TECHNICAL FIELD

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.

BACKGROUND OF THE INVENTION

Methods of promoting and enhancing the quantity and the nutritive quality of foodstuffs through the use of enzyme compositions have long been researched and applied in the agricultural and livestock industries. , Such enzyme 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 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.

It is also known to use enzymes in the production of dairy products. For example, 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 administering an enzyme composition to the livestock. This enzyme ccrposition may include cellulase and pectinase as well as ether enzymes. In addition, the use of the enzyme glucose oxidase in ensiling fcrage material has been described in U.S. Pa s.-.t Nc. 4,751,089. This patent discusses the depletion of

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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.

For example, 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.

S.U. Specification to an Inventor's Certificate No. 1,232,204 describes a method of preparing fodder for cattle from freshly mowed lucerne by treating ground lucerne with an enzyme preparation possessing alph≥-amylase, pectinase, xylanase ar.d cellulase activities. The enzyme preparation makes it possible to extract protein from the hydrolyzable green mass, and to disintegrate saponins in all products resulting from fracticning the freshly-mowed mass of lucerne.

None of the above discussed references, however, discloses a method for preserving the nutritive value of ensiled forage by treatment with an enzyme or enzymes with and without bacteria which act to prevent the growth of harmful

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bacteria which degrade forage components and produce undesirable compounds. For this reason, resulting silage is more palatable allowing maximum dry matter intake of feed.

These references also do not teach applying an enzyme composition to ensiled forage which, by breaking down digestive resistant substances present in. plant cells, "pre-digests" plant fiber, so releasing sugar for immediate use when consumed by ruminants. Such action also increases the surface area available for a ruminant's natural microorganisms present in the rumen to attach to and to digest forage thereby increasing the rate of digestion of treated forage. These references also do not disclose the improved capture of ammonia generated from plant non-protein nitrogen (NPN) in the rumen by rumen microorganisms, so increasing the production of digestible microbial protein, resulting in a lower requirement for additional dietary protein from other sources. This activity reduces the amount of ammonia excreted via the blood as blood urea nitrogen (BUN) requiring a significant use of dietary energy for the conversion of ammonia in the liver to urea. Another related area of interest has been the improving of storage and preservation of foodstuff for ruminant animals. Methods of storing and preserving feed for cattle during the winter season have long been desirable because forage crops do not grow or else have limited productivity during the winter months, and ruminants cannot graze fields and pastures at this time.

The ensiling of surplus forage grown and harvested over the summer and fall has proven to be a useful and convenient method to preserve feedstuffs for cattle fcr feeding during the winter months. Ensiling is the packing cf direct

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cut green fodder or herbage into an airtight pit, bunker, or tower for its preservation. Tne exclusion of air with consequent fermentation of carbohydrates in the forage produces organic acids, mainly lactic acid, which act as a preservative. Grasses, legumes and corn are typical of crops which may be stored in this manner. The major crops fed as silάge during the winter are alfalfa, grasses (ryegrass, timothy, orchardgrass, bluegrass, bermudagrass) , and 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.

During the ensiling period, however, 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. During the course of their metabolism, lactic acid bacteria convert glucose to lactic acid which reduces the pH of the ensiled forage to a level sufficient to prevent cr inhibit the growth of other microorganisms. The level at which a certain pH will inhibit growth of harmful

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microorganisms such as clostridia depends on several variables, including water activity or buffering capacity. However, in practice the following relationship between water content of crop and pH is acceptable: (a) 70 - 80% crop moisture content (m.c), pH 4.0 - 4.1; (b) 60 - 70% m.c, pH 4.2 - 4.4; and (c) 50 - 60% crop m.c, pH 4.7 - 4.9.

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 available. If these are used up before the inhibiting pH is achieved, clostridia can metabolise both sugars and lactic acid producing butyric acid, carbon dioxide and hydrogen. Such by¬ products will cause pH to rise, proteins will be degraded and the resulting silage will be odorous, unpalatable and of low quality.

The growth of such microorganisms is prevented because the high osmotic pressure brought about by the low pH prevents the growth of the harmful microorganisms. Inhibition of growth by lactic acid is caused by the increase in hydrogen ion concentration and also by the undissociated acids themselves. Consequently, the original forage composition is better preserved since the harmful microorganisms are net allowed to develop. The lactic acid bacteria, on the other hand, are able to survive at the low pH and high osmotic pressure levels present in the ensiled forage.

However, continued preservation of forage by this lactic acid fermentation process is dependent upon the sustained production of lacrxc acid to stabilize the silage at a low pH. Since lactic acid production is ften constrained by

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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.

These adverse results have typically been forestalled or mitigated by the addition of certain additives to the forage before, during, or after ensiling. The class of additives that has received the most interest is the industrial additives, including sulfuric acid, hydrochloric acid, phosphoric acid, and formic acid. The application of large quantities of these acids to the forage brings the pH down to about 4.0 and thereby essentially blocks all fermentation, eliminates fermentative loss, and preserves the nutrients in the ensiled forage. These additives are typically added before ensiling. However, use of these additives is undesirable because their corrosive properties are harmful to humans and farm equipment. There are also problems associated with the commercial production, availability and economic cost of these additives. Other additives which have been used or experimented with to a limited extent include salts, antibiotics, urea, ammonia, ammonium salts and formaldehyde.

Alternatively, the water soluble carbohydrates required by the lactic acid bacteria for the lactic acid fermentation process, -ay 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

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of fermentable carbohydrate which aids lactic fermentation. However, there are a number of difficulties associated with this approach. Generally, a large amount of sugar addition is needed, i.e. 10 - 20 kilograms εugar/tn. Molasses requires a usage rate of between 80 and 100. lbs/ton crop and it is difficult to apply this high quantity of a viscous material to the crop. Application of dry form additives evenly on grass is also difficult. Such application might also be uneconomical, especially if sugar is used. Starch cannot be used effectively by lactic acid bacteria unless amylase is present to convert the starch to sugar. Sugars such as lactose and sucrose, although effective, are uneconomic at the required use rate levels.

Finally, the lactic acid bacteria themselves may be introduced into the ensiled forage to lower the pH of the ensiled forare down to the desired level of between 4.0 and 5.0, depending on crop moisture content. However, this technique also suffers frcm the lack of water soluble carbohydrates available in the ensiled forage which are necessary to ensure the continued production of lactic acid by the bacteria ar.d the sustained low pH levels needed to protect the forage against degradation by harmful microorganisms.

Accordingly, improved methods for preserving the nutritive quality of forage during ensiling are desirable. Finding new ways to increase the efficiency at which ruminant farm animals are able to obtain nourishment from forage is also currently under active investigation. Ruminant farm ar.'.mals are most usually raised in order to produce -'.ilk a meat for human consumption. However, the rate ^~ .~- which such farm animals can produce milk and meat i . limited by the

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fairly low digestibility of forages resulting from the structural composition of the plant material. The large bulkiness of forage also constrains the ability of ruminant farm animals to consume forage since the volume of forage intake is limited by the gut size of the farm animals. The volume of forage intake can be expressed as fill value. This in turn limits the ability of the ruminant to obtain the required energy for milk and meat production.

This problem may be partially overcome by the use of feed supplements containing concentrated, easily digested nutrients. Such feed concentrates may supplement dietary energy and may contain corn and other cereals, or protein supplements containing soybean or other high protein sources. Generally, these food concentrates are used but they are a more expensive source of energy. However, 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 digestibility of lower cost forage for ruminants are highly desirable.

