WO1992003933A1 - Additifs pour fourrage ensile - Google Patents

Additifs pour fourrage ensile Download PDF

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Publication number
WO1992003933A1
WO1992003933A1 PCT/GB1991/001438 GB9101438W WO9203933A1 WO 1992003933 A1 WO1992003933 A1 WO 1992003933A1 GB 9101438 W GB9101438 W GB 9101438W WO 9203933 A1 WO9203933 A1 WO 9203933A1
Authority
WO
WIPO (PCT)
Prior art keywords
additive
silage
inhibitor
cysteine
crop
Prior art date
Application number
PCT/GB1991/001438
Other languages
English (en)
Inventor
Jane Ann Wetherall
John Andrew Rooke
Original Assignee
Imperial Chemical Industries Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Imperial Chemical Industries Plc filed Critical Imperial Chemical Industries Plc
Priority to SK13993A priority Critical patent/SK13993A3/sk
Priority to AU84390/91A priority patent/AU658368B2/en
Priority to RO93-00266A priority patent/RO110194B1/ro
Priority to BR919106792A priority patent/BR9106792A/pt
Publication of WO1992003933A1 publication Critical patent/WO1992003933A1/fr
Priority to BG97480A priority patent/BG60635B1/bg
Priority to CZ93277A priority patent/CZ27793A3/cs
Priority to NO93930712A priority patent/NO930712L/no
Priority to FI930896A priority patent/FI930896A/fi

Links

Classifications

    • 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
    • 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

Definitions

  • This invention relates to a silage additive and to a method for the production of silage.
  • Silage is produced by the fermentation of crops such as grasses; cereal grains, e.g. maize, wheat, barley and sorghum; legumes, e.g. clovers, peas and lucerne; and rice.
  • crops such as grasses; cereal grains, e.g. maize, wheat, barley and sorghum; legumes, e.g. clovers, peas and lucerne; and rice.
  • the fermentation is a natural fermentation brought about by native lactic acid-producing bacteria present on the crop when it is harvested.
  • the fermentation can however be improved by the addition of silage additives, which can be chemical or bacterial i.e. containing selected lactic acid producing bacteria to the crop.
  • silage additives results in improved preservation and increased stability of the silage product with reduced in-silo losses and improved performance from animals fed on the product.
  • Silage additives can be applied to harvested crops either as liquid suspensions applied using suitable applicators or as solid compositions comprising an active ingredient mixed with carriers.
  • silage During ensilage, plant proteins are extensively hydrolysed to peptides and amino acids by plant proteases and subsequently deaminated by microbial activity.
  • the nitrogenous constituents of silage therefore, consist predominantly of amino acids, short peptides and ammonia, in contrast to herbage which contains approximately 750-850 g true protein nitrogen/kg total nitrogen.
  • Silage nitrogen is poorly utilised as a substrate for rumen microbial protein synthesis. It is therefore necessary to supplement silage diets with costly protein-rich feedingstuffs such as soya-bean meal and fishmeal.
  • silage additive comprising an active ingredient for the ensilage of a crop and an inhibitor for cysteine or aspartic protease enzymes.
  • SUBSTITUTE SHEET Further according to the present invention we provide a method for the production of silage in which a crop is treated with an inhibitor for cysteine or aspartic protease enzymes.
  • Cysteine and aspartic proteases may be identified according to the recommendations of Storey and Wagner, Phytochemistr , 25 No 12, 2701 to 2709 (1986) which identifies properties of the proteases according to IUPAC-IUB Recommendations.
  • an inhibitor for cysteine protease enzymes should be used.
  • Any suitable active ingredient may be included in the silage additive of the invention. It may be a chemical ingredient such as an acid, e.g. formic or sulphuric acids or mixtures thereof, or an ingredient of bacterial origin.
  • Bacterial ingredients which are very suitable include lactic acid-producing strains of Lactobacillus Streptococcus, and Pediococcus acidilactici and Pediococcus pentosaceus especially strains of Lactobacillus plantarum.
  • a particularly useful strain for ensiling many materials is Lactobacillus plantarum strain MTD1, a culture of which has been deposited at the National Collections of Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive,
  • Lactobacillus amylophilus Lactobacillus caseii, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus curvatus, Lactobacillus brevis, Streptococcus lactis, Streptococcus thermophilus, Streptococcus faecium, Streptococcus faecalis, Lactobacillus amylophilus, Streptococcus lactis, Streptococcus thermophilus and in particular Lactobacillus amylophilus strain No. NCIMB 11546, Streptococcus lactis strain No. NCIMB 6681 and Streptococcus thermophilus strain No. NCIMB 8510. These strains are available to the public. Any suitable cysteine protease inhibitor may be included in the silage additive of the invention. Preferred inhibitors are generally of biological origin and include the cystatins,
  • Peptide epoxides such as trans-epoxy- succinyl-L-leucyl-amido (4-guanidino) butane (E-64).
  • the crop to be ensiled is treated with an aspartic protease inhibitor in addition to the cysteine protease inhibitor in order to obtain an enhanced inhibitory effect.
  • Suitable aspartic protease inhibitors include pepstatin A.
  • E-64 can be made according to the paper by Hanada et al, Agric. Biol. Chem. 42 (3) 529 to 536 (1978). Cystatin was described by Fossum et al, Arch. Bioche . Biophys. 125, 367, 1968. It was further purified and characterised by Sen et al, Arch. Biochem. and Biophysics 158, 623, (1973). Antipain is described in Suda et al, J. Antibiotics. 25_ 263 (1972) and by Umezawa et al, J. Antibiotics 2 ⁇ 267 (1972). Pepstatin is described by Umezawa et al, J. Antibiotics :23_ 259 (1970).
  • the cysteine protease inhibitor will generally be applied to the crop as part of a silage additive together with an active ingredient for the ensilage of the crop.
  • the cysteine protease inhibitor can also be applied separately, either before or after, the active ingredient.
  • cysteine and aspartic protease inhibitors are both used it is preferred that they are applied at the same time.
  • the cysteine protease inhibitor is applied to the crop to be ensiled in an amount in the range 10 to 130 ⁇ mol/kg crop.
  • the inhibitor is applied at a pH in the range 3 to 7.5.
  • the two materials are most suitably applied together although they can also be applied separately in either order.
  • the aspartic protease inhibitor is applied in an amount in the range 1 to 60 mg/kg crop.
  • Ensilage is suitably carried out under conditions such that any supply of oxygen is restricted or oxygen is substantially excluded.
  • the silage additive of the invention can be applied as a liquid or a particulate solid.
  • the cairier will comprise water, and optionally for example a lower alcohol for example propan-1-ol, whilst when it
  • SUBSTITUTE SHEET is to be applied as a solid the carrier will be a solid material.
  • additives to be applied as solids will be supplied to users as complete formulations including the carriers.
  • the additive is to be applied as a liquid the user will generally be supplied with the active ingredient to be suspended in an appropriate volume of water before use.
  • any suitable material may be used as a carrier in the silage additive of the invention.
  • suitable carriers include cereals such as ground corn cobs, ground barley and wheat and other materials such as limestone, clay, chalk, magnesite and talc.
  • the silage additives will generally contain further materials added for a variety of reasons. Any further material added may be included as another component in the active ingredient of the invention. Further materials include nutrients (which can be sugars such as sucrose, lactose, etc); growth factors which are necessary for the growth of some bacteria (including yeast extract, corn steep liquor, vitamins and amino acids); materials added to protect the viability of the bacteria; anti-oxidants; materials to assist with oxygen uptake; and oils and other materials to reduce dusting tendencies of the additives or improve adhesion to crops.
  • nutrients which can be sugars such as sucrose, lactose, etc
  • growth factors which are necessary for the growth of some bacteria (including yeast extract, corn steep liquor, vitamins and amino acids)
  • materials added to protect the viability of the bacteria including yeast extract, corn steep liquor, vitamins and amino acids
  • the relative proportions of carrier and the active ingredient included in a silage additive depend upon the additive and its activity.
  • the additive of the invention When applied as a solid the additive of the invention is suitably supplied to forage at a rate within the range 0.1 kg to 5 kg per tonne of the forage to be treated, preferably within the range 0.2 kg to 1 kg per tonne.
  • the additive When applied as a liquid the additive is suitably supplied at a rate within the range 0.5 litres to 5 litres per tonne of the forage to be treated, the active ingredient being added to water in an amount within the range 1 g to 100 g per litre.
  • Any suitable ' applicator may be used to apply the additive of the invention either as a solid or in a liquid.
  • the silage additive of the invention can be used to make silage out of any suitable material including the crops mentioned
  • Ryegrass extracts were purified 60-fold by ammonium sulphate precipitation, gel filtration and ion exchange chromatography. It was found that the enzyme activities so purified were heterogeneous but however shared similar patterns of inhibition by different protease inhibitors (Table 1). Substantial inhibition was found only with p-mercuribenzoate, a cysteine protease inhibitor. Inhibition was also found, but to a much lesser extent with pepstatin, an inhibitor of aspartic proteases. No inhibition was detected with phenylmethylsulphonyl fluoride, a serine protease inhibitor. From these data it was concluded that the major class protease present in ryegrass are cysteine proteases. TABLE 1
  • PCMB Para ercuribenzoate
  • IODAA Iodoacetic Acid
  • Example 1 was repeated using buffered ryegrass extracts. The extracts were incubated at different pHs in the presence of a cocktail of antibiotics, included to prevent fermentation during the incubations.
  • the protease inhibitors used were specific to different classes of proteases and were primarily microbial in origin. The results are summarised in Table 2. Inhibition of autolysis was less marked than that observed with the semi-
  • Perennial ryegrass (Dry matter (DM), 279 g/kg; Water soluble carbohydrate, 113 g/g DM; Total N, 26.5 g N/kg DM; soluble N, 433 g/kg N) was ensiled.
  • the grass was ensiled with or without treatment with formic acid (1.5 g/kg herbage) with the aim of ensiling the grass at pH 6.25 or pH 5.
  • Six treatments (set out in Table 3) were imposed on the grass at each pH.
  • protease inhibitors were used: E-64, a cysteine protease inhibitor; Pepstatin, an aspartic protease inhibitor; An aqueous extract of potatoes which contained both serine and cysteine protease inhibitors; Formaldehyde which reduces proteolysis by forming reversible cross links between proteins.
  • a total of 36 silos were filled there being triplicate silos per treatment.
  • the silos comprised 100 ml measuring cylinders equipped with bunsen valves and 70 g grass were ensiled in each silo. The silos were opened after 82 days. All appeared well ensiled with no visible spoilage.
  • the silage analyses are given in Tables 4 and 5.
  • protease inhibitors also had little effect upon the silage fermentation. When compared with the non-formaldehyde treated silages adding potato extract did not affect protein breakdown in the silo (Table 5). Neither did the application of pepstatin at ensilage influence silage quality. However, E-64, the cysteine protease inhibitor significantly (P ⁇ 0.001) reduced the quantities of soluble N (0.80 of control soluble N) and of ammonia-N (0.73 of control ammonia-N) produced during ensilage.
  • cysteine proteases are the major type of protease implicated in protein breakdown in the silo and that addition of specific inhibitors of cysteine protease inhibitors, microbial in origin, can reduce the extent of Proteolysis which takes place in the silo without adversely affecting the production of lactic acid therein.
  • Lactate Lactate
  • WSC Water Soluble Carbohydrate.
  • EXAMPLE 4 Perennial ryegrass was chopped to about 2 cm lengths and 20 ml of water or an aqueous solution as described below was added per 70 grams of grass. The mixture was compressed and packed into closed silos sealed from the access of air and maintained at room temperature for the duration of the experiment.
  • the ryegrass contained dry matter 190 g/kg, water soluble carbohydrate 241 g/kg of the dried matter, total nitrogen content 42.5 g/kg of dry matter and soluble nitrogen conte: . 487 g/kg of nitrogen. The remaining nitrogen being present substantially as protein.
  • the 20 ml of water or solution contained where indicated sufficient of the enzyme inhibitor and/or bacterial preparation indicated to give the quantities in micrograms per gram of fresh grass shown in the following table.
  • 'ECOSYL' is a commercially available silage additive made for aqueous suspension made by Imperial Chemical Industries PLC.
  • 'ECOSYL' is a Trade Mark of Imperial Chemical Industries PLC.
  • the product comprises as its major active constituent freeze dried Lactobacillus Plantarium strain NCIMB 48027 together with nutrients.
  • Pepstatin is an aspartic protease inhibitor.
  • Antipain is an inhibitor of cysteine and serine proteases and Cystatin is an inhibitor of cysteine proteases. They are believed to bind equimolecularly with proteases.
  • non protein nitrogen soluble nitrogen
  • SE means standard error and in each table is for three observations; La means Lactate; Ac means Acetate and Eth means Ethanol. Lactate and Acetate are expressed as the free acids. Investigation of the products of ensilage showed that the
  • Non-protein N does not include ammonia-N.
  • the soluble N (non-protein N) content was reduced to 87% of that of the control sample N.
  • the soluble content was reduced to 75% of control sample's soluble N.
  • the ammonia N content of silages treated with E-64 at 45 ⁇ g/g grass was reduced to 70% of that of the control samples ammonia N.
  • SUBSTITUTE SHEET DM means dry matter in grams per kilogram and WSC means water soluble carbohydrate; La means lactic acid; Ac means acetic acid; Eth means ethanol;
  • dry matter is meant the residue after dehydration of the grass or silage as the case may be.
  • Pepstatin reduced the quantities of soluble N (81% of control N) .
  • the reduction in the quantity of soluble N in the silages was greater on application of pepstatin and E-64 together compared to each inhibitor applied alone.
  • the soluble N content was reduced to 68% of control soluble N.
  • the soluble N content of cystatin treated silages was reduced to 88% of control soluble N.
  • the soluble N content of silage treated with antipain was reduced to 81% of control soluble N.
  • SUBSTITUTE SHEET determined compared to that for each treatment applied separately. On addition of 'ECOSYL' and E-64 together the soluble N content of the silage was reduced to 65% of control soluble N.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Husbandry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Fodder In General (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On produit du fourrage ensilé en présence d'un inhibiteur de protéase de cystéine et/ou aspartique.
PCT/GB1991/001438 1990-08-31 1991-08-27 Additifs pour fourrage ensile WO1992003933A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
SK13993A SK13993A3 (en) 1990-08-31 1991-08-27 Silage additive
AU84390/91A AU658368B2 (en) 1990-08-31 1991-08-27 Silage additive
RO93-00266A RO110194B1 (ro) 1990-08-31 1991-08-27 Aditiv pentru furaj si procedeu pentru prepararea unui furaj continand acest aditiv
BR919106792A BR9106792A (pt) 1990-08-31 1991-08-27 Aditivo de silagem,processo para a producao de silagem e respectiva silagem
BG97480A BG60635B1 (bg) 1990-08-31 1993-02-25 Силажна добавка
CZ93277A CZ27793A3 (en) 1990-08-31 1993-02-25 Ensilage ingredient
NO93930712A NO930712L (no) 1990-08-31 1993-02-26 Ensilasje-additiv
FI930896A FI930896A (fi) 1990-08-31 1993-02-26 Ensilagetillsats

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9019024.0 1990-08-31
GB909019024A GB9019024D0 (en) 1990-08-31 1990-08-31 Silage additive

Publications (1)

Publication Number Publication Date
WO1992003933A1 true WO1992003933A1 (fr) 1992-03-19

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ID=10681446

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1991/001438 WO1992003933A1 (fr) 1990-08-31 1991-08-27 Additifs pour fourrage ensile

Country Status (17)

Country Link
EP (1) EP0546017A1 (fr)
JP (1) JPH06500692A (fr)
AU (1) AU658368B2 (fr)
BG (1) BG60635B1 (fr)
BR (1) BR9106792A (fr)
CA (1) CA2090603A1 (fr)
CZ (1) CZ27793A3 (fr)
FI (1) FI930896A (fr)
GB (1) GB9019024D0 (fr)
HU (1) HUT68374A (fr)
IE (1) IE64928B1 (fr)
NO (1) NO930712L (fr)
NZ (1) NZ239616A (fr)
RO (1) RO110194B1 (fr)
SK (1) SK13993A3 (fr)
WO (1) WO1992003933A1 (fr)
ZA (1) ZA916923B (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030177A1 (fr) * 1999-10-29 2001-05-03 Ewos Innovation As Aliment et procede d'utilisation de ce dernier

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE701217C (de) * 1938-08-10 1941-01-11 I G Farbenindustrie Akt Ges Mittel zur Kaltsilage von Gruenfutter
EP0144993A2 (fr) * 1983-12-09 1985-06-19 Genetic Diagnostic Corporation Croissance des animaux et des plantes au moyen d'inhibiteurs de protéase

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4833790A (en) * 1988-12-29 1990-08-01 Eighth Milieu Nominees Pty. Ltd. Improvements relating to manufacture of rice straw silage

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE701217C (de) * 1938-08-10 1941-01-11 I G Farbenindustrie Akt Ges Mittel zur Kaltsilage von Gruenfutter
EP0144993A2 (fr) * 1983-12-09 1985-06-19 Genetic Diagnostic Corporation Croissance des animaux et des plantes au moyen d'inhibiteurs de protéase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Canadian Journal of Plant Science, volume 61, no. 1, 1981 (CA), B.D. McKersie: "Proteinases and peptidases of alfalfa herbage", pages 53-60, see page 53, abstract; page 55, left-hand column, paragraph 2; page 55, right-hand column, last paragraph - page 57, left-hand column, first paragraph; page 59, left-hand column, last paragraph *
Phytochemistry, volume 25, no. 12, 1986 (Oxford, GB), R.D. Storey et al.: "Plant proteases: A need for uniformity", pages 2701-2709, see page 2704, right-hand column, last paragraph - page 2706 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030177A1 (fr) * 1999-10-29 2001-05-03 Ewos Innovation As Aliment et procede d'utilisation de ce dernier
AU760393B2 (en) * 1999-10-29 2003-05-15 Ewos Innovation As A feed and the use of such a feed

Also Published As

Publication number Publication date
CA2090603A1 (fr) 1992-03-01
CZ27793A3 (en) 1993-10-13
SK13993A3 (en) 1993-07-07
NZ239616A (en) 1994-02-25
BG60635B1 (bg) 1995-11-30
JPH06500692A (ja) 1994-01-27
IE64928B1 (en) 1995-09-20
AU658368B2 (en) 1995-04-13
EP0546017A1 (fr) 1993-06-16
FI930896A0 (fi) 1993-02-26
HUT68374A (en) 1995-06-28
BR9106792A (pt) 1993-06-29
NO930712D0 (no) 1993-02-26
HU9300450D0 (en) 1993-05-28
AU8439091A (en) 1992-03-30
ZA916923B (en) 1992-06-24
BG97480A (bg) 1994-03-24
IE913041A1 (en) 1992-03-11
NO930712L (no) 1993-04-26
RO110194B1 (ro) 1995-11-30
FI930896A (fi) 1993-02-26
GB9019024D0 (en) 1990-10-17

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