invention contains the enzymes xylanase and amylase. In another embodiment, the enzyme composition of the present invention contains the enzymes cellulase, amylase and pectinase.
  • the enzyme composition of the present invention contains the enzymes pectinase,
  • the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase and arabinosidase.
  • the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase, cutinase, and esterase.
  • the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase, cutinase, and lipase.
  • the enzyme composition of the present invention contains the enzymes cellulase, xylanase, and amylase, and an effective amount of homolactic bacteria.
  • the enzyme composition of the present invention contains the enzymes xylanase, amylase, pectinase, and an effective amount of homolactic bacteria.
  • the use of the enzyme composition on ensiled forage preserves and retains the nutritive value of ensiled forage by preventing the growth of harmful bacteria in the forage. While Applicant does not limit himself to any particular field of operation of the invention, it is believed that the action of the enzymes present in the enzyme composition generates an increase in the amount of fermentable substrates which can function as the water soluble carbohydrates required by the lactic acid bacteria in the production of lactic acid. The increased concentrations of lactic acid decrease the pH of the ensiled forage and thereby prevent the growth of harmful microorganisms which could degrade the ensiled forage. The original nutritive value of the ensiled forage is accordingly retained.
  • Lactic acid bacteria are present on all crops grown naturally and are of two types, homolactic and heterolactic, with the latter predominating in most areas. The total number of bacteria present are dependent on climatic conditions.
  • Heterolactic bacteria convert one mole of glucose to single moles of lactic acid, ethanol and carbon dioxide. Such inefficient conversion of sugar results in a slow and reduced production of lactic acid.
  • This enzyme composition also increases the rate of digestion and/or the digestibility of the forage. This is believed to be accomplished by removing portions of digestive- resistant or digestive-delaying substances present in the plant cells of the forage. Cuticle and waxes are examples of
  • Pre-digestion of the cell-wall matrix comprising cellulose, hemicellulose and pectins also occurs during the ensiling process which means that when treated forage is consumed, there is a quicker release of energy (and therefore reduced lag time) before rumen microbes can attach and start such fiber breakdown.
  • Pectic substances are a sort of clue present between cell walls and act to inhibit the attachment of rumen microbes in order to release their own cell wall
  • Pectinase is an enzyme which dissolves this cell wall "glue”. Hemicellulase in plants also may contain arabinose side linkages which restrict the activity of xylanase and the addition of arabinosidase will increase the effect of the first enzvme.
  • the "pre-digestion" activity of this enzyme is an enzyme which dissolves this cell wall "glue”. Hemicellulase in plants also may contain arabinose side linkages which restrict the activity of xylanase and the addition of arabinosidase will increase the effect of the first enzvme. The "pre-digestion" activity of this enzyme
  • composition will increase the surface area for attachment of rumen microorganisms and accordingly, digestion of such treated forage will occur at a faster rate and with greater efficiency than is possible with untreated .forage.
  • the ruminant can consume more and obtain more nutrients from forage with less requirement for feed concentrates.
  • Improved preservation of forage treated with an enzyme composition can improve protein utilisation by
  • the term "forage” refers to grasses, legumes, straw, whole crop cereals, corn sorghum, or other plant crop or agricultural by-products.
  • the term "effective amount" of the enzyme composition of the present invention refers to an enzyme
  • an "effective amount" of the enzyme composition of the present invention also refers to an enzyme composition having enzymatic activities sufficient to increase the rate of digestion and/or digestability of forage by
  • the term "effective amount" of homolactic bacteria when used in combination with certain enzymes, refers to an amount of homolactic bacteria sufficient to more rapidly reduce the pH and to achieve a lower pH, e.g., around about pH 4.0 - 4.5, than would be achieved in the absence of the bacteria.
  • the aboreviations used for the units of the various enzyme activities have the following definitions: (1) CMC IU - carboxy-methyl cellulose international units, (2) IU - international units (xylanase), (3) PGU - polygalacturonic units, (4) SGU-glucodmylase units, and (4) CFU - colony forming unit (bacteria).
  • forage may be harvested by a variety of methods.
  • Forage harvesting typically utilizes machinery which mows and chops the forage before ensiling.
  • D.M. dry matter
  • Corn silage on the other hand, may be harvested and ensiled as it is found on the field. Its dry matter content generally varies between 35-45%.
  • the chopping assists in enabling the forage to be tightly packed when ensiled and also liberates some of the water soluble carbohydrates present in plants which may then participate in lactic acid fermentation.
  • the harvested forage is then collected and prepared for ensiling.
  • the invention is directed to a method for preserving and enhancing the nutritive value of forage by adding an effective amount of a selected enzyme composition to the forage.
  • the application of the enzyme composition to the forage also improves the digestibility of the forage by ruminants.
  • the enzyme composition of the present invention may preferably be used in combination with the addition of an effective amount of homolactic bacteria.
  • Carbohydrates are the main repository of
  • photosynthetic energy in plants and comprise roughly 50 - 80% of the dry matter of forage and cereals.
  • the nutritive characteristics of these carbohydrates for animal feeding are variable depending on sugar compounds and linkages.
  • Carbohydrates can be divided into non-structural such as simple sugars and starch (reserve plant stores) easily digested by ruminants and structural carccnydrates slowly digested or not available to ruminants.
  • the plant cell wall is a thick complex structure which provides strength and rigidity to plants. It is composed mostly of cellulose, arranged in microfibrils and embedded in a matrix consisting largely of polysaccharides.
  • the plant cell wall is the most distinctive feature of all plant cells and is absent in animal cells. These resistant structures reduce the nutritive value of the forage plant.
  • the plant cell has two cell walls; a primary wall and a secondary wall.
  • the primary cell wall formed during the early stages of growth, is composed mostly of pectin, more broadly and accurately called pectic substances, and
  • hemicelluloses hemicelluloses.
  • Cellulose, lignin and a small amount of protein are also present.
  • Pectic substances consist essentially of a polygalacturonic acid chain substituted with araban and possibly galactan side-chains.
  • the acid groups are combined as calcium salts and as methyl esters.