It is therefore an object of the present invention to provide a method for preserving and enhancing the nutritive value of ensiled forage to produce a palatable feed with high dry matter intake potential and with minimal protein bound by the Maiilard reaction as a result of high silo temperature. It s another cb]ect of the present invention to develop a method to increase and improve the digestibility and

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rate of di es ion of forage so as to allow increased use of available energy and protein from forage in the diet, with resultant reductions in overall feed costs.

It is another object of the present invention to increase utilisation of plant protein, reducing requirement for additional dietary protein supplements and which also reduces amount of blood urea produced, amount of dietary energy required for ammonia conversion and amount of dietary nitrogen excreted in urine.

SUMMARY OF THE INVENTION

These and other objects are achieved by the present invention which is directed to a method for preserving and enhancing the nutritive value of ensiled forage by the application of an enzyme composition to the forage. 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. In one embodiment, the enzyme compesitier of the present invention contains the enzymes cellulase and amylase.

In another embodiment, the enzyme composition of the present invention coπ ain≤ the enzymes xylanase and amylase.

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In another embodiment, the enzyme composition of the present invention contains the enzymes cellulase, amylase and pectinase.

In yet another embodiment, the enzyme composition of the present invention contains the enzymes pectinase, cellulase, and arabinosidase.

In another embodiment, the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase and arabinosidase. In still another embodiment, the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase, cutinase, and esterase.

In still another embodiment, the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase, cutinase, and lipase.

In a more preferred embodiment, the enzyme composition of the present invention contains the enzymes cellulase, xylanase, and amylase, and an effective amount of homolactic bacteria. Most preferably, the enzyme composition of the present invention contains the enzymes xylanase, amylase, pectinase, a; d an effective amount of homolactic bacteria.

The use of the enzyme composition on ensiled forage preserves and retains the nutritive value of e.-.siled 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

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lactic acid bacteria in the production of lactic ac _t 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.

The maximum effect in the rapidity of pH fall and final pH is seen when certain amounts of homolactic bacteria are added to enzyme compositions containing certain enzymes, including xylanase, amylase, and pectinase; or xylanase and amylase alone. This enhanced effect when homolactic bacteria are added to the enzyme composition of this invention occurs because such bacteria are more efficient in converting monomer sugars to lactic acid. Lactic acid bacteria are present on all crops grown naturally and are of two types, homolactic and heterolact c, 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.

Homolactic bacteria, however, will produce 2 moles of lactic acid for each mole of glucose without by products. Sufficient homolactic bacteria are therefore added to the crop to reduce the activity of natural heterolactic organisms by competition fcr available substrate, resulting in more rapid and efficient lactic acid production. The rapid fall in pH also prevents cr.e eveicpmer.t or clostridia, entercoacteria ana other organisms which can cause poor quality silage.

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A rapid pH droD early in the ensiling period also limits heat production from extended fermentation and this minimises the risk of "bound protein" arising from a Maillard reaction. This occurs when high silo temperatures cause condensation of amino acids with plant carbohydrates followed by polymerization into an lignin-like matrix. This results in a permanently bound and indigestible protein and loss of both potentially available energy and protein from the diet.

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 substances which obstruct digestion of the plant material and which may be removed cr weakened by the enzyme composition of the present invention.

"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. Pect c 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 digesting enzymes. 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 en∑vme.

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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. Thus, the ruminant can consume more and obtain more nutrients from forage with less requirement for feed concentrates.

In addition to the above, the readily available energy from treated forage allows rapid growth of rumen microorganisms which also need nitrogen for growth. This results in improved capture of plant non-protein nitrogen (NPN) degraded in the rumen to ammonia and conversion to digestible icrobial protein. Improved utilisation of rumen ammonia means reduced levels absorbed across the rumen wall into the bloodstream where it is transferred to the liver for conversion into blood urea. Such conversion requires 12 Kcal/g of nitrogen so converted and represents a significant energy loss to the animal when dietary NPN is high. High ammonia/blood urea nitrogen (BUN) levels are also implicated in reproductive problems; excessive amounts can cause ammonia toxicity and death.

Improved preservation of forage treated with an enzyme composition can improve protein utilisation by (1) limiting silo temperatures, so reducing "bound" protein and (2) improve rumen capture of non-protein nitrogen. Both reduce the requirement for expensive purchased protein sources, which are subject to the vagaries of the market for such products e.g., higher prices -following drought conditions.

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DETAILED DESCRIPTION OF THE INVENTIO

As used herein, the term "forage" refers to grasses, legumes, straw, whole crop cereals, corn sorghum, or other plant crop or agricultural by-products. As used herein, (i.e., throughout the specification and the claims) , the term "effective amount" of the enzyme composition of the present invention refers to an enzyme composition having enzymatic activities sufficient to maintain the pH of the ensiled forage at a level sufficient to prevent or inhibit the growth of harmful microorganism, e.g., a pH around about 4.0 - 4.5, so reducing bound protein and protein degradation resulting in a palatable silage with high feed potential. The term 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 removing portions of digestive resistant or digestive-delaying substances present in the plant cells of forage.

As used herein, (i.e., througnout the specification and tne claims) , 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.

As used herein, (i.e., throughout the specification and the claims) , the aboreviations used for the units of the various enzyme --.ctivit es have the following definitions: (1) CMC I' - carboxy—methvl cellulose international units, 2^ IU - international units (xylanase), (3) ?GU - polygalacturonic

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units, (4) SGU-glucodmylase units, and (4) CFU •• colony forming unit (bacteria) .

Upon maturation, forage may be harvested by a variety of methods. Forage harvesting typically utilizes machinery which mows and chops the forage before ensiling. For example, legume fcrage is cut and then left to dry or undergo pre- wilting on the field to achieve a dry matter (D.M.) content of 25-50%. 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.

In accordance with the above-mentioned objects and others, 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.

Plant Structure

Carbohydrates are the main repository of photcsynthetic energy in plants and comprise roughly 5C - 30% of tne cry r.atter or fcrage and cereals. The nutritive characteristics cf these carbohydrates for animal feeding are

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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.

Plants possess a number of resistant structural features such as lignin and cutin which serve as natural barriers protecting the plant tissue against wind, disease, and predation and digestion by animals. Almost all plants also have a plant cell wall surrounding the plant cell outside the plasma membrane. 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. Cellulose, lignin ar.d a small amount c- 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 a d as methyl esters. (Van Soest, P.J., Nutritional Ecology of the Ruminant, Cornell University Press 1982) The polygalacturonic =cid chain is varied with interpolation of rha nose in the chain which causes a sharp

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bend in the molecular structure. 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 insoluble. They are however, soluble in hot water and are easily degraded by pectin enzymes.

Cellulose is the main polysaccharide in living plants and is basically a polymer of glucose. It forms the skeletal str-cture of the cell wall. Cellulose is a polymer of B-D- glucopyranosyl units which are linked together, with elimination of water, to form chains of 2000-4000 units. 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 s usually applied to those polysaccharides which are extractable by dilute alkaline solutions. The term has also been used to include all 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 h^lp bird the ceils together and direct water flow. In general,

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ne iceliu ose is more tiyiitly 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 digestibilit≤s. 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-xyloεe, 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 external surfaces of the plant epidermal cells secrete an impermeable cuticle. The cuticle of plants is a waxy layer over the outer surface of the epidermis cf plants. It contains cutin, a varnish-like material and also contains waxes, resins and fatty acids. Cutin is an insoluble polyester comσosed orincio ^* allv cf a C.l. and/or a C.lo_ family of fatty acid monomers. Most cf the primary hydroxyl groups in the polymer are esteπfied and some of t a secondary hydroxyl groups are in ester linkage. The cuticle protects the plant cell against water loss ar.d parasitic infections. The cuticle, itself embedded m waxes, possesses small openings, called stomata, for gas ?.-:chanc . The cuticle is attached to the epidermal

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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. These components further obstruct the digestion of plant material by animals, including ruminants.

