FIELD OF THE INVENTION

This invention relates to microorganisms and their use in treating animal feed and silage.

BACKGROUND OF THE INVENTION

The use of enzymes and organisms can improve or enhance the performance of animals and the value of the feed the animals receive. For example, WO-A-9210945 discloses such a combination for use in enhancing the value of prepared silage, and WO- A-9617525 relates to enhancement of animal performance using microorganisms. The efficacy of combining the use of enzymes together with organisms producing volatile fatty acids (VFA's) is also described. In this case, better preservation of the silage, better animal performance and a reduction in effluent production were demonstrated. WO-A-

9503396 demonstrates that some advantages may accrue when a desired VFA profile is produced during the silage fermentation; it has also been found that this does not produce the desired reduction in heating, on opening the silage clamp. The production of silage and the associated crop husbandry have over recent years developed to an extent that a number of different processes can be defined. These are: (i) the ensiling of young grass with particularly low dry matter, e.g. less than 25%, (ii) the ensiling of higher dry matter, more mature grasses, the ensiling of high dry matter but young grass achieved by wilting; and (iii) the ensiling of whole maize including stova and cob, usually at a dry matter concentration of about 35%, and whole crop cereals, e.g. wheat, at 45-50% dry matter. Particularly in cases (ii) and (iii), one major problem occurs on a regular basis. This is the phenomenon known as aerobic spoilage. Although there are many differing opinions, the process of aerobic spoilage can be divided into phases. Thus, there is an initial phase in which yeasts and sometimes acetic acid bacteria start to respire the preserving organic acids. After an initial rise in pH, there is a secondary phase in which the activity of bacilli is apparent, and is associated with increasing temperature. A further phase includes activity of various microorganisms including fungi. In those silages which contain a substantial content of dry matter, i.e. over 30%, the problem of spoilage is particularly acute. Spoilage is seen to a greater or lesser extent once a silage clamp is opened and exposed to air. US patents 6,326,037 and 6,699,514 (both expressly incorporated herein by reference) further provides an organism of the species Lactobacillus buchneri alone and in combination with other materials for the purpose of further preventing aerobic spoilage in silage.

While the methods and compositions of US patents 6,326,037 and 6,699,514 have been effective in improving the situation, further advances in the treatment of silage to improve digestibility, reduce effluent production, and reduce spoilage would be advantageous. . In particular, it would be of considerable advantage to have a method that would reliably allow drier feed materials (e.g., hay harvested at 15 - 40% moisture; whole grains harvested at 15 - 25% moisture) to be stored without significant growth of molds and the risk of spontaneous combustion due to microbial growth.

SUMMARY OF THE INVENTION

The invention comprises methods and compositions for the treatment of silage to prevent spoilage and/or limit the growth of aerobic spoilage organisms such as bacteria yeast and molds.

In one aspect the invention relates to a method for treating silage to prevent or reduce aerobic spoilage which comprises adding to said silage a composition consisting of a combination of cellulase and glucose oxidase. In a further embodiment the silage is miantained closed for a period of at least 15 days, and preferably 30 days and said composition has an increased aerobic stability as comparedto untreated silage. In a further embodiment the treatment retards the developmnet of microbial colonies in the treated silage as compared to untreated silage under the same conditions. The silage to be treated may be composed of forage includeing but not limited tio the following sources: traditional grass maize; Lucern,; wilted grass;whole crop cereal. The traditional grass may be any traditional grass including rye grass. The crop cereal may be any crop cereal including wheat or barley.

The composition of the invention which comprises the enzymes glucose oxidase and cellulase may be present and applied in either liquid or powdered (dry) form. In a prefreed embidment the composition is in liquid form. In a further embodiment, the composition of the nvnetion further included Lactobacillus buchneri. In yet another mebodiment the composition further comprises probiotic yeast.