  • pectic substances arp alpha 1-4 linked which causes non-linearity and coiling of the polygalacturonic chain.
  • the majority of pectic substances are thus long chains of alpha-D-galacturonic acids, with side chains composed of other sugars.
  • the proportion of pectic substances present in the plant material varies depending on the type of forage under consideration. Alfalfa has a high amount of pectic substances (5-10%), while grasses have only about 1-2%. Other legumes also have a fairly high content of pectic substances. in general, pectic substances are
  • Cellulose is the main polysaccharide in living plants and is basically a polymer of glucose. It forms the skeletal stracture of the cell wall. Cellulose is a polymer of B-D- glucopyranosyl units which are linked together, with
  • Cellulose occurs within plant cell walls mainly in the form of microfibrils arranged side by side to form layers, or lamellae, which provide support and rigidity to the cell structure.
  • Hemicellulose is a term used to describe another group of polysaccharides present in the cell wall of plants. The term is usually applied to those polysaccharides which are extractable by dilute alkaline solutions. The term has also been used to include al l the polysaccharides components of the cell wall other than cellulose. Hemicellulose is closely associated with cellulose in tne plant cell wall.
  • Lignin consists of aromatic polymerized compounds. Lignin combines with hemicellulose materials to help bird the cells together and direct water flow. In general, hemicellulose is more tightly bound to lignin chemically and/or physically than cellulose, especially after the plant matures. The presence of lignin is the main reason why older plants have reduced digestibilites.
  • the secondary cell wall is formed of highly ordered microfibrils and has three layers. It is laid down on the inside of the primary wall as the plant matures and after the cell has ceased to grow.
  • the secondary wall may vary greatly in structure and composition. It contains more cellulose than the primary wall and may contain substantial amounts of lignin and other substances.
  • a structure termed the middle lamella is present between the primary cell walls of two adjacent plant cells.
  • the middle lamella is composed of pectic substances. Lesser amounts of other sugars including L-fructose, D-xylose, and D- galactose, are also present. Pectic substances present in the middle lamella serve to cement together the primary cell walls of adjacent plant cells.
  • the cuticle of plants is a waxy layer over the outer surface of the epidermis of plants. It contains cutin, a varnish-like material and also contains waxes, resins and fatty acids. Cutin is an insoluble polyester composed principally of a C 16 and/or a C 18 family of fatty acid monomers. Most of the primary hydroxyl groups in the polymer are esterified and some of the secondary hydroxyl groups are in ester linkage.
  • the cuticle protects the plant cell against water loss and parasitic infections.
  • the cuticle, itself embedded in waxes, possesses small openings, called stomata, for gas exchange.
  • the cuticle is attached to the epidermal plant cell walls by various pectinaceous materials. In addition to protecting plants against harmful micro-organisms and regulating cuticular transpiration, the cuticle also obstructs the digestion of plant material by animals.
  • the structurally resistant components of plant cells material described above not only provide protection against natural phenomenon such as wind, rain, and drought, but also afford a defense against disease and parasitic infection.
  • Ruminants are a particular type of grazing mammal possessing a modified stomach in which the anterior portion is enlarged to form the rumen, essentially a fermentation vat in which microorganisms, such as bacteria, yeasts, and protozoa, break down the otherwise indigestible cellulose of the animal's vegetable diet. The products of this fermentation are thus available for the nutrition of the animal. Accordingly, ruminants have an advantage over monogastric swine or poultry in that they have the ability to utilize forage and roughage, such as straw, seed hulls, and corn stover. Cattle, goats and sheep are examples of domesticated ruminants which are useful in the agricultural industry.
  • ruminants when graz ing or feeding on forage, ruminants swallow the plant material rapidly and transport it to the rumen where it remains for some time. Later the rumen contents are regurgitated and masticated more thoroughly before being returned to the rumen. The mastication begins only some time after the completion of the feeding period. Microbial fermentation in the rumen begins disrupting the cellulose structure of the plant material which is helped by mastication. Rumen microbial fermentation thus releases digestible energy mainly in the form of acetic, propionic and butyric acids, from the fibrous cellulose material of forage and at the same time a microbial mass with a high protein content is synthesized. While ruminants are therefore able to overcome the natural barriers presented by plant structure to some extent, significant constraints still persist on the ruminants ability to digest forage and obtain the required energy for growth, maintenance, and reproduction.
  • the ruminant's ability to consume and digest forage is limited by the size of rumen and the rate at which the rumen fermentation process may take place.
  • rumen microbes have limited access to the structurally resistant components of the plant material, both before and after mastication occurs. Accordingly, the amount of energy which a ruminant is able to obtain per unit volume of forage consumed and the utilization of non-protein nitrogen is restricted.
  • the present invention provides a method for overcoming these difficulties and is directed to the
  • the enzyme composition will:
  • the enzymes which may be used in the enzyme composition of the present invention are pectinase, cellulase, xylanase, amylase, arabinosidase, cutinase, lipase, and esterase.
  • the enzyme composition of the present invention contains the enzymes cellulase and amylase.
  • the enzyme composition of the present invention contains the enzymes xylanase and amylase. In another embodiment, the enzyme composition of the present invention contains the enzymes cellulase, amylase and pectinase.
  • the enzyme composition of tne present invention contains the enzymes pectinase,
  • the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase and arabinosidase.
  • the enzyme composition of the present invention contains the enzymes pectinase,
  • the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase, cutinase, and esterase.
  • composition of the present invention contains the enzymes xylanase and amylase, and an effective amount of homolactic bacteria.
  • the enzyme composition of the present invention contains the enzymes cellulase, xylanase, amylase, pectinase, and an effective amount of homolactic bacteria.
  • the enzyme composition of the present invention may be added to the forage at any time prior to, during, or after the ensiling of the forage.
  • the enzyme comptsition is, however, preferably added to the forage just prior to the time of ensiling.
