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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • A23K3/035—
• • A23K1/007—
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
  • A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
  • A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
  • A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K40/00—Shaping or working-up of animal feeding-stuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
  • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C12R1/225—
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
  • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
  • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

• the present description relates to silage. More specifically to silage inoculants and method of use of silage inoculants for enhancing aerobic stability of silage.
• Silage is fermented, high-moisture forage to be fed to ruminants, such as cud-chewing animals 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, alfalfa, fescue, corn (maize) 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 example oatlage for oats, haylage for alfalfa are sometimes used when this is done. Sometimes a mixture is used, such as oats and peas.
• silage and the associated crop husbandry have over recent years developed to an extent that a number of different processes can be defined. These are: (i) the ensiling of young grass with particularly low dry matter, e.g. less than 25% (ii) the ensiling of higher dry matter, more mature grasses, 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.
• 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 bacterial 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. Pat. No. 6,326,037 to Mann et al. provides methods and compositions for improving this situation.
• 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 ingress 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 secondary fermentation, akin to that of composting (the primary fermentation being the ensiling process). In this fermentation stage, yeast and moulds predominate.
• Mann teaches spoilage prevention by using treatment organisms that, at least in the first instance, inhibit microorganisms 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 13 Feb. 1996. Its accession number is 40788.
• a method for treating silage comprises adding to the silage a silage inoculant comprising a silage preserving effective amount of Lactobacillus hilgardii .
• the silage inoculant is effective to prevent or reduce aerobic spoilage.
• a silage inoculant comprising a silage preserving effective amount of Lactobacillus hilgardii.
• the silage inoculant further comprises a carrier.
• a silage comprising a silage preserving effective amount of Lactobacillus hilgardii.
• the Lactobacillus hilgardii is at least one of Lactobacillus hilgardii , strain SIL51, having accession number CNCM I-4784 filed on Jun. 26, 2013 and Lactobacillus hilgardii , strain SIL52, having accession number CNCM I-4785 filed on Jun. 26, 2013, or genetic equivalents thereof.
• Said strains have been deposited by Lallemand SAS 19 rue des Briquettiers, 31702 Blagnac Cedex, France.
• the silage inoculant further comprises a carrier.
• an isolated strain of Lactobacillus hilgardii having accession number CNCM I-4785 filed on Jun. 26, 2013 or genetic equivalents thereof.
• lactic acid bacteria have been isolated and purified which improve the aerobic stability of ensiled forage. More specifically, Lactobacillus hilgardii have been shown to enhance aerobic stability of silage. Furthermore, when inoculated on silage, the Lactobacillus hilgardii strains produce silage that is well preserved and in which the onset of secondary fermentation associated with aerobic spoilage 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 identified as Lactobacillus hilgardii and given the prototype number SIL51 and SIL52.
• the present description provides silage inoculants and method of use of silage inoculants for enhancing aerobic stability of silage.
• silage preserving effective amount when used herein will be understood to refer to an amount which is at least sufficient to preserve the silage.
• 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.
• anobic 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° C. above the ambient temperature.
• a method for treating silage 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.
• 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 hilgardii.
• the strain of Lactobacillus hilgardii may be an isolated strain of Lactobacillus hilgardii CNCM I-4784 filed on Jun. 26, 2013 (SIL51), CNCM I-4785 filed on Jun. 26, 2013 (SIL52), or genetic equivalents thereof. It is understood that mutants or genetic equivalents of strains CNCM I-4784 filed on Jun. 26, 2013 (SIL51) and CNCM I-4785 filed on Jun. 26, 2013 (SIL52) which retain the functional activity of improving aerobic stability of forage as described in the present description are also contemplated.
• the critical issue is that they function to improving aerobic stability of silage as described for the parent species and/or strain.
• the present description includes mutations resulting in such minor changes as, for example, minor taxonomical 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 present description may comprise a suitable carrier or may be used as is.
• the silage incoculant may comprise solid carrier.
• the suitable carrier may be in aqueous or non-aqueous liquid form or in solid form.
• aqueous or non-aqueous liquid form carrier include water, oils and parafins.
• solid form carrier include organic or inorganic carrier such as, for example, malto-dextrin, starches, calcium carbonate, cellulose, whey, ground corn cobs, and silicone dioxide.
• the solid composition can be applied directly to the forage in the form of a light powder dusting, or if it is disbursed in a liquid carrier 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.
• 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 suitable 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, including 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 spoilage.
• 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, poultry or ruminants.
• Silage was made with fresh cut sugar cane from plants that were approximately 12 months old.
• the sugar cane was manually harvested and chopped using a laboratory-type chopper (Pinheiro, model: PP-47) to an approximate 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 compacted 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. plantarum are commonly used as silage inoculant) and the Lactobacillus hilgardii strains SIL 51 (CNCM I-4784 filed on Jun. 26, 2013) and SIL 52 (CNCM I-4785 filed on Jun. 26, 2013) as inoculants.
• the Lactobacillus plantarum and the Lactobacillus hilgardii strains were isolated from sugarcane silage and identified with 98% sequence identity. Silage without any inoculants was used as a control.
• the inoculants were cultured according to Avila et al.
• the weights of the empty and full silos were recorded. After sealing, the silos were maintained at room temperature (average of 25° C.) 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.
• b Dry matter content
• c Fesh matter
• d neutral detergent fibre
• e water-soluble carbohydrates
• f lactic acid bacteria.
• the dried samples were ground in a Willey-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, D.C., USA Association of Official Analytical Chemists), water-soluble carbohydrates (WSC) by the phenol method (Dubois M, Gilles K A, Hamilton J K, Rebers P A, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350-356.) and neutral detergent fibre (NDF) as described by Holden (Comparison of methods of in vitro dry matter digestibility for ten 565 feeds. J Dairy Sci 82:1791-1794; 1999), using an Ankom Fiber Analyser (ANKOM Technology Corporation, Fairport, N.Y., USA) and expressed on a DM basis.
• the levels of ethanol, 1,2-propanediol and lactic, acetic, propionic and butyric acids were measured by HPLC according to Carvalho et al. (Effects 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 calibration method.
• the HPLC apparatus (Shimadzu model LC-10Ai; Shimadzu Corp., Tokyo, Japan) was equipped with a dual detection system consisting of an ultraviolet detector (UV-VisSPD-10Ai) and a refractive index detector (RID 10A).
• An ion exclusion column from Shimadzu (Shim-pack SCR-101H; 7.9 mm ⁇ 30 cm) operated at 50° C. was used for the chromatographic separation.
• the mobile phase consisted of a 100 mM perchloric acid solution with a flow rate of 0.6 mL min-1. The acids were detected by UV absorbance (210 nm). Ethanol and 1,2-propanediol were identified using the refractive index detector.
• the pH values were measured with a potentiometer (Expandomatic Beckman SS-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.
• 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.
• the plates were incubated at 30° C. for 48 h. Yeast and filamentous 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.
• DRBC Dichloran Rose Bengal Chloramphenicol Medium
• the mini-silos were opened, and triplicate samples of approximately 3 kg were removed from each mini-silo and placed in 5-kg plastic buckets 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° C. ( ⁇ 1.5° C.).
• the silage temperature was recorded every 8 h.
• the ambient temperature was measured using a thermometer located close to the buckets. Aerobic stability was defined as the number of hours that the silage remained stable before rising more than 2° C. above the ambient temperature.
• the temperature of the control silage was stable for approximately 21.3 h, while that of the silage inoculated with the SIL 51 SIL 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 SL 52 strains resulted in silage with superior temperature stability to the SIL 34 and the control silage.
• 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 population, lower ethanol content and less DM losses.
• Corn silage was produced in micro-silos as described in Example 1 using the Lactobacillus buchneri , NCIMB 40788 (U.S. Pat. No. 6,326,037 to Mann et al.), the Lactobacillus plantarum SIL 34 and the Lactobacillus hilgardii strains SIL 51 (CNCM I-4784 filed on Jun. 26, 2013) and SIL52 (CNCM I-4785 filed on Jun. 26, 2013) as inoculants. Silage without any inoculants was used as a control. The inoculants were cultured as described in Example 1.
• 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 Lactobacillus buchneri NCIMB 40788 was stable for 60.5 h.
• the SIL 51 and SIL 52 strains resulted in silage with superior 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 superior temperature stability to the NCIMB 40788 silage.

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