When the ccmposition o fthe invention is applied, the enzymes are each preferably applied at a rate greater than 100,000 IU, preferably greater than 200,000 IU per ton of forage, more preferably greater than 250,00 IU per ton of forage, even more preferably greater than about 300,00 IU per ton of forage. In one embodiment the enzymes are both applied at about 350,000 IU per ton of forage. .

DESCRIPTION OF THE INVENTION

In one aspect, the invention provides a mixture or enzyme system comprising two enzymes, cellulase, to generate glucose, and glucose oxidase (GO), to convert the glucose to gluconic acid.

Cellulase:

Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze the cellulolysis (or hydrolysis) of cellulose. However, there are also cellulases produced by other types of organisms such as plants and animals.

Several different kinds of cellulases are known, which differ structurally and

mechanistically. The EC number for this group of enzymes is EC 3.2.1.4.

In the most familiar case of cellulase activity, the enzyme complex breaks down cellulose to beta-glucose. This type of cellulase is produced mainly by symbiotic bacteria in the ruminating chambers of herbivores. Aside from ruminants, most animals (including humans) do not produce cellulase in their bodies, and are therefore unable to use most of the energy contained in plant material.

Cellulase generally causes the hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, lichenin and cereal beta-D-glucans. Other names for enzymes exhibiting cellulose activity include: 'endoglucanases', which are: endo-l,4-beta-glucanase, carboxymethyl cellulase (CMCase), endo-l,4-beta-D-glucanase, Beta-l,4-glucanase, Beta-l ,4-endoglucan hydrolase, Celludextrinase. The other types of cellulase belong to Excocellulases. Beta glucosidases can also be considered as yet another group of cellulases. The expression 'Avicelase' refers to the total cellulase activity of a given sample of the enzyme(s). The cellulase may be the result of the action of more than one type of enzymes.

In a preferred embodiment of the invention, the cellulase provided is

Trichoderma reesei. Trichoderma reesei is a mesophilic and filamentous fungus. It is an anamorph of the fungus Hypocrea jecorina. T. reesei has the capacity to secrete large amounts of cellulolytic enzymes (cellulases and hemicellulases). Several industrially useful strains have been developed and characterised, e.g. Rut-C30, RL-P37 and MCG- 80. The genome of this organism was released in 2008. [Martinez D, Berka RM, Henrissat B, et al. (May 2008). "Genome sequencing and analysis of the biomass- degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)". Nat. Biotechnol. 26 (5): 553-60.]

Glucose Oxidase:

The enzyme glucose oxidase (GO) (EC 1.1.3.4) binds to beta-D-glucopyranose (a hemiacetal form of the six-carbon sugar glucose) and aids in breaking the sugar down into its metabolites. GO is a dimeric protein that catalyzes the oxidation of beta-D- glucose into D-glucono-l ,5-lactone, which then hydrolyzes to gluconic acid. When produced commercially, GO can be extracted from Aspergillus niger. GO at the surface of a material can reduce atmospheric O ₂ to hydrogen peroxide (H ₂ O ₂ ), which in turn can act as an antimicrobial barrier. Specific Embodiments:

In certain embodiments, the invention can be provided in a mixture having both a cellulase and a glucose oxidase. The mixture can have a pH of approximately 3.0 to 6.5, and more preferably, approximately 4.0 to 5.0. The mixture can also have an activity level expressed in Carboxymethyl Cellulose substrate (CMC units/g), in which one CMC of activity liberates 1 μιηοΐε of reducing sugars (expressed as glucose equivalents in one minute, under assay conditions, of at least about 1000, preferably at least about 1500, more preferably at least about 2000, still more preferably at least about 2500, still more preferably at least about 3000, and most preferably at least about 3500.