  • a first enzyme composition containirg at least one enzyme selected from the group
  • a second enzyme composition consisting essentially of pectinase, amylase, cutinase, lipase, and esterase may be initially added to the forage, and a second enzyme composition, containing at least one enzyme selected from the group consisting essentially of cellulase, xylanase and arabinosidase, is then subsequently added to the forage.
  • pectinase is a particularly preferred component of the enzyme composition.
  • Pectinase also called polygalacturonase, is a hydrolase which cleaves the alpha-1,4- galacturonide linkages in pectic substances. When applied to plant material, pectinase thus acts to dissolve and remove a portion of the pectic substances which constitute the plant cell binding middle lamella. This assists in breaking up the normally tightly bound plant cells and thus increases the surface area available to ruminant enzymes and rumen
  • the degradation of forage in the rumen is also able to start with a shorter lag time and the overall rate of digestion is thereby quickened.
  • the pectinase in the enzyme composition also removes some of the pectic substances present in the primary cell wall. This serves to weaken or break down the cell wall, thus making it more amenable to digestion by the ruminant.
  • Cellulase is a generic name for a group of enzymes capable of degrading cellulose.
  • the enzymology of cellulose degradation is a relatively new topic in biochemistry and its development has been limited by the lack of availability of pure enzymes.
  • cellulase catalyses the hydrolysis of cellulose to glucose and to cellcoiose, a glucose [B1-4] gluccside disacharide. The application of cellulase to forage thus increases the
  • Xylanases are enzymes which catalyze the breakdown of hemicellulose.
  • the digestion of hemicellulose is a complex matter since hemicellulose is a composite of various sugars and glycosidic linkages.
  • the character of hemicellulose differs among various forages and types of plant cell walls.
  • xylanases have the ability to cleave a variety of sugar and glycosidic linkages, especially those that are associated with the glycosidic chain of hemicellulose. By undermining the structural coherence of plant cell walls in this manner, xylanase contributes to enhanced digestibility of enzymatically treated forage.
  • pectins classify pectins with hemicellulose in one group as non-cellulosic cell wall polysaccharides.
  • the distinction between pectin and hemicellulose is by no means clear.
  • pectinases to catalyze the
  • Arabinosidases are enzymes which catalyze the breakdown of arabinose, a 5 carbon sugar common in forages which invariably occurs as a furonoside in glycoside linkages. Such linkages are thought to limit the activity of xylanases and removal by arabinosidases can enhance the breakdown of plant hemicellulose.
  • Amylases are enzymes which catalyze the breakdown of starch. Alpha-amylase cleaves starch chains randomly while beta-amylase is an exoenzyme cleaving units from the ends of cha ins. These enzymes are particularly important with legumes such as alfalfa which use starch at a major storage
  • the enzyme cutinase is capable of degrading cutin, the principal constituent of the protecting layer of cuticle present on the external surfaces of plant epidermal cells.
  • Lipases are enzymes which hydrolyze lipid esters , liberating fatty acids . When incorporated into the enzyme composition of the present invention, lipases degrade some of the waxes of the cuticle in which the cutin is embedded, thus weakening the cuticle and making the cutin and other layers underneath more accessible to degradation by cutinase or other enzymes and rumen microbes.
  • Esterases are enzymes which break down ester linkages. Since ester linkages are widespread in the
  • esterases in the enzyme composition of the present invention serves to undermine and weaken the forage material, thus increasing its digestibility by enzymes and rumen microbes.
  • the above-mentioned enzymes can be used separately, but are preferably used in some combination of two or more.
  • the enzyme composition contains singly the enzyme cellulase at an activity ranging from about 40,000 - 1,000,000 CMC lu/tn.
  • cellulase most preferably has an activity of about 500,000 CMC lu/tn.
  • this enzyme composition contains singly the enzyme xylanase at an activity ranging from about 400,000 - 3,000,000 lu/tn.
  • xylanase most preferably has an activity of about 1,000,000 lu/tn.
  • this enzyme composition contains singly the enzyme pectinase at an activity ranging from about 100,000 - 50,000,000 PGU/tn.
  • pectinase When used alone in the enzyme composition of this embodiment, pectinase most
  • the enzyme composition contains the enzymes cellulase and amylase.
  • This enzyme composition preferably has activities of about 40,000 - 1,000,000 CMC lu/tn of cellulase and about
  • the most preferred enzyme composition of this embodiment has activities of about 500,000 CMC lu/tn of cellulase and 42,000 SGU/tn of amylase.
  • the enzyme composition contains the enzymes xylanase and amylase.
  • This enzyme composition preferably has activities of about 400,000 - 3,000,000 lu/tn of xylanase and about 10,000 - 50,000 SGU/tn of amylase. The most preferred enzyme
  • the enzyme composition contains the enzymes xylanase and amylase, and an effective amount of homolactic bacteria.
  • This enzyme composition preferably has activities of about 400,000 - 3,000,000 lu/tn of xylanase, about 10,000 - 50,000 SGU/tn of amylase, and a level of bacterial addition ranging from about 100,000 - 1.000.000 CFU LAB/g crop.
  • the most preferred enzyme composition of this embodiment has activities of 1,000,000 lu/tn xylanase, 42,000 SGU/tn amylase, and a level of bacterial addition of about 500,000 CFU LAB/g crop.
  • the enzyme composition contains the enzymes
  • This enzyme composition preferably has activities of about 400,000 - 3,000,000 lu/tn of xylanase, about 10,000 - 50,000 SGU/tn of amylase, about 100,000 - 50,000,000 PGU/tn of pectinase, and a level of bacterial addition ranging from about 100,000 - 1,000,000 CFU LAB/g crop.
  • the most preferred enzyme composition of this embodiment has activities of 700,000 lu/tn of xylanase, 25,000 SGU/tn of amylase, 200,000 PGU/tn pectinase, and a level of bacterial addition of about 1,000,000 CFU LAB/g crop.
  • the enzyme composition contains the enzymes
  • This enzyme composition preferably has activities ranging from about 40,000 - 1,000,000 CMC lu/tn of cellulase and about 400,000 - 3,000,000 lu/tn of xylanase.
