MXPA97009355A - Bacterial inoculants to preserve ensil - Google Patents

Abstract

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

Description

BACTERIAL INOCULANTS FOR PRESERVING SILAGE DESCRIPTION OF THE INVENTION This invention relates to a method for preserving agricultural products which are used to feed animals after storage under anaerobic conditions. Specifically, this invention relates to a method for preserving silage after storage under anaerobic conditions in such a way that the degree and speed of silage digestibility is improved. The use of silage additives has become a widely accepted practice in most of the agricultural world. During the process of forming the silage, aerobic respiration begins immediately after the silage is milled. During this early phase, the soluble carbohydrates in the plant tissue are oxidized and converted to carbon dioxide and water. This process continues until the oxygen level decreases or the water-soluble carbohydrates are depleted under ideal conditions, with adequate packing and sealing of the ensiled material, breathing lasts only a few hours. The growth of microorganisms during this period is limited for those that are tolerant to oxygen. Typically, this includes aerobic bacteria, yeasts and molds. These organisms are generally recognized as being negative for the system since they metabolize sugars to carbon dioxide, heat, and water. Other important chemical change that occurs during this early phase is the cleavage of the plant protein by plant proteases. The 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 can be decomposed during this process depending on the speed with which the pH decreases in the silage. Once the anaerobic conditions are established, the anaerobic bacteria proliferate. Enterobacteriaceae and heterofermentative lactic acid bacteria are generally the first populations that become established. These organisms produce mainly acetic acid, ethanol, lactic acid, and carbon dioxide from the fermentation of glucose and fluctuous. Once the pH begins to decrease, there is a marked increase in the population of lactic acid bacteria such as fermentative which produces mainly lactic acid. The rapid increase in the level of lactic acid results in the pH decrease to approximately around 4. At this point, the ensiled mass will remain totally generally stable in storage if it is not disturbed. In summary, when the material is initially packed in an oxygen limiting structure, such as a covered silo, the pH decreases, the residual oxygen is used and the material can 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 the air and the process is no longer anaerobic. The microflora in the same silage or contaminants can begin to oxidize the acids present. This oxidation causes a mass loss - with dry bacteria from the feed and thus causes losses in the feed. In addition, the resulting pH and temperature increases are unacceptable to the animals and the feed will be rejected by the animals after it has begun to warm up. The incidence of aerobic instability observed in practice depends on the speed at which the ensiled material is removed from the silo and the time at which the material has been ensiled before the opening. If the silage is unloaded slowly then there is more time for deterioration to occur on the surface of the open silage. Silage formation times generally produce more stable ensilages as acid concentrations are higher and all microflora populations tend to decrease. In general, silage must be stable for at least five days after opening. This will allow adequate time for the silage to be removed. It has become known recently that bacterial inoculants help to conserve silage, including pasture silage, alfalfa silage, and corn silage. For example, inoculation with lactic acid bacteria during the fermentation phase may be beneficial to the fermentation process, see for example U.S. Patent No. 4,842,871 to Hill filed June 27, 1989, as well as the references of literature cited in it. The high stability of the maize to moisture increase is probably due to the improvement of the speed of the anaerobic fermentation and the decrease of the pH of the inoculants. This is beneficial since the oscillating losses caused by the aerobic microflora sensitive to pH in the initial stages are thus avoided. In other silages such as a whole plant of corn, pasture, alfalfa, etc., the inoculant may also have beneficial effects on the digestibility of silage causing an increase in the availability of fiber, and / or provide more nutrients by quantity of silage at a faster speed. Accordingly, it is an object of the present invention to develop a bacterial silage inoculant which is effective during the initial anaerobic stages.

It is a further object of the present invention to develop a silage inoculant that increases the silage digestibility rate, thereby making the nutrients available to an animal sooner. A further object of the present invention is to develop an inoculant for silage which increases the degree of digestibility or silage, thereby making the nutrients more available to an animal. The method and manner of carrying out each of the objects of the invention as well as others will become apparent from the detailed description which follows thereafter. In the present invention, silage, including silage from pasture, alfalfa and / or corn is conserved both during the initial anaerobic phase of the silage formation process. Conservation is carried out by mixing certain facultative lactic acid bacterial inoculants with the silage. The present inoculants also improve the degree and speed of silage digestibility, especially pasture silage. The inoculants are combinations of selected strains of Lactobacillus plantarum and Enterococcus faecium. The inoculants present are compatible with the other bacteria, and thus do not retard the process of forming the silage in any way. Specifically, the inoculants include FJ1: a combination of Lactobacillus plantarum 286 and Enterococcus faecium 321 having the number; FAC: a combination of Lactobacillus plantarum 286, Lactobacillus plantarum 432 and Lactobacillus plantarum 435, which have ATCC number; and AC: a combination of Lactobacillus plantarum 432 and Lactobacillus plantarum 435, which has ATCC number. The present invention further provides methods for treating silage which comprise applying to the silage a small but effective amount of the prototypes of inoculants present to preserve the silage. The inoculants of the present invention are particularly effective in improving the stability and digestibility of the pasture silage. The inoculants present are also effective particularly for preserving nutrients in the silage. The term "silage" as used herein is proposed to include all types of fermented agricultural products such as pasture silage, alfalfa silage, corn silage, sorghum silage, fermented grains and pasture mixtures, etc. All can be successfully treated with the inoculants of the present invention. As used herein, the term "pasture silage" includes both the wet pasture, which has approximately 20% dry matter, and dry pasture, which has approximately 30-35% dry matter. The present invention is particularly effective in improving the anaerobic stability of the pasture silage, the present invention is also particularly effective in improving the degree and speed of digestibility of the pasture silage. Additionally, the present invention is particularly effective in preserving the protein content of silage. A surprising aspect of this invention is that only certain combinations of certain strains of Lactobacillus plantarum and / or Enterococcus faecium will effectively function in the present invention. The addition of Lactobacillus to silage as a general subject is known, see for example U.S. Patent No. 4,981,705. However, the present invention is necessarily specific to the strain with respect to 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, ("FJI") a combination of Lactobacillus plantarum 286, Lactobacillus plantarum 432 and Lactobacillus plantarum 435 ("FAC") and a combination of Lactobacillus plantarum 432 and Lactobacillus plantarum 435 ("AC"). It will be understood, however, that the invention of the applicants, while specifying the species, proposes to cover these species and their genetic equivalents, or the effective mutants thereof, which demonstrates the desired properties of the named species and strains. Such genetic equivalents or mutants thereof are considered to functionally equivalent to the progenitor 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 produced intentionally by a variety of known techniques. For example, mutants can be elicited using chemical, radioactive, and recombinant techniques. Regardless of the way in which mutations or genetic equivalents are induced, - the critical issue is that it works to conserve silage as described by the species and / or progenitor strains. In other words, the present invention includes mutations resulting from such minor changes as, for example, minor taxonomic alterations. Typical compositions useful for the treatment of this invention may include inoculants present within ranges useful for treating silage products, for example, typically 108-1014 viable organisms / ton, preferably 109-1011 viable organisms / ton, in form most preferred 1010 viable organism / ton. The inoculant in preferred form comprises from about 20% to about 80% of each FJ1 and AC more preferably from about 40% to about 60%, most preferably about 50% of each 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 1/3 of each strain. The composition of the present invention may also include other common silage conservation organisms, such as, for example, Propionibacteria, Streptococcus, Lactococcus and Pediococcus, and certain fungal or bacterial enzymes, providing that they are not in an antagonistic form for the active organisms. Those of ordinary skill in the art will know of other suitable carriers and dosage forms, or will be able to accept such using routine experimentation. In addition, administration of various compositions can be carried out using standard techniques common to those of ordinary skill in the art, for example, sprinkling, sprinkling, etc. The foregoing description generally describes the present invention. A more detailed understanding can be obtained by reference to the following specific examples which are provided here for purposes of illustration only and are not proposed to be limiting, unless otherwise specified.

EXAMPLES In the example runs shown in the table below, the treatment, preparation and storage are carried out using the standard procedure. The inoculants used in the silage tests are compared with a control sample which does not contain any inoculants. The level of the inoculant is preferably about lxlO5 ufe per gram in equal amounts of each strain. This corresponds to approximately 9x100 organisms per ton. The treatments are applied as a liquid. Developed prototype inoculants consist of selected strains of Lactobacillus plantarum and Enterococcus faecium in the following combinations: the strain of Lactobacillus plantarum 286 and Enterococcus faecium 301 ("FJl"): the strain of a Lactobacillus plantarum 286 the strain of Lactobacillus plantarum 432 and the strain Lactobacillus plantarum 435 ("FAC"); and the strain of Lactobacillus plantarum 432 and Lactobacillus plantarum 435 ("AC"). The prototype combinations are mixed in a 50:50 ratio for FJl and AC, and in a 33 1/3: 33 1/3: 33 1/3 ratio for FAC and applied on cut, withered or non-withered pasture. The frozen dried cultures of inoculants are solubilized and used on the forage with a fixed 30cmc syringe with a 16-gauge needle in a ratio of one ml / lb of forage. The final inoculum level for all bacterial treatments is lxlO5 cfu / g of forage. The treated forage is divided into equal portions and packaged for a standard density using a hydraulic press, 4-by-14in (lO.lßcm X 35.56cm) experimental PVC slats. The silos are sealed at each end with rubber caps rigidly fastened by metal rings. One end is fixed with a pressure release valve in such a way that the gases can still exit and maintain the anaerobic gases. The experimental silos are stored at 20-25 ° C for 80-120 days before opening to simulate the conditions of farm silos The experimental silos are opened, the silage is removed to a clean container, mixed, and samples are taken for microbial, chemical and digestibility analysis. the remaining silage is placed in a plastic linear polyethylene chiller, a probe is placed in the center of the silage mass, and the temperature is measured every three hours for a week to determine the aerobic stability. When the silage is exposed to the air, large losses of nutrients can occur as a result of the consumption of sugars and fermentation products of the aerobic microorganisms in the silage. The sugars are brought to carbon dioxide and water, producing heat, in addition to the loss of highly digestible portions of the silage, some aerobic microorganisms produce toxins which affect the health of the animal.

Two measurements are used to determine silage stability after exposure to air. The time at which the silage temperature is 1.7 ° C above room temperature is referred to as "silage rot" This is a measurement of the time after silage is exposed to the air before the aerobic microorganisms start to grow causing the heat of silage.

The days cumulative degrees, or cumm dd "is the integration of the area between the real temperature curve and a line drawing and a 1.7 ° C line above the ambient temperature, this is a measurement of the total amount of heating. High temperatures increase the rate and amount of protein breakdown and decrease the digestibility of nitrogen, fiber and other fractions.The determination of ammonia nitrogen is carried out using standard procedures that involve the dissociation of ammonia by raising the pH, followed by by steam distillation of ammonia off silage The amount of ammonia nitrogen is measured quantitatively by titration The ammonia nitrogen level is an indicator of the fermentation rate The faster the fermentation rate, the lower the activity of the proteolytic enzymes, making it more available to proteins for an animal. The reduced ammonia nitrate values are indicative of this form of nutrient conservation. The measurement of the end point of the fermentation is the pH. A satisfactory pH for the pasture silage is less than 4.5. As the pH decreases, the proteolytic activity decreases: pH measurements are made with a Orien® model 701A pH meter calibrated with pH 4.01 and 7.00 buffers. The low pH values are also indicative of the conservation of nutrients. To determine the degree of digestibility, the samples are dried and crushed through a 0.5mm tamper, Wiley® for digestibility analysis. The samples are analyzed by scanning using near infrared reflectance spectroscopy (NIRS). The ends of the spectrum are selected in which the speeds and degrees of digestibility of the dry matter in vitro (IVDM) are determined. The speed of digestibility IVDM is determined using a system designed to simulate what happens in the rumen. The dry silage samples are combined with a buffer and a rumen fluid containing active microorganisms from the rumen. As soon as the rumen microorganisms digest the dry matter in the silage sample, the gas is produced. The digestibility rate is defined as the slope of the linear portion of the curve produced by the gas reproduction plot against time. This is expressed as one percent of a standard to count the variation in microbial populations between batches of rumen fluid. A faster rate of digestibility means that nutrients are being available to animals sooner allowing them to use them to produce more milk or meat. A possibility of how the inoculants cause this increase in them change the structure of the forage, making it more available for the rumen microorganisms, which in turn convert the forage to energy for animal use the total volume of gas produced over a fixed period Time refers to the degree of digestibility and is also expressed as a percentage of a standard. The degree of digestibility is an indicator of the total amount of nutrients that are available for fiber digestion. The speed and degree of digestibility of IVDM for the ends are added to the NIRS calibration equation and for the remaining samples it is predicted based on its spectrum. Silage treated with the present inoculants exhibit higher digestibility rates than the control silage. In this way, the nutrients from the inoculated silage are available to an animal faster than the non-inoculated silage nutrients. Inoculated silages also exhibit a higher degree of digestibility indicating that more nutrients are available to an animal. Inoculated silages also show lower nitrogen levels of ammonia than control silage, thus indicating a faster fermentation rate that leads to less protein loss. The pH values are totally acceptable (<4.5) with inoculated silages that have pH numerically better than the control silage. The inoculants also provide putrefaction and acceptable cumm dd values. The cumm dd values are all low, indicating the minimum total heating. The putrefaction values are all satisfactory. The cumm dd values are very low showing that the total heating is minimal. Animal digestion tests are also used to determine silage digestibility. The standard procedures are used. Twelve castrated sheep with similar body weights are distributed for weight treatment and placed in metabolism cages with access to fresh water and lots of salt / mineral every time. A voluntary digestion study is conducted for nine days to establish levels of ingestion to libitum to ensure that the animals are receiving more food than they can eat, the feeding levels are adjusted daily to provide enough silage for approximately 10 days. % rejection The animals are fed twice daily. The ingestion is cut to 90% of the voluntary ingestion established for each lamb for the collection period of day seven that runs from days 10-16 the amounts eaten for each lamb are recorded. The faeces and urine are collected on days 12-16 the digestibility parameters are measured, which include dry matter, crude protein, natural detergent fiber, fiber, acid detergent fiber and emicelulose. The digestibility coefficients are calculated taking the difference between the level of the particular parameter in the food that is consumed and the level that is excreted in the feces and urine. The results indicate that the animals that are fed with inoculated silage use more nutrients per amount of food for maintenance and production. Variations on the above embodiments are within the skill of a person of ordinary skill in the art, and such variations do not depart from the scope of the present invention as described in the following claims.