Further Aspects of the Invention:

Further aspects of the invention include, without limitation, a mixture or enzyme system including both cellulase and glucose oxidase in combination with high dry matter silage, high moisture dry baled hay or grain for storage a method of treating the silage, and a method of feeding an animal using silage treated with the mixture. In one embodiment, the enzyme system is diluted in water and applied to the silage at a minimum rate of about 4 liters per ton. Use of the described enzyme system to function in situations where conventional bacterial inoculants are challenged, e.g. dry baled hay, high moisture corn, dry corn and other grains going into storage, can be especially beneficial. While not wishing to be bound by any particular theory of operation, the enzyme system of the invention would offer protection in two ways: (1) by the removal of oxygen; and (2) by the production of hydrogen peroxide as an anti-microbial. In addition, the enzyme system of the invention could be co-formulated or used in silage treatment along with other silage inoculants or treatments such as those described in US patents 6,326,037 and 6,699,514. As certain of those inoculants also produce hydrogen peroxide, further synergetic effects could be realized. In particular, Lactobacillus buchneri, Lactobacillus kefir, Lactobacillus parakefir and Lactobacillus parabuchneri may be used as co-innoculants with the compositionsand in the methods of the invention Again, while not wishing to be bound by any particular theory of operation, Applicant suspects that the invention provides surprising results in the high dry matter materials described above at least in part because Applicant believes that these materials have a high level of entrained oxygen. Untreated, or even treated with conventional treatments, these materials are very prone to severe spoilage. The oxygen scavaging and hydrogen peroxide production aspects of the enzyme system described limits the growth of aerobic spoilage organisms (such as bacteria, yeasts and molds) under these circumstances and yields superior results.

In a further aspect, the invention can include a live probiotic yeast for the purpose of reducing or limiting aerobic spoilage. The live probiotic yeast can be used on its own, or in combination with the enzyme systems described above, or further in combination with the lactic acid bacteria described above. The live probiotic yeast and methods of delivering it in a different context are described in the attached published international application (WO 2009/097333 A2). Applicant believes that this approach will also be effective in addressing aerobic spoilage in high dry matter silage as discussed above. Examples

Methods and Materials

Mycogen corn, hybrid TMF2W726, was harvested at 32.7% whole-plant DM content (DM%) and chopped using a New Holland FP230 (New Holland, PA) pull-type harvester equipped with a mechanical processor. Treatments in this experiment were: a) 200 mL of water (control), b) experimental powdered enzyme blend, reconstituted with

200 mL of water, c) experimental liquid enzyme blend, reconstituted with 200 mL of water, d) Buchneri 500 (Lactobaccilus Buchneri) applied at the manufacturer's recommended dosage, diluted with 200 mL of water, e) the experimental powdered enzyme blend in combination with the manufacturer's recommended dosage of Buchneri 500, diluted with 200 mL of water, f) the experimental liquid enzyme blend in combination with the manufacturer's recommended dosage of Buchneri 500, diluted with 200 mL of water. Samples of freshly chopped forages were collected for laboratory analysis of starting material. Four replicate piles of freshly chopped corn were prepared for each treatment, and treated appropriately before ensiling in 2-gallon buckets. A total of 72 buckets were prepared. Four replicates per treatment will be opened after 15, 30 and 90 days of ensiling.

For each treatment and replicate, a 25 gram representative sample of fresh forage or silage was combined with 225 mL of sterile Ringer's solution (Oxoid BR0052G) and homogenized for 1 min in a Proctor-Silex 57171 blender (Hamilton Beach Proctor- Silex Inc., Washington, NC, USA). The pH of the sample was determined and recorded immediately. A portion was then filtered through Whatman 54 filter paper (Whatman, Florham, NJ, USA) and acidified with three drops of 50% H ₂ S0 ₄ , and the water extract was then frozen until further analysis. Another portion was filtered through a double layer of cheesecloth and collected for the enumeration of LAB, yeasts and molds. The numbers of viable lactic acid bacteria were determined by pour plating 10-fold serial dilutions of silage water extracts on de Man, Rogosa, and Sharpe agar (CM3651, Oxoid,