  • the most preferred enzyme composition of this embodiment has activities of about 300,000 CMC lu/tn of cellulase and about 1,000,000 lu/tn of xylanase.
  • the enzyme composition contains the enzymes pectinase, cellulase, and amylase.
  • This enzyme composition preferably has activities of about 100,000 - 50,000,000 PGU/tn of pectinase, 40,000 - 1,000,000 CMC lu/tn of cellulase, and about 10,000 - 50,000 SGU/tn of amylase.
  • the most preferred enzyme composition of this embodiment has activities of, about 200,000 PGU/tn of pectinase, about 250,000 CMC lu/tn of
  • the enzyme composition contains the enzymes
  • pectinase preferably has activities of about 100,000 - 50,000,000 PGU/tn of pectinase, 400,000 - 3,000,000 lu/tn of xylanase, and about 10,000 - 50,000 SGU/tn of amylase.
  • the most preferred enzyme composition of this embodiment has activities of about 200,000 PGU/tn of pectinase, 750,000 lu/tn of xylanase, and about
  • the enzyme composition contains the enzymes
  • This enzyme composition preferably has activities, per ton treated, of about 40,000 - 1,000,000 lu/tn pectinase, about 50,000 - 1,000,000 lu/tn cellulase (CMC) and about 400, 000 - 3,000,000 lu/tn xylanase.
  • the most preferred enzyme composition of this embodiment has activities of about 100,000 PGU/tn pectinase, 300,000 (CMC) lu/tn cellulase, and 700,000 lu/tn xylanase. In this
  • the enzymes pectinase, cellulases, and xylanases together display a particularly beneficial effect in enhancing silage quality and improving digestion of forage by ruminants.
  • the enzyme composition contains the enzymes pectinase, cellulase, xylanases and arabinosidase. This enzyme composition
  • the most preferred enzyme composition of this embodiment has activities of about 100,000 PGU/tn pectinase, 300,000 (CMC) lu/tn
  • this enzyme composition contains the enzymes pectinase, cellulase, and arabinosidase.
  • This enzyme composition preferably has activities ranging from about 100,000 - 5,000,000 PGU/tn of pectinase, 40,000 - 1,000,000 CMC lu/tn of cellulase, and about 10,000 - 100,000 lu/tn of arabinosidase.
  • This enzyme composition preferably has activities of about 2,000,000 PGU/tn of pectinase, 1,000,000 CMC lu/tn of cellulase, and about 50,000 lu/tn of
  • the enzyme composition contains the enzymes
  • pectinase preferably has activities of about 40,000 - 1,000,000 PGU/tn pectinase, 40,000 - 1,000,000 CMC lu/tn cellulase, 400,000 - 3,000,000 lu/tn xylanase, 50 - 1,000,000 lu/tn cutinase, 50 - 100,000 lu/tn esterase.
  • This enzyme composition preferably has activities of about 40,000 - 1,000,000 PGU/tn pectinase, 40,000 - 1,000,000 CMC lu/tn cellulase, 400,000 - 3,000,000 lu/tn xylanase, 50 - 1,000,000 lu/tn cutinase, 50 - 100,000 lu/tn esterase.
  • the most of the pectinase preferably has activities of about 40,000 - 1,000,000 PGU/tn pectinase, 40,000 - 1,000,000 CMC lu/tn
  • preferred enzyme composition of this embodiment has activities of about 100,000 PGU/tn pectinase, 100,000 CMC lu/tn cellulase, 700,000 lu/tn xylanase, 1,000 - 1C,000 lu/tn cutinase, and 1,000 - 20,000 lu/tn esterase.
  • the enzyme composition contains the enzymes pectinase, cellulase, xylanase, cutinase, and lipase.
  • This enzyme composition prererably has activities of about 40,000 - 1,000,000 PGU/tn pectinase, 40,000 - 1,000,000 CMC IU/tn cellulase, 400,000 - 3,000,000 IU/tn xylanase, 50 - 100,000 IU/tn cutinase, and 1,000 - 10,000,000 IU/tn lipase.
  • the most preferred enzyme composition of this embodiment has activities of about 100,000 PGU/tn pectinase, 100,000 IU CMC/tn cellulase, 700,000 IU/tn xylanase, 1,000 - 10,000 IU/tn cutinase, and
  • arabinosidase, pectinase, lipase, cutinase, and esterase of the present invention are manufactured preferably in a fermentation process.
  • cellulase hemicellulase, arabinosidase, esterase and pectinase belong to Trichoderma, Aspergillus, Penicillum or other
  • Cutinase can be obtained, for instance, from
  • the enzymes can be applied to the forage in a solution, preferably in a diluted solution to ensure even distribution, or as a dried powder preferably with a suitable carrier, e.g. starch or grain.
  • a suitable carrier e.g. starch or grain.
  • enzyme composition of the present invention may be applied to the forage at any time before, during, or after the ensiling of the forage, it is preferred to add the enzyme composition to the forage after harvesting just prior to the time the forage is ensiled.
  • Enzyme compositions are typically liquids which can be diluted with water before application to ensure good crop cover.
  • the enzymes, including homolactic bacteria when it comprises an additional component of the enzyme composition of the present invention can be applied as a coarse spray over the feed rollers of the forage harvester, into a blower when filling silos, on crop when filling bunker silos or as bunker silos are being filled.
  • Enzymes thus applied to the forage serve to improve the preservation quality of the forage, including palatability and to improve the nutritive value of the forage.
  • the preservation guality is improved by the enzymes cellulase, xylanase and pectinase, which liberate soluble sugars from polysaccharides.
  • Lactic acid bacteria ferment the sugars to organic acids, mainly lactic acid, decreasing the pH and thus inhibiting the growth of harmful bacteria.
  • the harmful bacteria like clostridia are more sensitive to low pH and osmotic pressure than the lactic acid bacteria are.
  • pectinase can be used to improve the accessibility of other enzymes and rumen microbes to the forage cell wall.
  • the enzymatic effects produced by the enzymatic composition accordingly act to weaken or remove certain substances which normally interfere with efficient ruminant digestion of plant material, thus increasing the rate of
  • pectin is a glue between the adjacent cell walls, the removal of pectin opens up the structure of the plant.
  • Ruminants which are fed forage treated with the enzyme composition of this invention may also be able to produce larger quantities of meat and milk when compared to ruminants fed with untreated forage. This is because the ruminant is able to more efficiently convert forage treated with the enzyme composition to energy and protein which can be used by the ruminant for meat and milk production.
  • the digestion of forage starts from the open cut ends of stem or leaf pieces while the cuticle surface is left untouched.
  • the ruminant animal starts the mastication of feed after a certain period of resting (e.g. half an hour).
  • barriers of digestion, including the surface cuticulum are weakened physically. This makes the structures under the cuticulum a little more available for digestion by rumen microbes, although the
  • cuticulum still covers much of the forage structures.
  • the cuticulum can be weakened or removed, thus making the forage more susceptible to digestion by other enzymes and/or rumen microbes.
  • the weakening of the structure partially acts synergistically with the mastication which then unorganizes the forage structures to a greater extent.
  • the enzyme composition of the present invention further provides an increase in the amount of digestible protein which is available to the ruminant. This leads to additional economic benefits which are associated with the present invention, i.e., a reduction of the amount of money that must be spent en purchases of supplementary dietary protein for the animal.
  • the extent of digestibility of forage in the rumen is also dependent on the rate of passage of undigested particles from the rumen to the intestine. Faster breakage of forage and improved digestibility occur as a consequence of cutinase and lipase application. In the cases where intake is increased, the improvement in digestibility is smaller. In any case, the digestible energy uptake of the animal from plant sources is increased.
  • Lactic acid bacteria are present on all crops grown naturally and are of two types, homolactic and heterolactic, with the latter predominating in most areas. The total number of bacteria present are dependent on climatic conditions.
  • Heterolactic bacteria convert one mole of glucose to single moles of lactic acid, ethanol and carbon dioxide. Such inefficient conversion of sugar results in a slow and reduced production of lactic acid.
  • plants contain 60 - 80% true protein mainly in the leaves and 20 - 40% non-protein nitrogen (NPN) in the form of nitrate and non-essential amino acids. Typical values of NPN for fresh forage are shown below.
  • ADF acid detergent fiber
  • Silage proteins are also classified by
  • solubility where soluble nitrogen will include all NPN and soluble true protein (2) insoluble but available nitrogen which can escape rumen degradation, and (3) unavailable bound nitrogen.
  • the proportion of each group can be drastically changed by a slow pH drop after ensiling.
  • the main form of nitrogen utilized by rumen microorganism is ammonia and it is into this common substance that much of the dietary non-protein nitrogen is converted.
  • the nitrogen supply will promote microbial growth up to the limit of the microbial nitrogen requirement. This requirement is set by the available fermentable carbohydrate, ATP yield, and the efficiency of conversion to microbial rells. It is this first factor, i.e., available carbohydrate that enzyme treated forage can readily supply, which maximizes protein and, in particular, NPN utilization by the ruminant.
  • a surplus in the level of ammonia which is in excess of microbial requirements is absorbed across the rumen wall into the blood stream where it is transported to the liver for conversion into urea.
  • Large inputs of dietary NPN can cause ammonia production beyond the conversion capacity of the liver, causing a rise in blood ammonia levels resulting in toxicity.
  • Death from ammonia poisoning which can arise from feeding urea results from the creation of levels of blood ammonia beyond which the buffering capacity of blood is exceeded, causing a rise in pH and impairment of the blood's capacity to expel carbon dioxide.
  • blood urea normally measured as Blood Urea Nitrogen (BUN)
  • BUN Blood Urea Nitrogen
  • High levels of dietary NPN may also be a factor in limiting the intake of poor quality silages where utilization is limited by the low digestibility of energy and, therefore, microbial protein yield.
  • the present invention assists in preventing some of these problems associated with the presence of excess ammonia in the blood.
  • the enzyme compositions of this invention breakdown plant fiber to supply sugars for conversion to lactic acid. The resulting rapid fall in pH in the forage will minimize prctein breakdown by clostridial oacteria (lower NPN values shown as NH 3 in silage analysis) and reduced heat production, so eliminating any Maillard reaction causing heat bound protein (ADF bound nitrogen).
  • the overall benefit to the ruminant is that more dietary nitrogen is retained and utilized by the animal. It is also possible that improved con ception rates are achieved when animal is fed forage which has been treated with the enzyme composition of the present invention.
  • Second cut alfalfa was field wilted for 4 to 6 hours before being ensiled at 50% dry matter.
  • the crop contained crude protein (CP) 18.1% in dry matter (dm), acid detergent fiber (ADF) 34.1% in dm, buffering capacity 53.1 m. equi. NaOH/100 g crop and water-soluble carbohydrates (WSC) 7.4% in dm.
  • CP crude protein
  • ADF acid detergent fiber
  • WSC water-soluble carbohydrates
  • silos Some 100 to 125 lbs crop were treated with appropriate treatment before ensiling in 4" diameter x 14" long PVC laboratory silos. Silo ends were sealed with rubber caps (one fitted with bunsen valve to release gases) after hydraulic compression to a common density. Three silos were filled for each treatment for opening at each timed interval, e.g., 0.5, 1, 2, 4, 7, 14 and 90 days (3 x 7 silos/treatment).
  • Lactic acid and pH levels were measured at each interval sampling; acetic acid, ethanol and ammonia contents were measured in addition at the 14 and 90 day sampling.
  • Table 1 shows the pH reduction against time achieved with the six treatments.
  • Table 2 shows fermentation quality of silage sampled after 90 days ensiling.
  • the aim of efficient ensiling is to rapidly produce lactic acid to reduce pH and to limit
  • Table 3 shows the pH reduction against time achieved with the six treatments.
  • Table 8 shows fermentation quality of silage after 90 days ensiling.
  • Enzyme addition showed small improvements in ammonia nitrogen levels over control indicating less protein
  • xylanase 250 ml/ton crop activity 5,000 IU/ml plus 1,400 IU CMC/ml, amylase 250 ml/ton, activity 170 SGU/ml and 60,000 IU/ml, and bacteria at 585,000 CFU LAB/g crop.
