NZ715997B2 - Stability of silage inoculants and methods for improving aerobic stability of silage - Google Patents

Abstract

There is provided a method for treating silage which comprises adding to the silage a silage inoculant comprising a silage preserving effective amount of Lactobacillus hilgardii. The silage inoculant being effective to prevent or reduce aerobic spoilage.

Description

STABILITY OF SILAGE H‘IOCULANTS AND S FOR IMPROVE‘IG AEROBIC ITY OF SILAGE The present description relates to silage. More specifically to silage inoculants and method of use of silage inoculants for enhancing aerobic ity of silage.

Silage is fermented, high-moisture forage to be fed to ruminants, such as cud-chewing s like cattle and sheep. The silage is fermented and stored in a storage silo, a process called ensilage. Silage is most often made from grass or cereal crops, including ryegrass, a, fescue, corn ) or sorghum. Silage is made from the entire plant, or part of it. Silage can also be made from many other field crops, including sugar cane, and other names such as, for e oatlage for oats, haylage for alfalfa are sometimes used when this is done. Sometimes a mixture is used, such as oats and peas.

The production of silage and the associated crop husbandry have over recent years developed to an extent that a number of ent 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, or the ensiling of high dry matter but young grass achieved by wilting, and (iii) the ensiling of whole maize including stover and cob, usually at a dry matter concentration of about 35%, and whole crop cereals, e. g. wheat, at 45-50% dry matter.

While these ses generally produce a good yield, they are not without their problems. Particularly in cases (ii) and (iii), one major problem occurs on a regular basis. This is the phenomenon known as aerobic spoilage. The process of aerobic spoilage occurs at opening of the silo, when the material is exposed to air. Itcan be divided into specific phases. First, there is an initial phase in which yeasts and sometimes acetic acid bacteria start to respire the preserving organic acids, raising the silage pH, and the temperature begins to rise. After an initial rise in pH, there is a secondary phase in which the ty of bacilli is apparent, and is associated with increasing temperature. A further phase includes activity of various rganisms 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.

Biological additives such as ial inoculants have been used widely to improve the silage process, primarily to increase the extent and rate of lactic acid production, and guard against aerobic spoilage. U.S. Patent No. 6,326,037 to Mann et al. es methods and compositions for improving this situation. In ular, there described is based at least in part on identifying the aerobic spoilage process as being closely related to heating in the clamp on exposure to the s of air. Subsequent examination of such silages showed high concentration of thermophilic Gram-positive bacteria, including bacilli, yeasts and molds. This apparently demonstrates the onset of a ary fermentation, akin to that of composting (the primary fermentation being the ensiling process). In this fermentation stage, yeast and moulds predominate. It appears that, in order to prevent ge, the three main categories of organisms that need to be killed or suppressed are spore-forming bacteria, yeasts and fungi. To eliminate only one category may lead to the eration of the remaining categories, so that spoilage is not prevented.

Accordingly, Mann teaches spoilage prevention by using treatment sms that, at least in the first instance, inhibit rganisms that initiate aerobic spoilage, notably yeasts and, at the surface of silage, fungi. An organism capable of doing this may also inhibit the development of other spoilage microorganisms, and may be identified by screening. An organism of the species Lactobacillus buchneri, which meets this requirement, has been deposited at the National Collection of Industrial and Marine Bacteria on 13th Feb. 1996. Its accession number is 40788.

While treatments using Lactobacillus buchneri reduce spoilage in silage, they do so to only a limited extent. Accordingly, there remains a need for an improved silage treatment, particularly for improving aerobic ity of silage while increasing the amount of dry matter recovered or to provide the public with a useful choice.

Any sion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common l dge in the field.

Unless the t clearly requires otherwise, the words "comprise", "comprising" and the like, throughout the description and the claims, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to".

[FOLLOWED BY PAGE 2a] SUMMARY OF THE DISCLOSURE In an aspect, there is provided a method for treating . The method comprises adding to the silage a silage inoculant comprising a silage preserving effective amount of acillus dii. The silage inoculant is effective to prevent or reduce aerobic spoilage.

In a particular aspect, the present invention provides a silage inoculant sing a silage preserving effective amount of Lactobacillus dii, wherein said silage preserving effective amount improves aerobic stability of silage while increasing the amount of dry matter recovered, and wherein aerobic stability refers to the number of hours that the temperature of the silage ed stable before rising more than 2 °C above the ambient temperature; and wherein the Lactobacillus dii is at least one of Lactobacillus hilgardii, strain SIL51, having accession number CNCM 1-4784 filed on June 26, 2013 or acillus hilgardii, strain SIL52, having accession number CNCM 1-4785 filed on June 26, 2013..