Basingstoke, UK). Plates were incubated aerobically at 32°C for 48 to 72 h. Yeasts and molds were determined using Petrifilm Yeast and Mold Count plates (3M). These plates were also incubated at 32°C for 48 to 72 h. Aerobic stability was determined on silages by placing two kilograms of well-mixed silage from each silo into clean silos and placing a thermocouple wire into the geometric centre of each silage mass. The thermocouple wires were connected to a data logger (model number CR10X, Campbell Scientific, Inc., Logan, UT, USA) that recorded the temperature every 10 min, and then averaged the temperature over a half-hour period. Aerobic stability was defined as the time it took for the temperature in the silage masses to raise 2 °C above ambient temperature.

Both the enzyme products (Liquid and Powdered Enzyme Blends) used in the example were a combination of cellulase (EC 3.2.1.4, from Trichoderma reesei) and glucose oxidase (EC 1.1.3.4, from Aspergillus niger). Both were applied at a rate of 350,000 IU per ton of forage (for both activities). Results

Fresh Forage:

Powdered Liquid

L. buchneri

Control Enzyme Blend Enzyme PE + LB LE + LB

(LB)

(PE) (LE)

Yeasts, log cfu/g 7.06 7.16 7.60 7.53 7.34 7.32

Molds, log cfu/g 2.84 4.43 3.52 2.94 5.86 3.08

DM, % 33.0 32.3 32.3 33.3 32.8 32.5 pH 5.41 5.31 5.27 5.27 5.13 5.09

Density, lb 13.3 12.6 12.3 13.4 12.9 12.9

DM/cu ft

Silage after 15 days of ensiling:

Powdered Liquid

L. buchneri PE +

Control Enzyme Enzyme LE + LB

(LB) LB

Blend (PE) (LE)

Yeasts, log cfu g 3.10 1.38 2.90 2.28 2.16 2.71

DM, % 31.6 32.0 32.5 33.5 32.6 32.7 pH 3.78 3.78 3.89 3.90 3.82 3.87

Aerobic Stability, hr 25.5 25.4 25.1 96.9 65.3 107

Silage after 30 days of ensiling:

Powdered Liquid

L. buchneri PE +

Control Enzyme Enzyme LE + LB

(LB) LB

Blend (PE) (LE)

Yeasts, log cfu/g 2.75 2.31 1.94 3.10 3.44 2.86

Molds, log cfu/g 1.17 2.73 2.25 2.22 3.21 2.67

DM, % 32.3 32.2 31.5 32.8 31.9 32.2 pH 3.59 3.58 3.77 3.77 3.64 3.68

Aerobic Stability, hr 46.1 61.6 62 94.3 84 106.8

The data indicates some interesting and unexpected results.

First it appears that the enzyme blend (whether in liquid or dry format) did not significantly increase aerobic stability at 15 days of ensiling as compared to control. This is in stark contrast to silage treated with L. buchneri (X5jwhich showed a significant increase in aerobic stability at 15 days. When a liquid formulation of the composition of the invention (glucose oxidase and cellulase) was further combined with LB a further increase in aerobic stability was seen at 15 days.

Second, it appears that the Liquid Enzyme blend of cellulose and glucose oxidase performed the same as the powdered enzyme blend when it came to aerobic stability of silage at 15 and 30 days of storage but was superior at 30 days in reducing mold and yeast growth in the silage.

Finally, it is the case that the Liquid Enzyme blend of glucose oxidase and cellulase in combination with L. Buchneri was superior to both the enzyme blend alone and the L Buchneri alone when it comes to increasing aerobic stability at both 15 days and 30 days of ensiling. This is especially unexpected in light of (a) the lack of any increase in aerobic stability at 15 days when either the powdered or liquid enzyme blends were added to the silage ; and (b) the lack of a difference in increase aerobic stability between the powder or liquid enzyme blend formulations.

What is claimed is: