Field of the Invention

This invention relates to a method of preserving agricultural products which are used for animal feed after storage under anaerobic conditions. Specifically, this invention relates to a method of preserving silage after storage under anaerobic conditions such that the extent and rate of digestibility of the silage are improved.

Background of the Invention

The use of silage additives has become a widely accepted practice throughout much of the agricultural world. During the ensiling process, aerobic respiration begins immediately upon chopping of silage. During this early phase, soluble carbohydrates in the plant tissue are oxidized and converted to carbon dioxide and water. This process continues until either the oxygen level is depleted or the water soluble carbohydrates are exhausted. Under ideal conditions, with adequate packing and sealing of the ensiled material, respiration lasts only a few hours. The growth of microorganisms during this period is limited to those that are tolerant of oxygen. Typically, this includes aerobic bacteria, yeasts and molds. These organisms are generally recognized as being negative to the system because they metabolize sugar to carbon dioxide, heat, and water.

Another important chemical change that occurs during this early phase is the breakdown of plant protein by plant proteases. Proteins are degraded to amino acids and further metabolized to ammonia and amines. It has been reported that up to 50% of the total proteins may be broken down during this process depending on the rate of pH decline in the silage. Once anaerobic conditions are established, anaerobic bacteria proliferate. Enterobacteria and

heterofermentative lactic acid bacteria are generally the first populations to become established. These organisms produce primarily acetic acid, ethanol, lactic acid, and carbon dioxide from the fermentation of glucose and fructose. Once the pH begins to decline, there is a marked increase in the homofermentative lactic acid bacteria population which produces primarily lactic acid. The rapid increase in the lactic acid level results in the decline of the pH to around 4. At this point, the ensiled mass will generally remain stable throughout storage if undisturbed.

In summary, when the material is initially packed in an oxygen-limiting structure, such as a covered silo, the pH is reduced, the residual oxygen is utilized and the material is said to undergo a lactic acid fermentation. The material will remain stable and can be stored for many months in this condition.

When the silage is ready to be fed, the top cover is removed and the silo is opened for feeding. The material is then exposed to air and the process is no longer anaerobic. Microflora in the silage itself or airborne contaminants can begin to oxidize the acids present. This oxidation causes a loss in mass or dry matter of the feed and thus causes feeding losses. In addition, the resultant pH and temperature increases are objectionable to the animals and the feed will be refused by the animals after it has begun to heat. The incidence of aerobic instability observed in practice depends on the rate at which the ensiled material is removed from the silo and the length of time that the material has been ensiled before opening. If the silage is unloaded slowly then more time is allowed for deterioration to occur on the surface of the opened silage. Longer ensiling times produce generally more stable silage as the acid concentrations are higher and all microflora populations tend to decrease. In general the silage should be stable for at least five days after opening. This will allow for adequate time for the silage to be removed.

Recently it has become known that bacterial inoculants help preserve silage, including grass silage, alfalfa silage and corn silage. For example, inoculation with lactic acid bacteria during the fermentation phase can be beneficial to the fermentation process, see e.g. U.S. Patent 4,842,871 of Hill issued June 27, 1989, as well as the literature references cited therein. For high moisture alfalfa stability, this increase is probably due to the inoculants' enhancing the rate of anaerobic fermentation and pH decrease. This is beneficial because oxidative losses caused by aerobic pH-sensitive microflora in the initial stages are thus avoided. In silages such as whole plant corn, alfalfa, etc. the inoculant can also have beneficial effects on the digestibility of the silages by causing an increase in the availability of the fiber, and/or providing more nutrients per amount of silage at a faster rate.

Accordingly, it is an objective of the present invention to develop a bacterial silage inoculant that is effective both during the initial anaerobic stages and during the initial aerobic stages when a silo is opened to air.

It is a further objective of the present invention to develop a silage inoculant that increases the rate of digestibility of the silage, thereby making nutrients available to an animal sooner.

A further objective of the present invention is to develop a silage inoculant that increases the extent of digestibility or the silage, thereby making more nutrients available to the animal being fed. The method and manner of accomplishing each of the objectives of the present invention as well as others will become apparent from the detailed description which follows hereinafter.

SUMMARY OF THE INVENTION

In the present invention silage, including grass, alfalfa and/or corn silage, is preserved both during the

initial anaerobic phase of the ensilage process and during the initial phases of aerobic conditions after a silo is opened. Preservation is accomplished by mixing certain facultative bacterial inoculants. The present inoculants improve the extent and rate of digestibility of silage, especially alfalfa silage. The inoculants are combinations of selected strains of Lactobacillus plantarum and Enterococcus faecium. The present inoculants are compatible with the other bacteria, and thus do not retard the ensilage process in any way. Specifically, the inoculants include TJ1: a combination of Lactobacillus plantarum 347 and

Enterococcus faecium 301, having ATCC number ; ST: a combination of Lactobacillus plantarum 346 and Lactobacillus plantarum 347, having ATCC number ; and FS: a combination of Lactobacillus plantarum 286 and Lactobacillus plantarum 346, having ATCC number . The present invention further provides methods of treating silage which comprise administering to the silage a small but ensilage preserving effective amount of the present inoculant prototypes. The inoculants of the present invention are particularly effective in improving the digestibility of alfalfa silage.

DETAILED DESCRIPTION OF THE INVENTION The term "silage" as used herein is intended to include all types of fermented agricultural products such as grass silage, alfalfa silage, corn silage, sorghum silage, fermented grains and grass mixtures, etc. All can be treated successfully with the inoculants of the present invention. The present invention is particularly effective in improving the extent and rate of digestibility of alfalfa silage.

A surprising aspect of this invention is that only certain combinations of certain strains of Lactobacillus plantarum and/or Enterococcus faecium will function effectively in the present invention. The addition of Lactobacillus to silage as a general matter is known, see

for example U.S. Patent No. 4,981,705. However, the present invention is necessarily strain specific with regard to the

Lactobacillus plantarum and Enterococcus faecium. In particular, the inoculants found to work in the present invention are: Lactobacillus plantarum 347 in combination with Enterococcus faecium 301 ("TJ1") , Lactobacillus plantarium 346 in combination with Lactobacillus plantarum 347 ("ST") , and Lactobacillus plantarum 286 in combination with Lactobacillus plantarum 346 ("FS") . It is to be understood, however, that applicants' invention, while species specific, is intended to cover these species and their genetic equivalents, or the effective mutants thereof, which demonstrate the desired properties of the named species and strains. Such genetic equivalents or mutants thereof are considered to be functionally equivalent to the parent species. It is well known to those of ordinary skill in the art that spontaneous mutation is a common occurrence in microorganisms and that mutations can also be intentionally produced by a variety of known techniques. For example, mutants can be produced using chemical, radioactive, and recombinant techniques.

Regardless of the manner in which mutations or the genetic equivalents are induced, the critical issue is that they function to preserve the silage as described for the parent species and/or strain. In other words, the present invention includes mutations resulting in such minor changes as, for example, minor taxonomical alterations.

Typical compositions useful for treatment of this invention may include the present inoculants within the ranges useful for treating ensilage products, i.e. typically 10 ⁸ -10 ¹⁴ viable organisms/ton, preferably 10 ⁹ -10 viable organisms/ton, more preferably 10 ¹⁰ viable organisms/ton. A mixture of the two strains ranging from about 75% to about 25% of each strain is preferred. A mixture of about 50% of each of the two strains per inoculant is particularly preferred.

The composition of the present invention can also include other common silage preservation organisms as, for example, Propionibacteria, Streptococcus, Lactococcus and Pediococcus, and certain enzymes from fungi or bacteria, providing they are in no way antagonistic to the active organisms.

Those of ordinary skill in the art will know of other suitable carriers and dosage forms, or will be able to ascertain such, using routine experimentation. Further, the administration of the various compositions can be carried out using standard techniques common to those of ordinary skill in the art, i.e. spraying, dusting, etc,

The above disclosure generally describes the present invention. A more detailed understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only and are not intended to be limiting, unless otherwise specified.

EXAMPLES ^• In the examples shown in the tables below, the treatment, preparation and storage were conducted using standard procedures. The inoculants used in the silage trials, which were conducted in the years 1992, 1993 and 1994, were compared to a control sample which did not contain any inoculant. The level of inoculant was 1 x 10 ⁵ viable organisms per gram of forage in a 50:50 mixture. This corresponds to 9 x 10 ¹⁰ organisms per ton. Treatments were applied as a liquid. The prototype inoculants developed consisted of selected strains of Lactobacillus plantarum and Enterococcus faecium in the following combinations: Lactobacillus plantarum strain 347 and

Enterococcus faecium strain 301 ("TJ1") ; Lactobacillus plantarum strain 286 and Lactobacillus plantarum strain 346

("FS") ; and Lactobacillus plantarum strain 3 6 and Lactobacillus plantarum strain 347 ("ST") .

Prototype combinations were mixed in a 50:50 ratio and applied on wilted, chopped alfalfa in a liquid form at a

rate of 1 x 10 ⁵ cfu/g forage. Treated forage was divided into equal portions and packed to a standard density using a hydraulic press into 4" x 14" experimental PVC silos. Silos were sealed at each end with rubber caps held tightly by metal rings. One end was fitted with a pressure release valve so that gases could escape and still maintain anaerobiosis. Experimental silos were stored at 20-25°C for 80-120 days prior to opening to simulate farm silo conditions. Experimental silos were opened, silage removed into a clean container, mixed, and samples taken for microbial, chemical and digestibility analysis. The remaining silage was placed in a plastic lined polystyrene cooler, a probe placed in the center of the silage mass, and temperature measured every 3 hours for one week to determine aerobic stability. When silage is exposed to air, large losses of nutrients can occur as the result of aerobic microorganisms' consuming sugars and fermentation products in the silage. The sugars are respired to carbon dioxide and water, producing heat. Besides the loss of highly digestible portions of the silage, some aerobic microorganisms produce toxins which affect an animal's health.

Two measurements were used to determine the stability of silage upon exposure to air. The hour at which silage temperature went 1.7°C above ambient temperature was referred to as the "rot" of the silage. It is a measure of time after the silage is exposed to air before the aerobic microorganisms start to grow causing the silage to heat. Cumulative degree days, or "cumπi dd" is the integration of the area between the actual temperature curve and a line drawn by ambient temperature. It is a measure of the total amount of heating. Elevated temperatures increase the rate and amount of protein breakdown and reduce the digestibility of nitrogen, fiber, and other fractions. Ammonia nitrogen determination was conducted using standard procedures involving dissociation of the ammonia ion by raising the pH, followed by steam distillation of the

ammonia out of the silage. The amount of ammonia nitrogen is quantitatively measured by titration. The level of ammonia nitrogen is an indicator of the rate of fermentation. The faster the rate of fermentation, the lower the activity of proteolytic enzymes, thereby making more proteins available for an animal.

The fermentation endpoint measurement is pH. A satisfactory pH for alfalfa silage is less than 4.5. As the pH decreases, proteolytic activity decreases. The pH measurements were made with an Orien® model 701A pH meter calibrated with pH 4.01 and 7.00 buffers.

To determine the extent and rate of digestibility, samples were dried and ground through a 0.5mm Wiley® mill screen for digestibility analysis. All samples were scanned by near infrared radiation spectroscopy (NIRS) . Extremes in spectra were selected on which to run in vitro dry matter (IVDM) rates and extents of digestibility. IVDM rate of digestibility was determined using a system designed to simulate what happens in the rumen. Dried silage samples are combined with a buffer and rumen fluid containing live cellulytic microorganisms. As the cellulytic microorganisms digest the fiber in the silage sample, gas is produced. The rate of digestibility was defined as the slope of the linear portion of the curve produced by plotting gas production vs. time. It was expressed as a percent of a standard to account for the variation in microbial populations between batches of rumen fluid. A faster rate of digestibility means nutrients are being made available to the animal sooner allowing it to utilize them to produce more milk or meat. One possibility of how the inoculants are causing this increase is that they are changing the structure of the forage, making it more available to the rumen microorganisms, which in turn convert the forage to energy for use by the animal. The total volume of gas produced over a set period of time was referred to as the extent of digestibility and was also expressed a percent of a

standard. The extent of digestibility is an indicator of the total amount of nutrients made available by the digestion of the fiber. The IVDM rate and extent of digestiblities for the extremes were added to the NIRS calibration equation and values for the remaining samples were predicted based on their spectra.

Tables 1,2 and 3 below summarize the trials conducted. "Control" indicates uninoculated silage.

Table 1 summarizes the data from a 1992 trial. Table 1 indicates that TJ1, FS and ST all have higher rates of digestibility than the control silage. Thus, the nutrients from silages inoculated would be available to an animal faster than the nutrients from uninoculated silage. TJ1- and FS-inoculated silages also show lower ammonia nitrogen levels than control silage, thus indicating a faster fermentation rate leading to lower protein loss. The pH values were all acceptable (<4.5) with the inoculated silages having numerically better pH's than the control silage. Table 2 summarizes data from 1993 covering seven trials for pH, rot, cumm_dd, extent of digestion, and rate of digestion. Five trials were conducted for ammonia nitrogen.

TJ1, FS, and ST all show significantly (P≤.l) higher rates (nutrients available faster) and extents (more nutrients available) of digestibility and lower (P<.1) ammonia nitrogen levels (less protein loss) than uninoculated silage. The inoculants also provide better rot and cumm_dd values than control silage indicating better aerobic stability. Better rot values indicates less loss of nutrients due to aerobic heating. Cumm_dd values were all very low indicating minimal total heating. The rot values were all satisfactory as over 6 days passed before aerobic microorganisms started growing and causing heating. Cumm_dd values were very low showing that total heating was minimal. Table 3 summarizes data from five trials conducted for TJ1 and four trials conducted for ST in 1994. The data

indicate that TJl and ST have higher rates and extents of digestibility than control silage. Inoculated silages also had significantly (P≤.20) better pH values than control silage (which had a pH above 4.5) .

Table 1: 1992 Alfalfa Trial Summary

Treatment E5 Rε •. of dig. NH ₃ - -N (% total N)

Control 4.35 107.9 10.58

FJ1 4.30 123.0 8.88

FS 4.21 126.3 7.82 ST 4.17 124.6

Table 2: 1993 Alfalfa Trial Summary

Treatment pH Rot Cu m dd Extent Rate NH ₃ N of dig. of dig. (% total N)

Control 4.44 134.8 22.2 80.7 87.3 7.71

TJl 4.46 146.5 27.5 89.3 95.3 6.66

FS 4.43 157.1 1.6 89.2 92.0 5.36

ST 4.45 160.0 0 88.5 94.8 5.20

Table 3: 1994 Alfalfa Trial Summary

Treatment Rot Cumm dd Extent Rate of dig. of dig.

Control 152.9 11.4 88.81 87.23

ST 149.4 23.1 94.75 93.39

TJl 151.8 11.4 92.61 94.21