Claims (20)

1. CLAIMS 1. A method for preserving silage, the method characterized in that it comprises treating the silage with a small but effective amount of inoculant to preserve the silage 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. 2. The method according to claim 1, characterized in that the ensiled silage is silage pasture.
3. 3. The method according to claim 2, characterized in that the inoculant is a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof and Enterococcus Faecium 301, or the genetic equivalent thereof;
4. 4. The method according to claim 2, characterized in that 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. 5. The method according to claim 2, characterized in that the inoculant is a combination of Lactobacillus plantarum 432 or the genetic equivalent thereof, and Lactobacillus plantarum 435 or the genetic equivalent thereof.
6. 6. The method according to claim 2, characterized in that the inoculant is applied in a ratio of about 108 to about 1014 viable organisms per ton.
7. The method according to claim 6, characterized in that the inoculant is applied in a ratio of about 109 to about 1012 viable organisms per ton.
8. 8. The method according to claim 7, characterized in that the inoculant is applied in a ratio of approximately 1010 organisms per ton.
9. 9. A silage preservative characterized in that it is selected from the group consisting of the microorganism Lactobacillus plantarum 286, or the genetic equivalent thereof, and a small but effective amount of silage preservative of the microorganism, Enterococcus faecium 301, or the genetic equivalent of the same. A small but effective conservative amount of the silage of the Lactobacillus plantarum 286 microorganism, or the genetic equivalent thereof, a small but effective conservative amount of the Lactobacillus plantarum 432 microorganism silage or the genetic equivalent thereof, and a small but effective conservative amount of silage of the microorganism Lactobacillus plantarum 435, or the genetic equivalent thereof; A small but effective conservative amount of the silage of the microorganism Lactobacillus plantarum 432 or the genetic equivalent thereof, and a small but effective conservative amount of the silage of the microorganisms Lactobacillus plantarum 435.
10. 10. The preservative according to claim 9, characterized in that The preservative also contains a suitable culture carrier.
11. The preservative according to claim 10, characterized in that the 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. 12. The preservative according to claim 10, characterized in that the 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. 13. The preservative according to claim 10, characterized in that the preservative comprises, in combination, the microorganism Lactobacillus plantarum 432 or the genetic equivalent thereof, and the microorganisms Lactobacillus plantarum 435 or the genetic equivalent thereof.
14. 14. A method to improve the speed and degree of silage digestibility, the method characterized in that it comprises treating the silage with a small but effective conservative amount of the silage of an inoculant selected from the group consisting of: Lactobacillus plantarum 286, or the genetic equivalent of the silage. same 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, and Lactobacillus plantarum 435, or the genetic equivalent thereof.
15. 15. The method according to claim 14, characterized in that the preserved silage is silage pasture.
16. The method according to claim 15, characterized in that the inoculant is a combination of Lactobacillus plantarum 286, or the genetic equivalent thereof and Enterococcus faecium 301, or the genetic equivalent thereof.
17. 17. The method according to claim 15, characterized in that 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. 18. The method according to claim 15, characterized in that the inoculant is a combination of Lactobacillus plantarum 432 or the genetic equivalent thereof, and Lactobacillus plantarum 435 or the genetic equivalent thereof.
19. 19 The method according to claim 15, characterized in that the inoculant is applied in a ratio of about 108 to about 1014 viable organisms per ton. The method according to claim 19, characterized in that the inoculant is applied in a ratio of about 109 to about 1012 viable organisms per ton.