  • Table 5 shows the pH reduction against time achieved with the three treatments.
  • Table 6 shows fermentation quality of silage after 90 days ensiling.
  • Silage from the 55 gallon silos was fed ad-libitum to groups of 9 wether sheep. Feed was weighed for each group and the amount of silage consumed was measured to assess voluntary dry matter intake. Table 7 shows the results of silage analyses and the feed trial.
  • cellulase 100 ml/ton of crop activity 2,500 IU CMC/ml, xylanase 150 ml/ton, activity 5,000 IU/ml, amylase 150 ml/tn, activity 170 SGU/ml and 60,000 LU/ml, pectinase 100 ml/ton, activity 2,000 IU PG/ml and bacteria 1 million CFU LAB/g crop.
  • Table 8 shows the pH reduction against time achieved with the five treatments.
  • the alfalfa was highly buffered but, as shown in Table 8, displayed a sharp drop in the first 24 hours in pH from around 6.0 to around 5.0 with all treatments. Both treatments containing pectinase resulted in the lowest final pH after 90 days ensiling, and in general enzyme compositions of this invention with and without bacteria showed lower pH values at each interval compared with control or bacteria alone.
  • Table 9 shows fermentation quality of silage after 90 days ensiling.
  • Table 9 shows that enzyme compositions of this invention, except for amylase alone, increased the content of lactic acid and decreased ammonia nitrogen content (protein breakdown). Amylase addition again increased ethanol content when homolactic bacteria were not added.
  • silage was frozen with dry ice from each treatment and ground to pass a 4 mm screen in a Wiley Mill.
  • the ground forages were kept frozen until 24 hours before feeding rumen simulation fermenters.
  • the increase in digestible protein was estimated in rumen simulation fermenters. Such fermenters simulate rumen conditions (in-vitro) in the laboratory using actual rumen liquor. The fermenters were fed continuously with treated silage blended with grain in a 65:35 ratio to enhance
  • results show an increase in the yield of microbial protein of 0.5 1b/day when using silage treated with an enzyme composition of this invention. Furthermore, since the protein status of the trial was sub-optimal in terms of ruminally available protein, it is estimated that the increase in yield would be doubled with optimum protein status.
  • Table 11 shows that, in most parameters, treatment 5, consisting of the enzymes and bacteria achieved better results than the other treatments, and in six measurements the differences were statistically significant.
  • Table 12 shows that the enzyme compositions of this invention improved both rate and extent of NDF degradation over control .
  • Treatment 5 only showed a reduced lag time, i.e., with NDF degradation starting before first sampling at four hours and showed also maximum reductions at later sampling times.
  • fill values which are calculated as follows:
  • Second cut alfalfa was field wilted for up to 12 hours before being ensiled at 35% dry matter on a commercial farm. Crop was ensiled after one of the following treatments.
  • the formic mixture additive is designed to chemically protect protein to avoid rumen degradation which causes high urea nitrogen levels in the blood resulting in poor herd reproduction.
  • Table 14 shows that treatment with an enzyme composition of this invention resulted in lower ammonia concentration (less protein breakdown) and lower degradeability than both control and the formic product.
  • the silages were then fed in sequence to the dairy herd over periods of 30 days and, because of problems with herd reproduction, blood samples were taken from some animals at the end of each period. Samples were analyzed for blood urea nitrogen (BUN) and results are shown in Table 15.
  • BUN blood urea nitrogen
  • the enzyme composition of this invention has improved the utilization of NPN by supplying extra energy from digested fiber, resulting in BUN levels close to recommended levels of 16-17 mg/ml.
  • the reason for this reduction is due to the increased energy available for microbial growth resulting from enzyme activity on structural carbohydrates.
  • Increased microbial growth means enhanced capture of non-protein nitrogen (NPN) in the silage, a s well as reduced degradation of NPN to ammonia with a concomroitant lower requirement for excretion of this product in blood as urea (i.e., lower EUN levels).
  • NPN non-protein nitrogen
  • Second-cut alfalfa was field-wilted for up to 12 hours before being ensiled at 38% dry matter in tower silos. Crop was ensiled after one of the following treatments:
  • the silages were fed to four groups of 20 cows for the first 16 weeks of lactation. Newly-calved cows are used in this lactation trial when a high quality diet is required because dry matter intakes are reduced at a time when milk production is at a maximum.
  • Body fat may be mobilized to try to bridge the energy deficit. This is shown in loss of body condition or condition score and, if body fat is not utilized efficiently, this can lead to a condition known as ketosis. Fat utilization requires extra energy from carbohydrates and so increases the demand for dietary energy.
  • the diets for the groups were constructed as follows:
  • ground corn, soybean meal and minerals feed formulation based on NDF and non- structural content (NSC) of total feed ingredients in accordance with
  • GROUP II Normal NDF - enzyme-treated alfalfa, ground corn, soybean meal and minerals as for Group I - contains more alfalfa silage because of NDF reduction in silo.
  • NDF neutral detergent fiber
  • ADF acid detergent fiber
  • Normal NDF 0.71
  • High NDF 0.66 Mcal/lb.
  • those on high NDF diets must be obtaining the same energy as those on normal diet.
  • Protein arising from both soybean meal and ground corn is reduced by some 45%, whereas forage protein has been increased by over 50%. Although this increased use of forage would increase the supply of ruminally available protein, enhanced fiber digestion, and therefore energy supply, in the rumen due to enzyme activity allows efficient utilization of such NPN.
  • Body condition of each group was similar at the start of the trial but animals in this group very rapidly lost weight in an attempt to maintain milk production. Body condition score dropped from 3.7 to 2.7 in 3 weeks.
  • cows in Group III would mobilize and utilize reserves of back fat in order to maintain milk production.
  • Fat is the main energy reserve in such animals and under-feeding requires that a greater proportion of metabolized energy comes from fat than in adequately fed cows.
  • oxalacetate is essential, derived from carbohydrate precursors that are also in short supply at a low level of nutrition.
  • Group IV cows on the apparently low energy diet with alfalfa silage treated with an enzyme composition of this invention are expected to continue to produce similar amounts of milk to Group I and II cows without loss of body condition and without any cases of Ketosis.
  • the untreated diet is not expected to maintain levels of production recorded for other groups.
  • BUN levels in the other two groups (I and II) are also expected to be different although not as great as
  • cows eating treated silage will consume more feed and produce more milk with greater efficiency than cows on control silage.
  • the additional cost of using the enzyme composition i.e., $3.50/ton, is included in the actual cost of such treated silage. If some other treatment is used, the cost of this other treatment shculd be included in this comparison. It is also important to note that the untreated silage used in this trial is of a very good quality and is ensiled in a tower silo. If silage of average quality were fed to the ruminant, even greater economic benefits would be seen, such as would occur if a bunker silo were used.
  • the change in feed composition arising from the use of an enzyme composition of this invention indicates a daily saving of some 50 to 60 cents/cow or $50-$60/day for a 100 cow herd above the cost of treatment. This is a saving of 21% in total feed costs.
  • Timothy grass was harvested with a direct cut harvester, when the grass was 100* emerged.
  • the dry master of the crop was about 20%, the crude protein 12% of D.M., neutral detergent fiber (NDF) 63%, and acid detergent fiber (ADF) 36% of D.M.
  • the grass was ensiled in three different ways: (1) without an additive (i.e., the control); (2) with the addition of cellulase and xylanase enzymes; and (3) with the addition of cellulase, xyianase and pectinase enzymes.
  • the grass was ensiled in duplicate in 5 kg PVC laboratory tube silos.
  • the silos were opened up after a 5 month ensiling period in a temperature of 20-25oC.
  • the quality of the silages was then determined.
  • the digestibility of silages was then measured by the in sacco method.
  • This method involves the suspension of nylon bags (mesh size 40 um) containing a weighed sample of feed in the rumen of a cannulated dairy cow. Bags are suspended by nylon cords from the cannula top and can be withdrawn for sampling and analysis. Feed is incubated in the rumen contents allowing free access to rumen microorganisms. Samples were analyzed at Oh (zero hours, i.e., washing with water only), 3h,
  • Table 23 demonstrates the capability of different enzyme compositions to break down the ensiled forage .
  • digestibility of silage treated with the enzyme compositions of the present invention was significantly enhanced at the earlier incubation periods (Oh-6h) , as compared to untreated silage . This indicates that silage treated with the enzyme compositions of the present invention are degraded at a faster rate than silage which is not treated with such enzyme compositions .
  • the data also shows the synergistic effect of pectinase and cellulase and xylanase at the early stages of digestion.
  • the enzyme compositions of the present invention can therefore improve overall protein utilization of treated silage. This is of particular interest in protein-rich alfalfa silages.
  • Example a Another study similar to that made in Example a was made with forage containing about 35% Red Clover and 70% of mostly meadow fescue forages.
  • the dry matter content of raw material was about
  • Fermentation quality is judged by the amount and type of acids produced, final pH level and amount of protein
  • Treatment of crop with enzyme compositions of the present invention increased total acid production (sum of lactate, acetate, propionate and butyrate) by a factor of 2-3 times that in untreated control. This indicates the efficacy of substrate release to lactic acid bacteria by the enzyme compositions.
  • the type of acid produced is also a factor of fermentation quality with lactate preferred; enzyme treated silages contained between 81% and 89% of total acids as lactate compared with 33% for control. Butyrate is not wanted and is indicative of some degradation; enzyme treated silages
  • the pH is low for the enzyme treatment silages as a result of good homolactic fermentation. Lactic acid content increases when pectinase is added as a consequence of increased sugar production resulting from increased cellulase (treatment 4 vs. treatment 2) or cellulose and xylanase hydrolysis (treatments 5 or 6 vs. treatment 3). It can also be seen that the higher level of pectinase was more effective as the lactic acid content is higher with treatment 6 than with treatment 5. Also, values for volatile nitrogen were lower for silages treated with enzyme compositions of the present invention, indicating less protein breakdown during ensiling. This demonstrates that the silage treated with the various enzyme compositions of the present invention had an enhanced nutritive value when compared to untreated silage.
  • Example 9 The digestibility of silages was measured by the in sacco method using the same procedure followed in Example 8, except that the incubation periods were different. The measurement after three hours was omitted in Example 9. This method involves the suspension of nylon begs (mesh size 40 um) containing a weighed sample of feed in the rumen of a
  • cannulated dairy cow Bags are suspended by nylon cords from the cannula top and can be withdrawn for sampling and analysis. Feed is incubated in the rumen contents allowing free access to rumen micro-organisms. Samples were analyzed at Oh (zero hours, i.e., washing with water only), 6h, 12h, 24h, 48h, and 96h, washed under running water and the residue weighed after drying in an oven. Table 25 shows the results of this study.
  • Table 25 demonstrates the improved capability of the various enzyme compositions of the present invention to degrade the ensiled forage of Example 9.
  • the digestibility of silage treated with the enzyme compositions of the present invention was significantly enhanced at nearly every period of incubation measured as compared to untreated silage. This effect was more pronounced at the earlier incubation periods (0h-12h). This indicates that silage treated with the enzyme compositions of the present invention is degraded at a faster rate and to a more complete degree than silage which is not treated with such enzyme compositions.
  • pectinase gave improvements over cellulase or cellulase + xylanase alone.
  • the higher amount of pectinase was also more effective compared to the lower amount in this example.
  • Pieces of Timothy (Phleum pratense) stem were cut and incubated in 20 ml 0.05 M Na-Citrate buffer at pH 5.0, temperature 30o for 20 hours with and without lipase. After incubation, the samples were dried and the cuticulum was examined.

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Abstract

Le procédé décrit consiste à ajouter à un fourrage destiné à la consommation par des mamifères une composition enzymatique contenant au moins une enzyme choisie dans le groupe composé essentiellement de pectinase, de cellulase, de xylanase, d'amylase, d'arabinosidase, de cutinase, de lipase et d'estérase. Cette composition enzymatique peut de préférence être utilisée en combinaison avec l'addition d'une quantité efficace de bactéries homolactiques. L'addition de la composition enzymatique, avec ou sans les bactéries homolactiques, au fourrage conserve et accroît la valeur nutritive du fourrage, tout en améliorant sa digestibilité par les mammifères.
PCT/FI1991/000118 1990-04-18 1991-04-18 Fourrage traite par voie enzymatique pour la conservation en silo WO1991015966A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010945A1 (fr) * 1990-12-17 1992-07-09 Biotal Ltd. Formulation destinee au traitement de recoltes ensilees
WO1996017525A1 (fr) * 1994-12-07 1996-06-13 Biotal Ltd. Micro-organismes, enzymes et leur utilisation
US5720971A (en) * 1995-07-05 1998-02-24 Her Majesty The Queen In Right Of Canada, As Represented By The Department Of Agriculture And Agri-Food Canada Enzyme additives for ruminant feeds
EP0906952A2 (fr) * 1997-09-26 1999-04-07 Medipharm AB Souche bactérienne pour l'ensilage de paille
US5948454A (en) * 1992-04-10 1999-09-07 Ssv Development Oy Method for treatment of fibrous crops with a modified cellulase to improve feed values storage and other properties
EP1431692A2 (fr) * 2002-12-16 2004-06-23 Ekokraft Gotland AB Procédé de séchage de plantes de pâturage
CN110373431A (zh) * 2019-07-22 2019-10-25 徐州工程学院 一种无灰高热值生物质燃料的制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IE20040144A1 (en) * 1990-07-24 2004-05-19 Dsm Nv Cloning and expression of xylanase genes from fungal origin

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4247561A (en) * 1979-04-16 1981-01-27 Nelson R W Process and method of use for a stable emulsified edible liquid starch product
US4292331A (en) * 1976-08-17 1981-09-29 Ceva Process and composition for the preservation of vegetables
CH643988A5 (fr) * 1979-02-12 1984-07-13 Sanofi Sante Animale Sa Procede et composition pour la conservation de vegetaux en vert et de sous-produits humides des industries agro-alimentaires.
US4751089A (en) * 1982-11-02 1988-06-14 Suomen Sokeri Oy Composition and method for ensilaging fodder and grain
EP0311469A2 (fr) * 1987-09-02 1989-04-12 Plant Genetic Systems N.V. Bactéries lactiques transformées
EP0346909A2 (fr) * 1988-06-17 1989-12-20 Cultor Oy Procédé par fermentation et additif pour la conservation de fourrage
EP0369198A2 (fr) * 1988-11-14 1990-05-23 Valio Meijerien Keskusosuusliike Conservation de fourrage

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4292331A (en) * 1976-08-17 1981-09-29 Ceva Process and composition for the preservation of vegetables
CH643988A5 (fr) * 1979-02-12 1984-07-13 Sanofi Sante Animale Sa Procede et composition pour la conservation de vegetaux en vert et de sous-produits humides des industries agro-alimentaires.
US4247561A (en) * 1979-04-16 1981-01-27 Nelson R W Process and method of use for a stable emulsified edible liquid starch product
US4751089A (en) * 1982-11-02 1988-06-14 Suomen Sokeri Oy Composition and method for ensilaging fodder and grain
EP0311469A2 (fr) * 1987-09-02 1989-04-12 Plant Genetic Systems N.V. Bactéries lactiques transformées
EP0346909A2 (fr) * 1988-06-17 1989-12-20 Cultor Oy Procédé par fermentation et additif pour la conservation de fourrage
EP0369198A2 (fr) * 1988-11-14 1990-05-23 Valio Meijerien Keskusosuusliike Conservation de fourrage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
F.A. SKINNER et al., "Microbiology in Agriculture, Fisheries and Food", ACADEMIC PRESS, LONDON; NEW YORK; SAN FRANCISCO, see page 116. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010945A1 (fr) * 1990-12-17 1992-07-09 Biotal Ltd. Formulation destinee au traitement de recoltes ensilees
US5432074A (en) * 1990-12-17 1995-07-11 Biotal Ltd. Formulation for treating silage containing β-1,4-xylanase and β-1,3-xylosidase but essentially free of β-1,4-glucanase and β-1,4-cellobiohydrolase, and one or more lactic acid-producing bacteria
US5948454A (en) * 1992-04-10 1999-09-07 Ssv Development Oy Method for treatment of fibrous crops with a modified cellulase to improve feed values storage and other properties
WO1996017525A1 (fr) * 1994-12-07 1996-06-13 Biotal Ltd. Micro-organismes, enzymes et leur utilisation
US5720971A (en) * 1995-07-05 1998-02-24 Her Majesty The Queen In Right Of Canada, As Represented By The Department Of Agriculture And Agri-Food Canada Enzyme additives for ruminant feeds
EP0841859B2 (fr) † 1995-07-05 2009-02-18 Her Majesty the Queen in Right of Canada, representend by the Department of Agriculture and Agri-Food Canada Additifs a base d'enzymes pour aliments pour ruminants
EP0906952A2 (fr) * 1997-09-26 1999-04-07 Medipharm AB Souche bactérienne pour l'ensilage de paille
EP0906952A3 (fr) * 1997-09-26 1999-12-22 Medipharm AB Souche bactérienne pour l'ensilage de paille
EP1431692A2 (fr) * 2002-12-16 2004-06-23 Ekokraft Gotland AB Procédé de séchage de plantes de pâturage
EP1431692A3 (fr) * 2002-12-16 2006-08-02 Ekokraft Gotland AB Procédé de séchage de plantes de pâturage
CN110373431A (zh) * 2019-07-22 2019-10-25 徐州工程学院 一种无灰高热值生物质燃料的制备方法
CN110373431B (zh) * 2019-07-22 2023-02-24 徐州工程学院 一种无灰高热值生物质燃料的制备方法

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