In another particular aspect, the present invention provides a method for treating forage, comprising adding to the forage a silage inoculant sing a silage preserving effective amount of Lactobacillus hilgardii, wherein said silage preserving effective amount es c stability of silage while increasing the amount of dry matter recovered, and wherein aerobic stability refers to the number of hours that the temperature of the silage remained stable before rising more than 2°C above the ambient temperature.

In a yet further particular aspect, the present invention provides a silage comprising a silage preserving effective amount of Lactobacillus hilgardii, wherein said silage preserving effective amount improves aerobic stability of silage while increasing the amount of dry matter recovered, and wherein aerobic stability refers to the number of hours that the temperature of the silage remained stable before rising more than 2°C above the ambient temperature; and n the Lactobacillus hilgardii is at least one of Lactobacillus hilgardii, strain SIL51, having accession number CNCM 1-4784 filed on June 26, 2013 or Lactobacillus hilgardii, strain SIL52, having accession number CNCM 1-4785 filed on June 26, 2013.

In a yet further particular aspect, the present invention provides an isolated strain of Lactobacillus hilgardii, strain SIL51, having accession number CNCM 1-4784 filed on [FOLLOWED BY PAGE 2b] June 26, 2013.

In a yet further particular aspect, the present invention es an isolated strain of Lactobacillus hilgardii, strain SIL52, having accession number CNCM 1-4785 filed on June 26, 2013 In another , there is provided a silage inoculant comprising a silage preserving effective amount of Lactobacillus hilgardii.

[FOLLOWED BY PAGE 3] In an aspect of the silage inoculant, the silage ant further comprises a carrier.

In a further aspect, there is provided a silage comprising a silage preserving effective amount ofLactobacillus hilgardl'i.

In an aspect of the method, in the silage inoculant and the silage, the acillus hilgardil' is at least one ofLactobacillus hilgardii, strain SIL51, having accession number CNCM 1-4784 filed on June 26, 2013 and Lactobacillus hilgardiz', strain SILS2, having accession number CNCM 1-4785 filed on June 26, 2013, or genetic equivalents thereof. Said strains have been deposited by Lallemand SAS 19 rue des Briquettiers, 31702 Blagnac CedeX, France.

In an aspect of the method, the silage inoculant further comprises a carrier.

In a yet further aspect, there is provided an isolated strain of Lactobacillus hilgardii, strain SIL 51, having accession number CNCM 1-4784 filed on June 26, 2013 or genetic equivalents f In another aspect, there is provided an isolated strain of Lactobacillus hilgardii, strain SIL 52, having accession number CNCM 1-4785 filed on June 26, 2013 or genetic equivalents thereof DETAILED DESCRIPTION According to the present description, lactic acid bacteria have been isolated and purified which e the aerobic stability of ensiled forage. More ically, Lactobacillus dii have been shown to enhance aerobic stability of silage. Furthermore, when inoculated on , the Lactobacillus hilgardii strains produce silage that is well preserved and in which the onset of secondary fermentation ated with aerobic ge and heating is reduced or prevented.

The stains of the present description were isolated from sugarcane (Saccharum spp.) silage. After purification and isolation of the strains, taxonomic studies were done to identify the strains. Two of them were fied as Lactobacillus hilgardii and given the prototype number SIL51 and SIL52.

Therefore, the present description provides silage inoculants and method of use of silage inoculants for enhancing aerobic stability of silage.

The term e preserving effective amount" when used herein will be understood to refer to an amount which is at least ient to preserve the silage. Thus the amount is at least sufficient to improve the stability of silage, but preferably is an amount sufficient to improve the stability of silage while increasing the amount of dry matter recovered.

The term ic stability" when used herein will be understood to refer to the number of hours that the temperature of the silage remained stable before rising more than 2 CC above the ambient temperature.

Reference will now be made to the ments described . It is understood that no limitation of the scope of the disclosure is thereby intended. It is further understood that the t disclosure includes any alterations and modifications to the illustrated embodiments and includes further applications of the principles of the sure as would normally occur to one skilled in the art to which this sure pertains.

In an embodiment, there is provided a method for treating . The method comprises the step of adding to the silage a silage inoculant comprising a silage preserving effective amount of Lactobacillus hilgardii. The silage inoculant being effective to prevent or reduce aerobic spoilage.

There is also provided a silage inoculant comprises at least a strain of Lactobacillus hilgardii. More specifically, the silage inoculant comprises a silage preserving effective amount of the species Lactobacillus hilgardil'.

In an ment of the method and the silage inoculants described above, the strain of Lactobacillus hilgardl'i may be an isolated strain of Lactobacillus hilgardl'i CNCM 1-4784 filed on June 26, 2013 (SILSl), CNCM 1-4785 filed on June 26, 2013 (SIL52), or genetic equivalents thereof It is understood that mutants or genetic equivalents of strains CNCM 1-4784 filed on June 26, 2013 (SILSl) and CNCM 1-4785 filed on June 26, 2013 (SILS2) which retain the functional activity of improving aerobic stability of forage as described in the present description are also contemplated.

Regardless of the manner in which mutations or the c equivalents are induced, the critical issue is that they on to improving aerobic stability of silage as bed for the parent species and/or . In other words, the present description includes mutations resulting in such minor changes as, for example, minor mical alterations.

The silage inoculants according to the present description may be in either liquid of solid form and may comprises additional bacterial strains. The silage inoculants according to the t description may comprise a suitable carrier or may be used as is. In solid form, the silage incoculant may comprise solid carrier. The suitable carrier may be in s or non~aquecus liquid form er in solid form. Examples of aqueous or nonwaqueeus liquid form carrier include water, cils and parafinsr Examples of sclid ferrn carrier include crganic er nic carrier such as for example, inaltd—dextrin, starches, calcium carbonate, cellulose, whey, gidund ccrn cabs, and silicone dioxide; The solid composition can be applied directly to the forage in the form of a light powder dusting, er if it is disbursed in a liquid r it can successfully be sprayed on the forage. It is understood that any other suitable carrier for the purpose of the present description may be used It appears that the inhibitory substance may be a secondary metabolite. Therefore, its full effect may not be seen if, when used in silage, that silage is opened too soon. The silage is preferably kept closed for at least 30 days, and more preferably for at least 45 days. The optimum periods may depend inter alia on the size of the silage mass, and the nature of the ensiled material.

Materials that are le for ensiling in accordance with the present description are those susceptible to aerobic spoilage. The materials usually contain at least 20% by weight of dry matter. Such materials include, for example, rye or traditional grass, maize, ing high moisture corn, whole plant corn, Lucerne, wilted grass, wheat, legumes, sorghum, sunflower, barley, other whole crop cereal and other field crop such as sugarcane. The silage may be in bales (a form particularly susceptible to aerobic spoilage), oxygen limiting bags, bunkers, upright stave silos, oxygen limiting silos, bags, piles or any other suitable form of storage which may be susceptible to aerobic ge. In an embodiment, the silage incoculant of the present description may be used with any suitable animal feed, whether solid or liquid, for the purpose of feeding animals such as, for example, pigs, y or nts.

The following examples serve to r describe and define the invention, and are not intended to limit the invention in any way.

Example 1: Silage was made with fresh cut sugar cane from plants that were imately 12 months old. The sugar cane was manually harvested and chopped using a laboratory-type chopper (Pinheiro, model: PP-47) to an imate length of 30 mm. 3 kg of the chopped material was mixed with the inoculants and conditioned in PVC plastic buckets (mini-silos, 10 cm in diameter and 60 cm in length), which were sealed with tight lids containing Bunsen valves for gas release. The material in the silo was ted to a density of approximately 630 :: 19.9 kg m-3. The mini-silos were stored at room temperature and analyzed after 61 days of storage, and three replicates were prepared for each silo.

Silage was produced using the Lactobacillus plantarum SIL 34 (L. rum are commonly used as silage inoculant) and the Lactobacillus hilgardil' strains SIL 51 (CNCM I- 4784 filed on June 26, 2013) and SIL 52 (CNCM I-4785 filed on June 26, 2013) as inoculants.

The acillus plantarum and the Lactobacillus hilgardii strains were isolated from sugarcane silage and identified with 98% sequence identity. Silage t any inoculants was used as a control. The inoculants were cultured according to Avila et al. ts of an indigenous and a commercial Lactobacillus buchneri strain on quality of sugar cane silage, Grass Forage Sci 6:3 , 2009). After the final culture, the number of cells was counted on De Man Rogosa Sharpe agar (Oxoid CM361, Basingstoke, Hampshire, England), and the concentration of the culture was adjusted to 9 log cfu ml-l. The culture was mixed with 80 mL of sterile distilled water and sprayed onto the chopped sugar cane to a final concentration of 6 log cfu g-l e.

The control received the same amount of water without any bacteria. For each treatment, a separate sprayer was used to avoid cross-contamination.

The weights of the empty and full silos were recorded. After g, the silos were maintained at room temperature (average of 25 CC) and protected from sunlight and rain. After 61 days of ensiling, the full silos were weighed prior to opening. The loss of dry matter (DM) was calculated using the weight and DM content of the fresh forage and silage. ation with the Lactobacillus plantarum (SIL 34) resulted in silage with a lower DM content, a higher DM losses and a higher NDF compared to the other strains and the l. In the silage inoculated with Lactobacillus hilgardii strains SIL 51and SIL 52, lower DM losses were found compared to the SIL 34 and the control (Table 1). Inoculation with these same strains also resulted in silage with a higher DM content and lower NDF. The ants did not influence the pH value and soluble carbohydrate content of the silage.

Table 1 Chemical composition of sugarcane si1ages at day 61 of ensi1ing without inoculants and with different inoculants Silage treatments DMb Losses NDFd WSCe pH LABf Yeasts (g ngFMS DM (%) (g kg-l DM) Log cfu. g-1 246,2b 22,61a 617.9b 25.3 8,16a 5,38b SIL34 241.3b 26.4% 676% 24.6 6.93b 5.08a SIL 51 264.8a 14.90b 598.0b 23.4 3.61 8.36a 4.61b CNCM 1-4784 filed on June 26; 2013 CNCM 1-4785 filed on June 26; 2013 b-Dry matter content; c-Fresh matter; d-neutra1 detergent fibre; e-water-soluble carbohydrates; f: lactic acid bacteria.

Mean values with different s in a column are significantly different (p<0.05) Analytical procedures On the opening day; two samples were d from each mini-silo; and all of the contents of the mini-silo were homogenized. One of the samples was weighed and dried in a fan- assisted oven at 55 CC for 96 h; another sample was used to make a water extract to determine the pH value; evaluate the microbial population and detect fermentation end products.

The dried samples were ground in a -type grinder using a 30-mesh sieve and stored in labeled plastic pots. The samples were analyzed for DM content (AOAC (1990) Official methods of analyses. 15th edition. Washington; DC; USA: Association of Official Analytical Chemists); water-soluble ydrates (WSC) by the phenol method s M; Gilles KA; Hamilton JK; Rebers PA; Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem -356.) and neutral detergent fibre (NDF) as described by Holden ( Comparison of s of in vitro dry matter digestibility for ten 565 feeds. J Dairy Sci 82:1791-1794; 1999); using an Ankom Fiber Analyser (ANKOM Technology ation; Fairport; NY; USA) and eXpressed on a DM basis.

The levels of ethanol, opanediol and lactic, acetic, propionic and butyric acids were measured by HPLC according to Carvalho et al. ts of propionic acid and Lactobacillus buchneri (SIL72) addition on fermentative and microbiological characteristics of sugar cane silage treated with or without calcium oXide. Grass Forage Sci doi: 10.1111/j.1365- 2494.2012.00863.X). The acids, ethanol and 1,2-propanediol were identified by comparing their retention times with the retention times of known standards. The concentrations of the identified compounds were determined by the external ation method. The HPLC apparatus (Shimadzu model LC-10Ai, Shimadzu Corp., Tokyo, Japan) was equipped with a dual ion system consisting of an ultraviolet detector sSPD-lOAi) and a refractive indeX detector (RID 10A). An ion exclusion column from Shimadzu (Shim-pack SCR-lOlH, 7.9 mm X 30 cm) operated at 50 CC was used for the chromatographic separation. The mobile phase consisted of a 100 mM oric acid solution with a flow rate of 0.6 mL min-1. The acids were detected by UV absorbance (210nm). Ethanol and 1,2-propanediol were identified using the refractive indeX detector. The pH values were ed with a potentiometer domatic n SS-2).

As shown in Table 2, the silage inoculated with the SIL 51 and SIL 52 strains with the lowest loss of DM had a lower concentration of ethanol than the SIL 34 and the control. The SIL 34 strain that resulted in the silage with the greatest loss of DM produced the highest amount of lactic acid. In the silages inoculated with SIL 51 and SIL 52 strains, higher concentrations of acetic acid and 1,2-pronanediol were also noted compared to the SIL 34 and the control. The propionic acid levels were similarly low, consistent with the SIL 34 and the control silage.

Table 2 , acetic, propionic and butyric acids and ethanol and 1,2-propanodiol of sugarcane si1ages at day 61 of ensi1ing without inoculants and with Lactobacillus hilgardii inculants Silage Lactic Acetic Propionic Butyric Ethanol 1 ,2- tretaments Acid Acid Acid Acid (g kg-1 propanodiol (g kg-1 (g kg-1 (g kg-1 (g kg-1 DM) (g kg-1 DM) DM) DM) DM) DM) —————m_17b SIL 51 34.6b 19.7a 4.0b 1.2b 39.9c 3.27a CNCM I- 4784 filed on June 26, SIL 52 31.4b 22.5a 4.1b 1.3b 44.4c 3.98a CNCM I- 4785 filed on June 26, iological is Samples (70 g) of fresh forage and sugar cane silage after 61 d of incubation were mixed with 630 mL of 0.1 % sterile peptone water and stirred in an orbital mixer with 120 rpm for 20 min. Subsequently, 10-fold dilutions were prepared to fy the different microbial groups.

Lactic acid bacteria (LAB) were enumerated using MRS agar (De Man Rogosa Sharpe, Difco, Detroit, MI, USA) containing 0.1 % cysteine HC1 (Merck, Dasmstadt, Germany) and 0.4 % cycloheximide (0.4 %) (Sigma) after anaerobic incubation (AnaeroGen; Oxoid, Basingstoke, UK). The plates were incubated at 30 °C for 48 h. Yeast and ntous fungi were enumerated on Dichloran Rose Bengal Chloramphenicol Medium (DRBC, Difco, Becton Dickinson, Sparks, MD, USA) after incubating the plates at 28 °C for 72 h. For all of the microorganisms, only plates containing between 30 and 300 cfus were enumerated. ment of aerobic stability of si1ages After 90 d of ensi1ing, the mini-silos were opened, and triplicate samples of approximately 3 kg were removed from each mini-silo and placed in 5-kg plastic s to assess their aerobic stability. A thermometer was inserted into the silage mass to a depth of 10 cm for 7 d. The containers were kept in a room with a controlled temperature of 26 CC (:: 1.5 °C).

The silage temperature was ed every 8 h. The ambient temperature was measured using a thermometer d close to the buckets. Aerobic ity was defined as the number of hours that the silage remained stable before rising more than 2°C above the ambient temperature.

Table 3. Aerobic ity of sugarcane silages with inoculants Silage Aerobic stability Maximum temperature Time for maximum treatments (hours) (0C) Temperature(hours) control 21.3::4.6 43.7::0.6 45 3-:12.2 SIL 34 24020.0 43721.4 37.3:—9 2 SIL 51 :4.6 :1 0 :92 CNCM 1-4784 filed on June 26, SIL 52 CNCM 1-4785 filed on June 26, As shown in table 3, the temperature of the control silage was stable for approximately 21.3 h, while that of the silage inoculated with the SIL SISIL and 52 strains lost temperature stability after 26.7 and 21.3 h respectively, after the opening of the silo. The time to reach maximum temperature was longer for both SIL 51 and 52 strains. Therefore, SIL 51 and SIL 52 strains resulted in silage with superior temperature stability to the SIL 34 and the control silage.

The silage ated with the Lactobacillus plantarum strain SIL 34 that produced lactic acid lost temperature stability after 24h. r, the SIL 34 strain resulted in silage with a higher content of ethanol, higher yeast counts and greater DM losses. The SIL 51 and SIL 52 strains provided better characteristics to silage, such as a smaller yeast tion, lower ethanol content and less DM losses.

Example 2: Aerobic stability of corn silages Corn silage was produced in micro-silos as described in Example 1 using the Lactobacillus buchneri, NCIlVfl3 40788 (US. Patent No. 6,326,037 to Mann et al.), the Lactobacillus rum SIL 34 and the Lactobacillus dil' strains SIL 51 (CNCM 1-4784 filed on June 26, 2013) and SIL52 (CNCM 1-4785 filed on June 26, 2013) as inoculants. Silage without any inoculants was used as a control. The inoculants were cultured as bed in Example 1.

After 90 d of ensiling, the mini-silos were opened, and samples of approximately 3 kg were removed from each mini-silo and placed in 5 kg plastic buckets to assess the aerobic stability. A data logger was ed into the silage mass, at a depth of 10 cm, for 7 days. The t temperature was measured using a data logger located close to the buckets. The data on c stability of silages are shown in Table 4.

Table 4. Aerobic stability of corn silages with inoculants. cfu/g_reatment Dose Maximum Time to reach maximum Aerobic stability temperature (0C) temperature (h) —_average average average Control 34. 2 34.2 72.3 72.3 42.7 42.7 SIL 34 38. 0 37.2 37.4 21.8 17.4 38.8 37.7 13.0 SIL 51 31. 0 133.1 138.3 73.8 73.05 CNCM I-4784 30.8 143. 5 72.3 filed on June 26, 2013 SIL 52 30.8 32.05 132.2 112.3 49.0 53.35 filed on June 26, 2013 NCIMB 40788 1 33.7 33.1 85.5 101.1 42.8 60.5 2 32.5 116.7 77.7 Dose 1 = 10 cfu/g; 2 = 10' cfu/g As shown in table 4, the temperature of the control silage was stable for approximately 42.7 h, while that of the silage inoculated with the SIL 51 and 52 strains lost temperature stability after 73.05 and 53.35 h respectively, after the opening of the silo. The silage inoculated with SIL 34 was stable for 17.4 hours. The silage inoculated with the Lactobacz'llus buchneri NCIlVfl3 40788 was stable for 60.5 h. The SIL 51 and SIL 52 strains resulted in silage with or temperature stability to the silage inoculated with SIL 34 and the control silage. The SIL 51 and SIL 52 strains also resulted in silage with or temperature stability to the 3 40788 silage.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of r modifications and this description is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and ing such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and as may be d to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims (12)

1. A silage inoculant comprising a silage ving effective amount of Lactobacillus hilgardii, wherein said silage preserving ive amount improves aerobic stability of silage while sing the amount of dry matter recovered, and wherein aerobic stability refers to the number of hours that the temperature of the silage remained stable before rising more than 2 °C above the ambient temperature; and wherein the acillus hilgardii is at least one of Lactobacillus hilgardii, strain SIL51, having accession number CNCM 1-4784 filed on June 26, 2013 or Lactobacillus hilgardii, strain SIL52, having accession number CNCM 1-4785 filed on June 26, 2013.

2. A method for treating , comprising adding to the forage a silage inoculant comprising a silage preserving effective amount of Lactobacillus hilgardii, wherein said silage preserving effective amount improves aerobic stability of silage while increasing the amount of dry matter recovered, and wherein c stability refers to the number of hours that the temperature of the silage remained stable before rising more than 2°C above the ambient temperature.

3. The method according to claim 2, wherein the Lactobacillus hilgardii is at least one of Lactobacillus hilgardii, strain SIL51, having accession number CNCM 1- 4784 filed on June 26, 2013 and Lactobacillus hilgardii, strain SIL52, having accession number CNCM 1-4785 filed on June 26, 2013.

4. The method according to claim 2 or 3, wherein the forage is traditional grass, maize, e, wilted grass, crop cereal or sugarcane silage.

5. The method according to any one of claims 2 to 4, wherein the forage is in a bale, a bag, a bunker, a stave silo or a silo.

6. A silage comprising a silage preserving effective amount of Lactobacillus hilgardii , wherein said silage preserving ive amount improves aerobic stability of silage while increasing the amount of dry matter recovered, and wherein aerobic stability refers to the number of hours that the temperature of the silage remained stable before rising more than 2°C above the ambient temperature; and wherein the Lactobacillus hilgardii is at least one of Lactobacillus hilgardii, strain SIL51, having accession number CNCM 1-4784 filed on June 26, 2013 or Lactobacillus hilgardii, strain SIL52, having ion number CNCM 1-4785 filed on June 26, 2013.

7. An isolated strain of Lactobacillus dii, strain SIL51, having accession number CNCM 1-4784 filed on June 26, 2013.

8. An isolated strain of Lactobacillus hilgardii, strain SIL52, having accession number CNCM 1-4785 filed on June 26, 2013.

9. The silage inoculant according to claim 1, substantially as herein described with reference to any one of the es thereof.

10. The method according to claim 2, substantially as herein described with reference to any one of the Examples thereof.

11. The silage according to claim 6, substantially as herein described with reference to any one of the Examples thereof.

12. The isolated strain ing to claim 7 or claim 8, substantially as herein described with reference to any one of the Examples thereof.