BACTERIAL INOCULANTS TO PRESERVE SILAGE

Abstract: Silage is preserved by treatment with a small but silage preserving effective amount of an inoculant selected from: a combination of the microorganism Lactobacillus plantarum 286, or the genetic equivalent thereof, and the microorganism Enterococcus faecium 301, or the genetic equivalent thereof; a combination of the microorganism Lactobacillus plantarum 286, or the genetic equivalent thereof, the microorganism Lactobacillus plantarum 432, or the genetic equivalent thereof, and the microorganism Lactobacillus plantarum 435, or the genetic equivalent thereof; and a combination of the microorganism Lactobacillus plantarum 432, or the genetic equivalent thereof, and the microorganism Lactobacillus plantarum 435, or a genetic equivalent thereof. The present inoculants are particularly effective in improving the anaerobic stability of grass silage as well as rate and extent of digestibility.

Description: BACTERIAL INOCULANTS TO PRESERVE SILAGE 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, yeast and molds. These organisms are gen¬ erally recognized as being negative to the system because they metabolize sugars 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 corn 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 other silages such as whole corn plant, grass, 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 during the initial anaerobic stages. 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 which increases the extent of digestibility or the silage, thereby making more nutrients available to an animal. 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. Preser- vation is accomplished by mixing certain facultative lactic acid bacterial inoculants with the silage. The present inoculants also improve the extent and rate of digestibility 4 of silage, especially grass 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 FJ1: a combination of Lactobacillus plantarum 286 and Enterococcus faecium 301, having ATCC number ; FAC: a combination of Lactobacillus plantarum 286, Lactobacillus plantarum 432 and Lactobacillus plantarum 435, having ATCC number ; and AC: a combination of Lactobacillus plantarum 432 and Lactobacillus plantarum 435, having ATCC number . The present invention further provides methods of treating silage which comprises applying 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 stability and digestibility of grass silage. The present inoculants are also particularly effective in preserving nutrients in the 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. As used herein, the term "grass silage" includes both wet grass, having about 20% dry matter, and dry grass, having about 30-35% dry matter. The present invention is particularly effective in improving the anaerobic stability of grass silage. The present invention is also particularly effective in improving the extent and rate of digestibility of grass silage. Furthermore, the present invention is particularly effective in preserving the protein content of the ensiled forage 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: a combination of Lactobacillus plantarum 286 and Enterococcus faecium 301 ("FJ1") ; a combination of Lactobacillus plantarum 286, Lactobacillus plantarum 432 and Lactobacillus plantarium 435 ("FAC") , and a ' combination of Lactobacillus plantarum 432 and Lactobacillus plantarum 435 ("AC") . 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 6 -10 14 viable organisms/ton, preferably lO^lO 11 viable organisms/ton, more preferably 10 10 viable organisms/ton. The inoculant preferably comprises from about 20% to about 80% each of FJ1 and AC, more preferably from about 40% to about 60%, most preferably about 50% each of FJ1 and AC. For FAC, the inoculant preferably comprises from about 20% to about 50% of each strain, more preferably from about 25% to about 40%, most preferably about 33 3% of each strain. 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 example runs shown in the table below, the treatment, preparation and storage are conducted using standard procedure. The inoculants used in the silage trials are compared to a control sample which does not contain any inoculant. The level of inoculant is preferably about 1 x 10 5 cfu per gram in equal amounts of each strain. This corresponds to about 9 x 10 10 organisms per ton. Treatments are applied as a liquid. The prototype inoculants developed consist of selected strains of Lactobacillus plantarum and Enterococcus faecium in the following combinations: Lactobacillus plantarum strain 286 and Enterococcus faecium strain 301 ("FJ1") ; Lactobacillus plantarum strain 286, Lactobacillus plantarum strain 432 and Lactobacillus plantarum strain 435 ("FAC") ; and Lactobacillus plantarum strain 432 and Lactobacillus plantarum strain 435 ("AC") . Prototype combinations are mixed in a 50:50 ratio for FJ1 and AC, and in a 33 1 / 3 :33 1 / 3 :33 1 / 3 ratio for FAC and applied on wilted, or unwilted, chopped grass. Freeze-dried cultures of inoculants are solubilized and applied on the forage with a 30cc syringe fitted with a 16 gauge needle at a rate of 1 ml/lb forage. The final inoculum level for all bacterial treatments is 1 x 10 5 cfu/g forage. Treated forage is divided into equal portions and packed to a standard density using a hydraulic press into 4" x 14" experimental PVC silos. Silos are sealed at each end with rubber caps held tightly by metal rings. One end is fitted with a pressure release valve so that gases can escape and still maintain anaerobiosis. Experimental silos are stored at 20- 25°C for 80-120 days prior to opening to simulate farm silo conditions. Experimental silos are opened, silage removed into a clean container, mixed, and samples taken for microbial, chemical and digestibility analysis. The remaining silage is 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 are used to determine the stability of silage upon exposure to air. The hour at which silage temperature goes 1.7°C above ambient temperature is 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 "cumm dd" is the integration of the area between the actual temperature curve and a line drawn 1.7°C above 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 is 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. Lower ammonia nitrate values are thus indicative of nutrient preservation. The fermentation endpoint measurement is pH. A satisfactory pH for grass silage is less than 4.5. As the pH decreases, proteolytic activity decreases. The pH measurements are made with an Orien® model 701A pH meter calibrated with pH 4.01 and 7.00 buffers. Low pH values are also indicative of nutrient preservation. To determine the extent and rate of digestibility, samples are dried and ground through a 0.5mm Wiley® mill screen for digestibility analysis. All samples are scanned by near infrared reflectance 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 is 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 rumen microorganisms. As the rumen microorganisms digest the dry matter 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 per cent 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 is referred to as the extent of digestibility and was also expressed a per cent of a standard. The extent 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 are added to the NIRS calibration equation and values for the remaining samples were predicted based on their spectra. Silage treated with the present inoculants exhibit higher rates of digestibility than the control silage. Thus, the nutrients from silages inoculated are available to an animal faster than the nutrients from uninoculated silage. Inoculated silages also exhibit a higher extent of digestibility indicating that more nutrients are available to an animal. 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 are all acceptable (<4.5) with the inoculated silages having numerically better pH' s than the control silage. The inoculants also provide acceptable rot and cumm_dd values. Cumm_dd values are all quite low indicating minimal total heating. The rot values are all satisfactory. Cumm_dd values are very low showing that total heating is minimal. Animal digestion trials are also used to determine the digestibility of silage. Standard procedures are used. Twelve wether lambs with similar body weights are allotted to treatment by weight and placed in metabolism crates with access to fresh water and salt/mineral blocks at all times. A voluntary intake study is conducted for nine days to establish ad libitum intake levels. To ensure that animals are receiving more food than they can eat, feeding levels are adjusted daily to provide enough silage for approximately 10% refusal. Animals are fed twice daily. Intake is cut to 90% of the established voluntary intake for each lamb for the seven day collection period running from days 10-16. Amounts eaten by each lamb are recorded. Feces and urine is collected on days 12-16. Digestibility parameters measured include dry matter, crude protein, neutral detergent fiber, acid detergent ' fiber and hemicellulose. Digestibility coefficients are calculated by taking the different between the level of the particular parameter in the feed being consumed and the level being excreted in the feces and urine. Results indicate that the animals being fed inoculated silage utilize more nutrients per amount of feed for maintenance and production. Variations on the above embodiments are within the ability of one of ordinary skill in the art, and such variations do not depart form the scope of the present invention as described in the following claims.

Claims: What is claimed is: 1. A method of preserving silage, said method comprising treating silage with a small but silage preserving effective amount of an inoculant selected from the group consisting of: a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof, and Enterococcus faecium 301, or the genetic equivalent thereof; a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof, Lactobacillus plantarum 432, or the genetic equivalent thereof, and Lactobacillus plantarum 435, or the genetic equivalent thereof; and a combination of Lactobacillus plantarum 432, or the genetic equivalent thereof, and Lactobacillus plantarum 435, or the genetic equivalent thereof. 2. The method of Claim 1 wherein the silage preserved is grass silage. 3. The method of Claim 2 wherein the inoculant is a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof, and Enterococcus faecium 301, or the genetic equivalent thereof. 4. The method of Claim 2 wherein the inoculant is a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof, Lactobacillus plantarum 432, or the genetic equivalent thereof, and Lactobacillus plantarum 435, or the genetic equivalent thereof. 5. The method of Claim 2 wherein the inoculant is a combination of Lactobacillus plantarum 432, or the genetic equivalent thereof, and Lactobacillus plantarum 435, or the genetic equivalent thereof. 6. The method of Claim 2 wherein the inoculant is applied at a rate of from about 10 8 to about 10 14 viable organisms per ton. 7. The method of Claim 6 wherein the inoculant is applied at a rate of from about 10 9 to about 10 12 viable organisms per ton. 8. The method of Claim 7 wherein the inoculant is applied at a rate of about 10 10 organisms per ton. 9. A silage preservative selected from the group consisting of: a small but silage preserving effective amount of the microorganism Lactobacillus plantarum 286, or the genetic equivalent thereof, and a small but silage preserving effective amount of the microorganism, Enterococcus faecium 301, or the genetic equivalent thereof; a small but silage preserving effective amount of the microorganism Lactobacillus plantarum 286, or the genetic equivalent thereof, a small but silage preserving effective amount of the microorganism Lactobacillus plantarum 432, or the genetic equivalent thereof, and a small but silage preserving effective amount of the microorganism Lactobacillus plantarum 435, or the genetic equivalent thereof; and a small but silage preserving effective amount of the microorganism Lactobacillus plantarum 432, or the genetic equivalent thereof, and a small but silage preserving effective amount of the microorganisms Lactobacillus plantarum 435. 10. The preservative of Claim 9 wherein said preservative further contains a suitable culture carrier. 11 . The preservative of Claim 10 wherein said preservative comprises, in combination, the microorganism Lactobacillus plantarum 286, or the genetic equivalent thereof, and the microorganism Enterococcus faecium 301, or the genetic equivalent thereof. 12. The preservative of Claim 10 wherein said preservative comprises, in combination, the microorganism Lactobacillus plantarum 286, or the genetic equivalent thereof, the microorganism Lactobacillus plantarum 432, or the genetic equivalent thereof, and the microorganism Lactobacillus plantarum 435, or the genetic equivalent thereof. 13. The preservative of Claim 10 wherein said preservative comprises, in combination, the microorganism Lactobacillus plantarum 432, or the genetic equivalent thereof, and the microorganism Lactobacillus plantarum 435, or the genetic equivalent thereof. 14. A method of improving the rate and extent of digestibility of silage, said method comprising treating silage with a small but silage preserving effective amount of an inoculant selected from the group consisting of: Lactobacillus plantarum 286, or the genetic equivalent thereof, in combination with Enterococcus faecium 301, or the genetic equivalent thereof; a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof, Lactobacillus plantarum 432, or the genetic equivalent thereof, and Lactobacillus plantarum 435, or the genetic equivalent thereof; and Lactobacillus plantarum 432, or the genetic equivalent thereof, in combination with Lactobacillus plantarum 435, or the genetic equivalent thereof. 15. The method of Claim 14 wherein the silage preserved is grass silage. 16 . The method of Claim 15 wherein the inoculant is a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof, and Enterococcus faecium 301, or the genetic equivalent thereof. 17. The method of Claim 15 wherein the inoculant is a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof, Lactobacillus plantarum 432, or the genetic equivalent thereof, and Lactobacillus plantarum 435, or the genetic equivalent thereof. 18. The method of Claim 15 wherein the inoculant is a combination of Lactobacillus plantarum 432, or the genetic equivalent thereof, and Lactobacillus plantarum 435, or the genetic equivalent thereof. 19. The method of Claim 15 wherein the inoculant is applied at a rate of from about 10 8 to about 10 14 viable organisms per ton. 20. The method of Claim 19 wherein the inoculant is applied at a rate of from about 10 9 to about 10 12 viable organisms per ton.

Inventors: DENNIS SCOTT M, HINDS MARK A, ROGERS MICHAELA G, RUTHERFORD WILLIAM M, ZIMMERMAN CINDI S, DENNIS, SCOTT, M., HINDS, MARK, A., ROGERS, MICHAELA, G., RUTHERFORD, WILLIAM, M., ZIMMERMAN, CINDI, S.
Applicants: PIONEER HI BRED INT, PIONEER HI-BRED INTERNATIONAL, INC.
Application year: 1996
Main classification: A23K3
IPC codes: A23K1, A23K3