B. Digestion by Ruminants

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 n the agricultural industry. hen gra ing or feeding en forage, ruminants swallow the plant r.aterial rapidly and transport it to the rumen where it remains for some time. Later the rumen contents are regurgitated and masticated more tr.c-cuςhly before being r°f:rned ~ the rumen. The mastication begins only some time after the completion of the feeding period.

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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.

For example, 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. Furthermore, because of the rigid and cohesive nature of the forage, 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.

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C. The Present Invention

The present invention provides a method for overcoming these difficulties and is directed to the application of an effective amount of an enzyme composition with or without bacteria to forage.

The enzyme composition will:

1) preserve the crop by rapid pH fall, limiting nutrient losses and temperature rises causing 'bound' protein, resulting in palatable forage with high feed intake potential. 2) "pre-digest" fiber in the silo allowing rapid release of energy when forage is consumed, so allowing rapid growth of rumen microorganisms, improved utilisation of NPN and greater production of microbial protein. Lower BUN levels will lead to improved reproduction rates and a lower energy requirement for urea production.

3) improve the rate of digestion by pre-digestion and improved attachment of rumen microorganisms, decreasing rumen turnover time, so allowing more forage to be fed. Thus, leading to lower feed costs from using cheaper feed with less need for purchased prot in supplements.

The enzymes which may be used in the enzyme composition of the present invention are pectinase, cellulase, xylanase, amylase, arabinosidase, cutinase, lipase, and

In one embodiment, the enzyme composition of the present invention contains the enzymes cellulase and amylase.

In another embodiment, the αnzy e composition of the present invention contains the enzymes xylanase and amylase.

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In another erabo inont _r e enzyme composition of the present invention contains the enzymes cellulase, amylase and pectinase.

In yet another embodiment, the enzyme composition of tne present invention contains the enzymes pectinase, cellulase, and arabinosidase.

In another embodiment, the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase and arabinosidase. In still another embodiment, the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase, cutinase, and lipase.

In still another embodiment, the enzyme composition of the present invention contains the enzymes pectinase, cellulase, xylanase, cutinase, and esterase.

In a more preferred embodiment, the enzyme composition of the present invention contains the enzymes xylanase and amylase, and an effective amount cf homolactic bacteria. Most preferably, 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 fcrage. The enzyme comp-sition is, however, preferably added to the forage just prior to the time of ensiling. Alternatively, a first enzyme composition contain rg at least one enzyme selected from the group consisting essentially of pectinase, amylase, cutinase, lipase,

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and esterase may be initially added to the foraαe. 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. The enzyme 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 microorganisms to act in digesting the forage. 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 enzy ology of cellulose degradation is a relatively new topic in biochemistry and its development has been limited by the lack of availability of pure enzymes. In general, however, it may be said that cellulase catalyses the hydrolysis of cellulose to glucose ar.d to cellcoiose, a glucose Bl-4] gluccside disacharide. The application of cellulase to forage thus increases the digestibility of plant material by degrading cellulose, the

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principal component responsible for the rigidity of plant cell walls.

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. Moreover, the character of hemicellulose differs among various forages and types of plant cell walls. In general, 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.

Some chemists classify pectins with hemicellulose in one group as nor.-cellulosic cell wall polysaccharides. The distinction between pectin and hemicellulose is by no means clear. However, the use of pectinases to catalyze the breakdown of pectic substances can be shown both to improve rapidity of pH drop and crop digestibility when added to xylanases.

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 a d removal by arabinosidases can enhance the breakdown of plant hemicellulose.

Ar.yla≤es are enzymes which catalyze the breakdown of starch. Alp a-amylase cleaves starch chains randomly while b ta-anyiase is an exccnzyme cleaving units from the ends cf r.ams. These enzymes are particularly important with legumes

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such as alfalfa which use stcrch ac a major storage polysaccharide.

The enzyme cutinase, as the name suggests, 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 polysaccharide components of which forage material is composed, the presence of 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.

In one embodiment of the present invention, the enzyme composition contains singly the enzyme cellulase at an activity ranging from about 40,000 - 1,000,000 CMC lu/tn. When used alone in the enzyme composition of this embodiment, cellulase most preferably has an activity of about 500,000 y.C lu/tn.

In another embodiment, this enzyme composition contains singly the enzyme xylanase at an activity ranging frcr.

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about 400,000 - 3,000,000 lu/tn. When used al "τ= ^» in the onzymc composition of this embodiment, xylanase most preferably has an activity of about 1,000,000 lu/tn.

In another embodiment, this enzyme composition contains singly the enzyme pectinase at an activity ranging from about 100,000 - 50,000,000 PGU/tn. When used alone in the enzyme composition of this embodiment, pectinase most preferably has an activity cf about 2,000,000 PGU/tn.

In one preferred embodiment of the present invention, 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 10,000 - 50,000 SGU/tn of amylase. 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.

In another preferred embodiment of the present invention, 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 composition of this embodiment has activities of about 1,000,000 Tu/tn of xylanase and about 42,000 SGU/tn of amylase.

In a more preferred embodiment of the present invention, the enzyme composition contains the enzymes xylanase and amylase, and an effective amount of homolactic bacteria. This enzyme compositicn 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 CFtJ LAB/g crop. The mcst preferred enzyme composition of this embodiment has activities

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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.

In the most preferred embodiment of the present invention, the enzyme composition contains the enzymes xylanase, amylase, pectinase, 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, 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. In another preferred embodiment of the present invention, the enzyme composition contains the enzymes cellulase and xylanase. 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.

In another preferred embodiment of the present invention, 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 cf cellulase, and about 10,000 - 50,000 SGU/tn of amylase. The most preferred enzyme composition of this embodiment has activities of, about

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200,000 PGU/tn of pectinase, about 250,000 CMC lu/tn of cellulase, and 25,000 SGU/tn of amylase.

In another preferred embodiment of the present invention, the enzyme composition contains the enzymes pectinase, xylanase and amylase. This enzyme composition preferably nas 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 25,000 SGU/tn of amylase.

In another preferred embodiment of the present invention, the enzyme composition contains the enzymes pectinase, cellulase, and xylanase. 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 embodiment, the enzymes pectinase, cellulases, and xylanases together display a particularly beneficial effect in enhancing silage quality and improving digestion of forage by ruminants. In another preferred embodiment, the enzyme composition contains the enzymes pectinase, cellulase, xylanases and arabinosidase. This enzyme composition preferably has activities, per ton treated, of about 40,0C0 - 1,000,000 lu/tn pectinase, about 50,000 - 1,000,000 lu/tn cellulase (CMC) , about 400,000 - 3,000,000 lu/tn xylanase, nd about 10,000 - 100,000 lu/tn of arabinosidase. The most

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preferred enzyme composition of this embodiment has activities of about 100,000 PGU/tn pectinase, 300,000 (CMC) lu/tn cellulase, 700,000 lu/tn xylanase, and 50,000 lu/tn of arabinosidase. In another embodiment, 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 arabinosidase.

In still another preferred embodiment of the present invention, the enzyme composition contains the enzymes pectinase, cellulase, xylanase, cutinase, and 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 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. In still another preferred embodiment of the present invention, the enzyme composition contains the enzymes pectinase, cellulase, xylanase, cutinase, and lipase. This enzyme composition prer≤rably 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

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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 50,000 IU/tn lipase.

The enzymes cellulase, xylanase, amylase arabinosidase, pectinase, lipase, cutinase, and esterase of the present invention are manufactured preferably in a fermentation process. The strains of production microorganisms of cellulase, hemicellulase, arabinosidase, esterase and pectinase belong to Trichoderma, Asperσillus, Penicillum or other strains. Cutinase can be obtained, for instance, from Streptomyces and Pseudomoπas strains, especially from Pseudomonas Putida. 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.

While the 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 cf 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.

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By loosening or disengaging the structural components of plant material, these enzymatic effects of the enzyme composition of the present invention produce an increase in the amount of fermentable substrates present in the ensiled forage.

5 These fermentable substrates are then available to lactic acid bacteria for conversion to lactic acid. Accordingly, when forage is treated with the enzyme composition of the present invention, lactic acid bacteria are able to produce lactic acid for a longer period of time which means that silage will be

1 _Q more palatable. Consequently, the pH remains lower and the growth of harmful microorganisms is prevented or hindered for a longer period of time. The forage is thus better preserved during the ensiling period and displays an improved nutritive value, when it is eventually fed to ruminants, as compared to ιc forage which has not been treated with the enzyme composition of the present invention.

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.

20 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

25 harmful bacteria like clostridia are more sensitive to low pH and osmotic pressure than the lactic acid bacteria are.

More important than preservation, the application cf pectinase can be used to improve the accessibility of other enzymes and rumen microbes to the forage cell wall. The

30 enzymatic effects produced by the enzymatic composition, as

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discussed above, accordingly act to weaken or remove certain substances which normally interfere with efficient ruminant digestion of plant material, thus increasing the rate of digestion of such forage by the ruminant. As pectin is a glue between the adjacent cell walls, the removal of pectin opens up the structure of the plant. This is important as there normally exists a lag phase before the digestion of the cell walls in the rumen by the rumen bacteria. As pectinase opens up more cell wall to the bacteria, a larger surface area is available to the bacteria and thus digestion begins and continues at a faster pace.

Since the rate of digestion is increased, the ruminant's total intake of forage may be increased. The ruminant is therefore able to grow faster or to produce more milk from forage and diets can be formulated to include more forage so reducing overall feed costs. 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.

Normally, the digestion of forage starts from the open cut ends of stem or leaf pieces while the cuticle surface is left untouched. After eating, the ruminant animal starts the mastication of feed after a certain period of resting (e.g. half an hour) . During this mastication, barriers of digestion, including the surface cuticulu , are weakened physically. This makes the structures under the cuticulum a little more

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available for digestion by rumen microbes, although the cuticulum still covers much of the forage structures.

As cuticulum covers much of the plant surface, it is a remarkable hindrance to rumen digestion. By applying cutinase and/or lipase or esterase to the forage prior to feeding, the cuticulum can be weakened or removed, thus making the forage more susceptible to digestion by other enzymes and/or rumen microbes. In addition, the weakening of the structure partially acts synergistically with the mastication which then unorganizes the forage structures to a greater extent.

As a result, faster and possibly more complete digestion of forage in the rumen occurs. When the digestion is faster, the animal is capable of consuming more feed per day since the physical limitation of intake is decreased. This is important in e.g. the early stages cf lactation by a cow, where the intake of feed and thus the milk production is limited due to rumen size. Also when low quality roughages which have low digestibility are fed to the ruminant, the higher rate of intake or increased digestibility are important for the improvement of the efficiency of animal production.

The enzyme composition of the present invention further provides an increase in the amount of digestible protein which is available to the 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 i: also dependent on the rate of passage of undigested particles

^S UBST ^I T

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.

When certain amounts of homolactic bacteria are added to enzyme compositions containing certain enzymes, including xylanase, amylase, pectinase, or xylanase and amylase alone, a more rapid fall in pH and a lower firal pH is achieved.

This enhanced effect when homolactic bacteria are added to the enzyme composition of this invention occurs because such bacteria are more efficient in converting monomer sugars to lactic acid. 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.

Homolactic bacteria, however, will produce 2 moles of lactic acid for each mole of glucose without byproducts. Sufficient homolactic bacteria are therefore added to the crop to reduce the activity of natural heterolactic organisms by competition for available substrate, resulting in more rapid and efficient lactic acid production. The rapid fall in pH also prevents the development of clostridia, enterobacteria and other organisms which can cause poor quality silsge.

^S UBSTITUTE S

35

Another problem associated with πvn nsn consumption of forage foodstuffs is the accumulation of excess ammonia (NH,) in the animal's blood. The presence of such excess ammonia in the blood has deleterious consequences for the ruminant's reproduction and other aspects of the animal's biology. The present invention is able to alleviate seme of these problems.

In general, 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.

% NON-PROTEIN NITROGEN, (NPN)

NITRATES

BASIC AMINO ACIDS

AMINO BUTYRIC ACID

CLUTAMINE

GLUTAMIC & ASPARTIC

ASPAAGINE

OTHER AMINO ACIDS

NON-AMINO ACID BASES

When crops are ensiled, the components of plant NPN will be replaced or added to by ammonia and amines, and the proportion of true protein and NPN will be reversed. Silage will contain only 20 - 40% true protein with 60 - 80% NPN. The extent of this change is cεpendent on ensiling efficiency. Rapid pH drop will minimize true protein breakdown to NPN, particularly that breakdown of true prctein caused by proteolytic clostridia bacteria.

In addition, a rapid pH drop early in the ensiling period will also limit heating caused by extended fermentation and this minimizes the risk of a Maillard reaction with

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36

protein. This occurs when high silo temperatures cause condensation of amino acids with carbonyls and dehydroreductones from carbohydrates followed by polymerization into a lignin-like matrix. This results in a permanently bound and indigestible protein which is recovered in acid detergent fiber (ADF) and which is known as "ADF bound".

Silage proteins are also classified by

(1) 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. As indicated earlier, the proportion of each group can be drastically changed by a slow pH drop after ensiling.

Typical ranges for each group for alfalfa silage are shown below. SILAGE PROTEIN - 17 - 25% d.πi. as crude protein

OF THIS PROTEIN: 30 - 60% will be soluble protein 15 - 40% bound prc'eln 0 - 50% insoluble but available protein The available but insoluble true protein will bypass rumen fermentation and be absorbed and utilized later in digestion. Utilization of soluble nitrogen (NPN and true protein) however is entirely dependent upon microbial conversion in the rumen. The main form of nitrogen utilized y 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

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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.

Some blood urea is recycled through saliva and this process is relatively independent of the metabolism of dietary nitrogen. All urea conversion by the liver in excess of this requirement is wasteful in terms of energy, as 12 K cal are required for each gram of nitrogen converted into urea. Furthermore, blood urea, normally measured as Blood Urea Nitrogen (BUN) , is normally excreted by the kidneys and thus represents a complete loss of dietary 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.

As mentioned above, high BUN levels associated with the feeding of silage are common in many agriculturally useful rumiπarts such as dairy cows and are linked with problems with ru inart reproduction. On a practical level, this result can be seen as infertility or delayed conception rates where

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multiple artificial inseminations are needed to make cattle pregnant which results in lower milk production.

Research work published by K. H. Lotthammer, Effect of Feed and Feed Production on the Health and Fertility of Dairy Cattle: TIERAR2TL. prax. (1979), 7: 425-428 shows that herds with recorded fertility disorders had BUN levels around 55 mg/100 ml 4 - 6 weeks pre-calving, dropping to 27 one week pre-calving and rising again five weeks post-calving. Herds without fertility problems had BUN levels of around 23 mg/100 ml which were maintained over all of the same period (6 weeks pre and post-calving) . Lotthammer also stresses the need or correct feeding during the dry period pre-calving as the incidence of clinical problems with the ovaries and uterus are increased by a factor of 2 or 3 post-calving. Correct feeding resulted in a 52% achievement of first insemination compared with 36% in the wrongly-fed group.

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 pr tein breakdown by clostridial oacteria (lower NPN values shown as NH^ in silage analysis) and reduced heat production, so eliminating any Maillard reaction causing heat bound protein (ADF bound nitrogen) .

Further breakdown of plant fiber in the silo (pre- digestion) , upon consumption by the ruminant, supplies the rumen w th readily available carbohydrates to enhance micrcbial growth and therefore microbial protein production.

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Such protein can supply over 50% of the amino acid requirements for the producing animal.

Lower BUN levels resulting from such improved NPN utilization was demonstrated in two commercial tests in crops ensiled with enzyme compositions of the present invention on two commercial farms with less energy lost in conversion to urea (12 Kcal/g) and less nitrogen excreted by the animal.

Thus, 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 prr-tsent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following examples, the present invention will be illustrated in more detail by describing the effects of treatment of forage (alfalfa and grasses) with assorted enzyme compositions. These examples should not be considered to restrict the scope of the invention, instead they are mere examples of the application of an enzyme composition to forage to preserve and enhance the nutritive value of forage and to improve its digestibility.

EXAMPLE 1 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 n. equi . NaOH/100 g crop and water-soluble carbohydrates (WSC) 7.4% in dm. Levels of naturally occurring bacteria c.-. crop were

SUBSTIT

recorded at 5.6 million colony forming units (CFU) of lactic acid bacteria (LAB) per g of crop.

Crop was ensiled after one of the following treatments: 1. without any additive (control)

2. Cellulase

3. Xylanase

4. Cellulase and amylase

5. Xylanase and amylase 6. Xylanase and amylase and bacteria

Levels of enzyme addition/activity were as follows: cellulase; 200 ml/ton, activity 2,500 IU CMC/ml; xylanase, 250 ml/ton, activity - 5,000 IU/ml plus 1,400 IU CMC/ml and amylase, 250 ml/ton, activity 170 SGU/ml and 60,000 LU/ml- Level of bacterial addition was 88,000 CFU LAB/g crop.

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.

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Table 1 shows the pH reduction against time achieved with the six treatments.

The results presented in Table 1 show that addition of cellulase or xylanase produced minor improvements in the rapidity of pH fall and final pH over untreated control. Addition of amylase with either cellulase or xylanase produced a more rapid pH fall and lower final pH. Maximum effect was seen, however, when homolactic bacteria was added to xylanase and amylase.

Table 2 shows fermentation quality of silage sampled after 90 days ensiling.

42

As explained earlier, the aim of efficient ensiling is to rapidly produce lactic acid to reduce pH and to limit

(1) production of by-products such as acetic acid and ethanol, and (2) protein breakdown seen as ammonia nitrogen. The data presented in Table 2 shows that enzyme addition has caused increased production of lactic acid, high lactic/ cetic acid ratios and reduced ammonia nitrogen levels. Ethanol production increased when amylase was added but this was corrected where homolactic bacteria were also added.

The effect of enzymes and bacteria were additive causing the most rapid preservation with minimal by-product production or protein breakdown when both enzymes (cellulase, xylanase, amylase) and bacteria were used.

EXAMPLE 2 Third cut alfalfa was field wilted for 4 hours before being ensiled at 40% dry matter. The crop contained

WSC 7.7% in dry matter and had a buffering capacity of 54.8 m. equ. NaOH/100 g crop.

Crop was ensiled after one of the following treatments:

1. Without any additive (control)

2. Cellulase 3. Xylanase

4. Cellulase and amylase

5. Xylanase and amylase

6. Xylanase, amylase and bacteria

Levels of enzyme addition and activity were as follows: cellulase, 200 ml/ton crop, activity 2,500 IU

CMC/ml; xylanase 250 ml/ton, activity 5,000 lU/ml plus 1400 IU CMC/ml and amylase, 250 ml/ton, activity 170 SGU/ml and 60,000 LU/ml. Level of bacterial addition was 106,000 CFU LAB/g crop. Laboratory silos and techniques were as described in the previous example.

Table 3 shows the pH reduction against time achieved with the six treatments.

TABLE_3 Treatment Davs from Ensiling

0. 5 1 2 4 7 14 90

The data presented in Table 3 shows that addition c: cellulase or xylanase produced minor impro%εmcr.ts in the

rapidity of pH fall and final pH over control. Addition of amylase with either cellulase or xylanase produced a more rapid pH fall and lower final pH. The most effective combination, however, was when xylanase, amylase and bacteria were added.

Table 8 shows fermentation quality of silage after 90 days ensiling.

TABLE 4

Treatment

The positive effect of adding enzymes can be seen in that data shown in Table 4 where there is an increased production of lactic acid following treatment and improvements in the lactic/acetic ratios. Ethanol production, indicative of inefficient conversion of sugars to lactic acid, decreased when cellulase or xylanase was added, increased when amylase was added to either, but were significantly decreased when homolactic bacteria were added with the enzyme mixture including amylase. Enzyme addition showed small improvements in ammonia nitrogen levels over control indicating less protein breakdown. Maximum effect was seen again with the bacterial/enzyme mixture.

EXAMPLE 3 Fifth cut alfalfa was ensiled after field drying 24 hours with a dry matter content of 52%, crude protein 23.3% in dm; WSC 9.6% in dm and buffering capacity of 60.1 m. equ. NaOH/100 g. Levels of naturally occurring bacteria on crop were recorded at 90,000 CFU LAB/g crop.

Crop was ensiled after one of the following treatments:

1. Without an additive (control) 2. Xylanase and amylase

3. Xylanase, amylase and bacteria Levels of addition/activity were as follows: 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.

The crop was ensiled both in 55 gallon capacity pilot silos for feed trial and laboratory tube silos as before.

Table 5 shows the pH reduction against time achieved with the three treatments.

Treatment

1. Without Additive

2. Xylanase & Amylase

3. Xylanase, Amylase & Bacteria

The effect of ensiling a highly buffered crop (resistant to acidification) can be seen in the data presented

46

in Table 5 where there is a small fall in pH over 90 days with control silages. Increasing substrate availability through enzyme addition alone marginally improved pH fall over 90 days. When bacteria and enzymes were added, however, acidification was improved dramatically from day 4.

Table 6 shows fermentation quality of silage after 90 days ensiling.

Treatment

1. Without Additive

2. Xylanase & Amylase

3. Xylanase, Amylase && Bacteria

Levels of lactic acid arising from treatment 3 in Table 6 (bacteria and enzymes) show the benefits of adding sufficient homolactic bacteria for efficient acid production and enzymes to provide adequate substrate Lactic/acetic ratios were also much higher following treatment 3; both ethanol and ammonia levels were much reduced ai~o.

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.

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47

TABLE 7

Feedin

The effect of xylanase/cellulase enzyme content on crop fiber can be seen in Table 7 with reduction in NDF (4.5%) and ADF (2.0%) levels over control. Both enzyme treatments increased dry matter intake but dry matter digestibilities of both treatments were slightly lower than control.

Calculation of digestible dry matter intake showed a 3.8% improvement from enzymes and 18.0% from bacterial and enzymes treatment. The latter major improvement in intake arises partly from more palatable feed (low ammonia nitrogen) and mere rapid rumen turnover following fiber digestion.

EXAMPLE 4

Sixth cut alfalfa was ensiled after 6 hours field drying with a dry matter content of 37%, WSC 5.3% in dm and buffering capacity of 63.9 m. equ. NaOH/100 g crop. Levels of natural background lactic acid bacteria were recorded at 3.3 million CFU/g crop.

Crop was ensiled after one of the following treatments.

1. Control

2. Bacterial inoculant

3. Amylase

4. Cellulase and amylase and pectinase

5. Xylanase, amylase and pectinase, and bacteria Levels of addition/activity were as follows: 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. Laboratory silos and techniques were as detailed in Example 1. Table 8 shows the pH reduction against time achieved with the five treatments.

TABLE 8

Treatments Davs from Ensiling

I 2 I £ 90

1. Control 5.03 4.99 4.97 4.95 4.91

2. Bacteria Alone 5.00 4.95 4.95 4.«4 4.88

3. Amylase Alone 5.04 4.94 4.92 4.91 4.90

4. Cellulase. Amylase - Pectinase 5.02 4.89 4.90 4. ^r O 4.SO

5. Xylanase. Amylase. Pectinase &

Bac teria 4 . 99 4 . 84 4 . S4 4 . 79 4 . 30

~ " ~"~ -~-

49

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.

On completion of the above trial, the remaining 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

50

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 utilization of imino acids and peptides in the forages.

The results show an increase in the yield of microbial protein of 0.5 lb/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.

Analyses of the silage component of diets are shown in Table 10.

TABLE 10

The data presented in Table 10 shows that enzyme compositions of this invention with and without bacteria decreased content cf NDF and ADF as before, maximum reduction in NDF occurred following treatment 5 (enzymes & bacteria) .

The results cf the fermenter studies are given in Table 11.

cr

TABLE 11

Cellulase & Amylase Enzymes & Control Bacteria Amylase & Pectinase Bacteria

6 .09 6.28

68.5 73.7 a 47 .0 50.6 a 51. 1 53.9 a

63 66

22 16 b

10 13 c

3 .0 4.2 b

6.71 6.99

26 .81 26.99 2.30 2.49

NOTE: TREATMENT SIGNIFICANTLY BETTER THAN ALL OTHER TREATMENTS : a - P <.03 b - P <.06 c - P <.02 MICROBIAL EFFICIENCY - g MIC.NAg digestible D.M.

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.

It has been suggested that this treatment increased the rate of ruminal fermentation of the diet. This is important as previous research has shown that increased rates of NDF digestion can increase milk production.

Protein degradeability and microbial growth were net significantly affected by treatments. Compared with control, however, lower ammonia and higher microbial efficiencies

indicate that enzyme treatments of this invention were supportive of the efficient use of protein and carbohydrate fractions in the forages. Highest estimated daily yield of microbial protein came from treatment 5 (enzymes & bacteria) . Supplementation of this diet with more degradeable protein would be expected to further increase microbial yield and nutrient digestion and would also support milk production through enhanced rumen digestion rather than using expensive by-pass sources of energy and protein. Samples of the original treated silages were also used for Ln sacco digestion trials. Weighed samples of each silage treatment were placed in small nylon bags (pore size 40 mm) . These bags were suspended by cords in the rumen of a dairy cow through a surgically formed permanent fistula. Bags were removed after 4, 8, 12, 16, 24, 30, 36 and 48 hours fermentation, washed and analyzed for NDF as shown in Table 12,

% NDF Degradation Treatments

1. Control

2. Bacteria alone

3. Amylase alone

4. Cellulase, Amylase - Pectinase 0 1 1 19 19 23 26 40

5. Xylanase, Amylase, Pectinase

& Bacteria 8 10 19 28 35 39 43 49

The data presented in 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 s ar ing before

first sampling at four hours and showed also maximum reductions at later sampling times.

The above measurements are used to estimate fill values, which are calculated as follows:

FILL VALUE = D . e" ^~ ' ~ + U

Kd + KS KS

Where D = potentially digestible fraction at 24 hours L = lag time Kd = digestion rate Ks = rate of passage

U = undigested fraction at 24 hours

Values for this trial were as shown in Table 13.

Treatments

1. Control

2. Bacteria

3. Amylase

4. Cellulase, Amylase & Pectinase 20.9

5. Xylanase, Amylase, Pectinase & Bacteria 18.5

Table 13 shows that treatment 5, an enzyr.e composition of the present invention, decreased fill value by 2.6 units, whereas other work has shown that a 3 unit decrease in this value for total dietary NDF resulted in significantly increased milk production.

-

54

EXAMPLE 5 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. 1. Without any additive (control)

2. Formic acid, molasses and formaldehyde mix at 1.75 gals/ton.

3. Xylanase, amylase, and bacteria.

Level of enzyme addition was xylanase, 200ml/ton, "LQ activity 5,000 IU/ml plus 1400 IU CMC/ml and amylase 100 ml/ton, activity 170 SGU/ml and 60,000 LU/ml. Crop was ensiled in three separate farm silos.

Analysis of silage after 120 days ensiling are shown in Table 14.

15

20

25

The formic mixture additive is designed to chemically protect protein to avoid rumen degradation which causes high 30 urea nitrogen levels in the blood resulting in poor herd reproduction.

By comparison, 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 ar.d, because of problems with herd _c 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.

TABLE 15 10 BUN Levels After 30 Days Feeding

1. Control range 30 - 35 mg/ml

2. Enzymes range 17 - 20 mg/ml

3. Formic Mix range 30 - 33 mg/ml

15 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

20 enzyme activity on structural carbohydrates. Increased microbial growth means enhanced capture of non-protein nitrogen (NPN) in the silage, a n 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). 5 This trial was repeated for another cycle of silages, and the trends of the earlier cycle were confirmed, except that the enzyme treated silage resulted in mean BUN levels of 25 mg/ml.

A similar result was noted on another commercial farm 0 using control and enzyme treated second cut alfalfa. A

reduction in BUN of 10 mg/ml was recorded in changing to enzyme silage from control.

EXAMPLE 6 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:

1. Without any additive (control) .

2. Xylanase, amylase, pectinase and bacteria. Level of enzyme addition was xylanase, 120 ml/ton, activity 5,800 IU/ml plus 2900 IU CMC/ml, amylase 50 ml/ton, activity 170 SGU/ml and 60,000 LU/ml, pectinase 100 ml/ton activity 2000 PGU/ l and bacteria, 1 million CFU LAB/g crop.

Analyses of silages after 90 days ensiling are shown in Table 16.

Table 16 SILAGE ANALYSES Control Enz me

The data presented in Table 16 shows that the enzyme composition of this invention reduced, as shown in previous examples, NDF by 5.7 units, ADF by 3.1 units. Samples of each silage were subjected to in-vitro techniques to assess digestibility of organic matter and NDF. This involves the anaerobic fermentation of the samples in filtered mmen liquor wiLh a buffer solution.

57

The time of batch fermentation is commonly 72 hours for estimation of digestibility. Values for the two silages are shown in Table 17.

DIGESTIBILITY, %

(a) ORGANIC MATTER Control Silage Enzyme Sil≥ge (-) FIBER

Control Silage Enzyme Silage

As the data of Table 18 shows, both organic matter and fiber digestibilities of enzyme treated silages were higher at each sampling time, indicating more rapid rate of rumen fermentation as seen in previous examples.

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.

Energy is normally limiting at this time and 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.

Incomplete oxidation of f:-t leads to the presence of ketones in blood which are excreted, representing further potential energy losses.

According to the procedure of this lactation trial, recently cut, very good quality untreated and enzyme treated alfalfa silage is ensiled in tower silos.

The diets for the groups were constructed as follows:

GROUP I - Normal NDF - untreated alfalfa silage, ground corn, soybean meal and minerals, feed formulation based on NDF and non- structural content (NSC) of total feed ingredients in accordance with recommendations of NRC.

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.

GROUP III - High NDF - Untreated silage + other feed ingredients. Higher levels of NDF included based on predictions from previous research work.

GROUP IV - High NDF - enzyme treated silage - even higher levels of silage included again because of NDF reduction in silo.

Details of the diets are given in Table 18.

TABLE 18

NORMAL NDF HIGH NDF

GROUP I GROUP II GROUP III GROUP IV

All groups were condition scored at weekly intervals and records of milk production and quality were taker..

The diets of Group I and Group IV support milk yields which in some cases are in excess of 100 lbs/day without loss of body condition.

Treatment with an enzyme composition of this 5 invention allows the use of 21% more neutral detergent fiber (NDF) and 30% more acid detergent fiber (ADF) than would be recommended by the National Research Council. With regard to the net energy for lactation (NEL) it is predicted that 7% more Mcal/lb would come from the total treated diet or 11% from the ■_ Q enzyme treated forages alone.

The calculation of net energy is made as follows:

Normal NDF = 0.71, High NDF = 0.66 Mcal/lb. As cows have similar performance, those on high NDF diets must be obtaining the same energy as those on normal diet. 5 0^66 _{χ 100 m 93%}

Therefore 7% more energy is being found in the high NDF diet.

Extra energy/lb = 0.71 - 0.66 = 0.05 Meal. 0 Therefore from treated silage only:

0.05 x 100 =

65.3 .076 Meal

Extra energy from silage « 0.076 x 100 ^~~ 10.7% 5 0.71

In terms of protein, the percentages arising from each feed ingredient in both diets are shown below in Table

19.

60

TABLE 19

Alfalfa silage Ground corn Soybean meal

Diet (% of Diet) (% of Diet) (% of Diet)

Untreated forage diet 44% 25% 31%

Enzyme treated forage 67% 16% 17%

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. This is demonstrated by Group III, the group of cows also on a high NDF diet but using untreated forage. Ratio of forage concentrate in this diet is lower at 58:42 and cows started to produce similar levels of milk to group IV cows. 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.

This was an expected result because the calculated energy of the diet for Group III at 0.66 Mcal/lb was 7% lower than required for cows on untreated silage as for Group I, i.e., 0.71 Mcal/lb. In order to cope with this energy inbalance, 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.

For the efficient conversion of such mobilized fat, oxalacetate is essential, derived from carbohydrate precursors that are also in short supply at a low level of nutrition. This leads to the incomplete conversion of fat leading to an increased pool of Ketone bodies in the blood. This leads to a condition known as Ketosis which causes loss of appetite and further energy inbalance.

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. Five of Group III cows on untreated alfalfa silage, to date, have required treatment for acute Ketosis and all cows have shown losses in body conditions. It is expected that all cows in this group will show lower levels of milk production than other groups over the period of the trial. When body reserves of fat become depleted, the untreated diet is not expected to maintain levels of production recorded for other groups. I is also expected that a similar scenario will be seen with protein utilization. The increased levels of silage in Group III and IV diets results in higher levels of soluble protein and non-protein nitrogen (NPN) in these diets. Increased levels of ru inally degradable protein associated with the reduced energy status of the Group III diet, means that more ammonia will be present in the rumen. It is expected th.it this will lead to poor utilisation by rumen microbes, leading to low production of microbial protein and increased losses of nitrogen as urea excreted from the cows. This will be reported as higher blood urea nitrogen (BUN) levels

62

(expected to be 30+ mg/100 ml) in this group compared with Group IV (below 20 mg/100 ml) following routine blood sampling planned for the lactation trial.

Such lower production of microbial protein on Group IV diets is expected to also reduce milk production on these diets. In addition to thse studies, further studies are being carried out on the same site with fistulated/cannulated cows to measure microbial protein production on all diets.

BUN levels in the other two groups (I and II) are also expected to be different although not as great as differences in the other groups because of the lower level of silage in these diets. It is expected that BUN levels in Group II cows on treated silage will be below.20 mg/100 ml whereas Group I cows will be between 20 and 30 mg/100 ml. This trial has been designed to snow that on normally designed diets according to National Research Council recommendations (Groups I and II) , cows eating treated silage will consume more feed and produce more milk with greater efficiency than cows on control silage. Diets for Groups III and IV have been formulated utilising the expected increase in energy and improvement in protein utilization following treatment of silage with an enzyme composition of this invention. Such cows on much higher levels of dietary silage and therefore high NPN inputs are only expected to maintain milk production at similar levels to GroupII cows when consuming treated silage.

EXAMPLE 7

There are also considerable economic benefits associated with use of the present invention. A provisional estimate of such economic benefits is made under the assumption that milk production from the lower NDF untreated group continues at a level similar to that from the high NDF enzyme treated group.

The following additional assumptions which have been made are presented in Table 20:

TABLE 20

Dry matter intake/day = 44 lbs

Untreated alfalfa silage/ton ^~ - $30.00 Treated alfalfa silage/ton ( includes treatment cost with enzyme composition) = $33 .50

Ground corn/ton ^~ - $112.00

Soybean meal , 48/ton = $220. 00

UNTREATED GROUP SllACE 60.41bs @ $30.00/t - 0.906

CORN 22.011blsl <§ $112/t - 1.232

SOYBEAN 6.61bs @ $220/t - 0.726

TOTAL FEED C0ST/C0W/DAY - $2.864

ENZYME_GROUP

SI1ΛGE 751bs ' $33.50/t - 1.256 CORN 121bs @ $112/t - 0.672

SOYBEAN 3 .01bs @ $220/t - 0 . 330

- $2 .258

The additional cost of using the enzyme composition, i.e., $3.50/ton, is included in the actual cost of such treated silαgc. If some other treatment is used, ^~ .he cost of this

64

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.

EXAMPLE 8 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. ^τhβ grass was ensiled in three different ways: (l) without an additive (i.e., the control); (2) with the addition of cellulase and xylanase enzymes; ?nd (3) with the addition of cellulase, xyianase and pectinase enzymes.

The levels of enzyme additions were as follows: cellulase 100 ml/tn (metric ton), activity-3,000 IU CMC/ml; xylanase, 200 ml/tn, activity-5,000 IU xylanase/ml; pectinase 500 ml/tn, activity-2,000 IU PG/ml.

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 20-25'C. The quality of the silages was then determined.

Table 21 shows the results of this study.

TABLE 21 Fermentation Quality of Silages in Example 1

o z TOTAL VOLATI E O -ι I

a

CODES:

Control: No additives, just plain grade

X Xylanase (ml/tn) c Cellulase (ml/tn)

P Pectinase (ml/tn)

Although final silage pH was similar with all treatments, early fermentation was faster when pectinase was added. This led to better prevention of the growth of harmful micro-organisms (clostridia) indicated by the lower values in Table 21 for butyrate and volatile nitrogen measurements in crop treated with cellulase, hemicellulase and pectinase.

The synergistic effect of this enzyme ccmposition can further be seen from the neutral detergent fiber (NDF) and acid detergent fiber (ADF) recovery figures as shown in Table 22.

Table 22

The Effect of Enzyme Additions on the Degradation of Cell Wall Fractions During Ensiling

% NDF % ADF HEMICELLULOSE

TREATMENT RECOVERED RECOVERED RECOVERED

Control 97.7 108.8 85.8

'100 + X 200 87.6 96.0 78.6

^100 ^{+ X} 200 85.5 94.5 76.4 ^500

CODES:

Control: No additives, just plain grade X: Xylanase (ml/tn)

C: Cellulase (ml/tn)

P: Pectinase (ml/tn) The digestibility of silages was then measured by the in sacco method. This method involves the suspension of nylon bags (mesh size 40 urn) 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

< ~ ^m ~"-~"

allowing free access to rumen microorganisms. Samples were analyzed at Oh (zero hours, i.e., washing with water only), 3h,

6h, 12h, 24h, 48h, and 96h and washed under running water.

The results of the above measurement are shown in Table 23.

Table 23

Degradability of Silage in the Rumen (In sacco)

Of a Cow During Different Incubation Periods

(% of Total Silage P.M. Degraded)

TREATMENT Oh 3h 6h 12h 24h 48h 96h

1 Control 32.6 35.4 45.7 58.9 64.0 76.4 82.6

2 C ₁₀₀ + X ₂₀₀ 43.3 41.0 47.6 54.5 64.2 75.8 81.0

^{3 C} 100 ^{+ C} 200 ^+P 500 ^{S 4} ' ^{2 8} - ^{8 56'6 60} - ^{7 72"5 81} -° CODES : Control: No additives, just plain grade

X: Xylanase (ml/tn)

C: Cellulase (ml/tn)

P: Pectinase (ml/tn)

Table 23 demonstrates the capability of different enzyme compositions to break down the ensiled forage. The digestibility of such enzyme treated silages, i.e., ensiled forage, was then determined after different periods of incubation in the rumen. As is shown in Table 23, the 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.

Release of nutrients from enzyme treated silage would be more rapid than other silages which would encourage earlier and more vigorous growth of rumen microorganisms, providing a quicker turnover of rumen contents. In addition, such rapid microbial growth would improve microbial capture of non-protein nitrogen with better production of microbial protein. This is an essential addition to dietary protein, reducing the o requirement for an expensive bypass protein addition to the diet. 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.

5

EXAMPLE 9

Another study similar to that made in Example c was made with forage containing about 35% Red Clover and 70% of mostly meadow fescue forages. 0 The dry matter content of raw material was about

18.3% and the protein content was about 16.0%.

The treatments were as follows (ml/metric ton) :

(1) control (no additive) ,

(2) cellulase 100 ml/tn 5 (3) cellulase 100 ml/tn + xylanase 200 ml/tn

(4) cellulase 100 ml/tn + pectinase 500 ml/tn,

(5) cellulase 100 ml/tn + xylanase 200 ml/tn + pectinase 50 ml/tn,

(6) cellulase 100 ml/tn + xylanase 200 ml/tn + 0 pectinase 500 ml/tn; and

Chopped forages were ensiled with the above treatments in duplicated one kilo PVC laboratory tube silos stored in a temperature range of 20-25'C. 5 kg of grass were ensiled in each silo. The silos were opened after a 5 month (133 days) ensiling period and the quality of the silage was analyzed according to the following procedure. Table 24 shows the results of this study.

Table 24

~ - ^* " o- ------ -

As is shown in Table 24, the silages treated with enzyme compositions of the present invention all displayed, for t _h e different parameters measured in Table 24, an enhanced fermentation quality when compared to the fermentation quality of the control treatment.

Fermentation quality is judged by the amount and type of acids produced, final pH level and amount of protein degraded as shown by the amount of volatile nitrogen expressed as a percentage of total nitrogen. 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 contained 0.4 - 0.6%-total acids as butyrate compared with 12% in control.

Finally, values for volatile nitrogen were lower for silages treated with enzyme compositions of the present invention indicating less protein breakdown during ensiling. 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 a d xylanase hydrolysis

_> n i*^re

(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. 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 urn) 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.

73

Table 25

Degradability of Silages in the Rumen (In sacco)

Of a Cow During Different Incubation Periods

(% of T'-tal Silage P.M. Degraded)

SILAGE TREATMENT (per ml per per tonne crop) Oh 6h_ 12h 24h 4Sh 96h

1 Control 37.6 45.2 58.3 71.8 83.3 85.4

2 C 49.2 53.4 67.4

100 76.8 83.5 86.7

52.0 56.6 69.8 78.1 84.7 87.1

⁴ ιoo ⁺ 58.5 60.2 65.4 77.2 83.4 87.5 ^r 500

53.8 59.3 65.7 76.2 83.8 86.2 2oo ^{+ r} 50

58.8 59.7 68.0 77.1 84.8 87.57

^{6 C} 100 ⁺ _p 200 ⁺ *500

CODES:

Control: No additives, just plain grade

X: Xylanase (ml/tn)

C Cellulase (ml/tn) P Pectinase (ml/tn)

" -~~ ^m "~- -*- - w- m -m

Table 25 demonstrates the improved capability of the various enzyme compositions of the present invention to degrade the ensiled forage of Example 9. As is shown in Table 25, 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 o is degraded at a faster rate and to a more complete degree than silage which is not treated with such enzyme compositions. Again, it can be seen that pectinase gave improvements over cellulase or cellulase + xylanase alone. The higher amount of pectinase was also more effective compared to the lower amount 5 in this example.

This confirms the results from Example 8 and would have the same benefits detailed previously in terms of speed of rumen turnover and improved protein utilization.

EXAMPLE 10 0

Pieces of Timothy (Phleum pratense) stem were cut and incubated in 20 ml 0.05 M Na-Citrate buffer at pH 5.0, temperature 30 ^* for 20 hours with and without lipase. After incubation, the samples were dried and the cuticulum was 5 examined.

When the pieces of Timothy (Phleum pratense) stem were subjected to the control treatment, i.e., incubation in 0.05 M Na-Citrate buffer at pH 5.0, temperature 30'C for 20 hours, and without lipase, the cuticulum was unbroken.

il

When the cuticulum of pieces of Timothy (Phleum pratense) stem were subjected to lipase treatment, i.e., incubation in 0.05 Mona-citrate buffer at pH 5.0, temperature 30*C for 20 hours, and with lipase, the cuticulum of lipase treated stem is flaked and uneven, in- contrast to the unbroken cuticulum associated with the control treatment. Due to the flaked and uneven surface of the lipase-treated cuticulum, the other layers of stem and cuticulum become more accessible to other enzymes and to the rumen microbes.

SUBSTITUTESHEET