AU658368B2 - Silage additive - Google Patents

Description

OPI DATE 30/03/92 AOJP DATE 14/05/92 APPLN. ID 84 1 390 91 PCT NUMBER PCT/GB91/01438 INTERNATiUNAL API'LICAIIUN PUBLISHELD UNDER THE PATENT COOPERATION TREATY (51) International Patent Classification 5 International Publication Number: WO 92/03933 A23K 3/00 Al (43) International Publication Date: 19 March 1992 (19.03.92) (21) International Application Number: PCT/GB9I/0 1438 (74) Agents: LOCKE, Timothy, John et al,; Imperial Chemical Industries plc, Legal Departmient: Patents, P.O. Box 6, (22) International Filing Date: 27 August 1991 (27.08.9 1) Bessemer Road, Welwyn Garden City, Herts AL7 IHQ

(GB).

Priority dati: 9019024.0 31 August 1990 (31.08.90) GB (81) Designated States: AT, AT (Euiropean patent), AU, BB, BE (European patent), BF (GAP! patent), BG, Ri (OAPI (71) Applicant bb aii dmgatr Stt- eme US)- lhtPERlAtI- patent), BR, CA, CF (OAPI patent), CG (OAPI patent), CIILNIICaLi INDUST:RIES PLC !GWGCBI; imperial CH, CH (European patent), CI (QAPI patent), CM CeIcai HMOus, Mhllbafl, 1AJMIIoII SwiF~j 3m (OAPI patent), CS DE, DE (European patent), DK, DK (European patent), ES, ES (European patent), Fl, (72) Inventors; and FR (European patent), GA (OAPI patent), GB, GB (Eu- Inventors/Applicants (for US only) :WETHERALL, Jane, ropean patent), GN (OAPI patent), GR (European pa- Ann [GB/GB]; 14 Strothers Terrace, High Spen, Row- tent), HU, IT (European patent), JP, KP, KR, LK, LU, land Gill, Tyne Wear NE39 2HL ROOKE, LU (European patent), MC, MG, ML (OAPI patent), John, Andrew [GB/GB]; 57 Denview Road, Potterton, MN, MR (OAPI patent), MW, NL, NL (European pa- Aberdeen AB23 8ZL tent), NO, PL, RO, SD, SE, SE (European patent), SN (GAPI patent), SU+,TD (OAPI patent), TG (OAPI patent), US.

0a tcc ITA eka-r~c-01 cY.Q flkith intern ational search report, (54) Title: SILAGE ADDITIVE (57) Abstract Silage is made in the presence of a cysteine and/or aspartic protease inhibitors.

See back of page PCT/GB 9 01 4 3 8 28 August 1992 Silage additive 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. In its simplest form 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. Use of such 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.

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.

To date little is known about the mechanism whereby plant proteins are hydrolysed during ensilage of grasses. It is however desirable that the nutritive value of silage is improved by the reduction or elimination of the proteolysis which occurs during ensilage.

According to the present invention a silage additive is 3 characterised by comprising an active ingredient for the ensilage of a crop and .a least one protease enzyme inhibitor selected Unitod Kingdom Patent Office SUBSTITUTE SHEE ltuernationl Application PCTT/8 9 1 0 14 38' 28 August 1992 2 from cysteine protease inhibitors and aspartic protease inhibitors, said inhibitor(s) being of biological origin.

Further according to the present invention a process for the production of silage is characterised by ensiling a crop in the presence of said at least one inhibitor.

Cysteine and aspartic proteases may be identified according to the recommendations of Storey and Wagner, Phytochemistry, No 12, 2701 to 2709 (1986) which identifies properties of the proteases according to IUPAC-IUB Recommendations.

It is preferred that an inhibitor for cysteine protease enzymes should be used.

Any suitable active ingredient may be included in the silage additive of the invention. It r .y 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, Aberdeen AB2 1RY, UK and has been given the accession No. NCIMB 40027. Deposit was on 12 November 1986 and it was accepted as a deposit for patent purposes on 20 July 1988, the International Form under the Budapest Treaty being issued on 25 July 1988.

Other suitable strains include strains of the species 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.

Preferred cysteine protease inhibitors include the cystatins, SUnited Kingdom Patent .Office SUBSTITUTE

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PCT International Application CI./3J 1 014 3t 12 antipain and particularly peptide epoxides puch as trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane In a preferred form of the invention 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. 42 529 to 536 (1978). Cystatin was described by Fossum et al, Arch. Biochem. 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 25 267 (1972). Pepstatin is described by Umezawa et al, J. Antibiotics 23 259 (1970).

In the process of the invention 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. However the cysteine protease inhibitor can also be applied separately, either before or after, the active ingredient. In many instances it is preferred to apply the cysteine protease inhibitor to newly cut grass before it is ensiled. When cysteine and aspartic protease inhibitors are both used it is preferred that they are applied at the same time.

Preferably the cysteine protease inhibitor is applied to the crop to be ensiled in an amount in the range 10 to 130 pmol/kg crop. Preferably the inhibitor is applied at a pH in the range 3 to 7.5. When the inhibitor is applied with an aspartic protease inhibitor the two materials are most suitably applied together although they can also be applied separately in either order. Preferably 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. When it is to be applied as a liquid the carrier will comprise water, and optionally for example a lower alcohol for example propan-1-ol, whilst when it SInaed K.dom Patent Office SBTITUTE JCl" n ational Application SUBSTITUTE

SHEET

r.

a;\ WO 92/03933 PCT/GB91/01438 4 is to be applied as a solid the carrier will be a solid material.

Generally additives to be applied as solids will be supplie to users as complete formulations including the carriers. However when 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.

When the additive of the invention is to be applied as a solid any suitable material may be used as a carrier in the silage additive of the invention. Examples of suitable carriers include cereals such as ground corn cobs, ground barley and wheat and other materials such as limestone, clay, chalk, magnesite and talc.

In addition to carriers, active ingredients and the protease inhibitors 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.

The relative proportions of carrier and the active ingredient included in a silage additive depend upon the additive and its activity.

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 kg per tonne of the forage to be treated, preferably within the range 0.2 kg to 1 kg per tonne. When applied as a liquid the additive is suitably supplied at a rate within the range 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 SUBSTITUTE

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PT/G 9 1 0 1 4 3 8' 2 August 1993 earlier in this specification. It is however particularly useful for ensiling grasses.

The invention is illustrated by the following Example,:- EXAMPLE 1 (diagnostic) 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 Substantial inhibition was found only with p-chloromercuribenzoate, 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 IInhibitor I "Target" IConcentration Inhibition SEnzyme I JPCMB Cysteine 1 mM 100 IIODAA I Cyst .ne 1 mM IPMSF i ,rine 1 mM 2 IPepstatin j Aspartic 5 g/ml I 17 Abbreviations in Table 1.

PCM Parachloromercuribenzoate, IODAA: lodoacetic Acid, PMSF Phenylmethylsulphonyl Fluoride.

EXAMPLE 2 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 f autolysis was less marked than that observed with the semi- WO 92/03933 PCT/GB91/01438 6 purified preparations in Example 1. However inhibition was again only detected using E-64 (a specific cysteine protease inhibitor) and pepstatin.

TABLE 2 I

I

)Inhibitor "Target" Concentration pH Inhibition SEnzyme E-64 1 Cysteine I 10 M 4 1 13 Pepstatin Aspartic 5 pg/ml 5 1 47 Aprotinin Serine 5.6 TIU 6 0 A-PMSF 1 Serine 130 pM 6 0 Abbreviations in Table 2 E-64 trans-Epoxy-succinyl-L-leucyl-amido (4-guanidino) butane, A-PMSF: (p-Amidinophenyl) Methylsulphonyl Fluoride, TIU Trypsin Inhibitor Unit.

EXAMPLE 3 Perennial ryegrass (Dry matter 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. Four differing 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 Reduction of the pH of the ensiled grass using formic acid had little effect on silage composition. Although, there were significant effects of formic acid treatment upon the SUBSTITUTE

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WO 92/03933 PCT/GB91/01438 7 concentrations of lactic and acetic acids, and ethanol, these differences arose from the poor fermentation quality of the silage ensiled after treatment with both formic acid and formaldehyde. The reasons for this are not clear.

The 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 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.

The results from these studies show that 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.

TABLE 3 Treatment Concentration Water 1 E-64 0 M Pepstatin 5 Pg/ml E-64 and Pepstatin as above Potato extract undiluted Extract Formalin I 3 g/kg grass SUBSTITUTE

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WO 92/03933 WO 923933PT/GB9I /01438 TABLE 4

I

ITreatment DM I pH IWSC ILac I Ac I Et I )(g/kg DM)) I Ensiled at pH1 6.2I Control 249 3.75 )30.1 )120 24.3 )5.37 IE-64 244 3.76 30.6 115 122.7 13.15 Pepstatin 239 I3.75 30.2 I126 122.2 I9.26 E-64 Pepstatin 247 13.76 30.1 1111 122.5 2.56 IPotato 244 3.72 I30.0 1124 23.1 7.22 IFormaldehyde 254 I3.76 I29.6 I115 I20.8 I2.67 Ensiled at pH 1control 250 13.69 30.2 1124 122.1 10.5 1E-64 249 I3.72 I30.2 I115 I19.6 I0.92 IPepstatin 251 13.72 129.9 1115 121.0 10.5 IE-64 Pepstatii 244 13.80 130.9 1102 I 17.5 12.25 Potato 247 I3.74 I30.8 I110 I24.2 I13.71 IFormaldehyde 252 4.49 I29.7 I51 I19.3 I18.5 1SE 0.5 10.086 10.60 17.0 1 1.16 11.061 Abbreviations in Table 4 SE :Standard Error of Mean for 6 observations, Lac: Lactate, Ac Acetate, Et Ethanol, WSC: Water Soluble Carbohydrate.

SUBSTITUTE. SHEET WO 92/03933 PCT/GB91/01438 9 TABLE I Treatment Total N (gN/kg DM) Ensiled at pH 6.2 Control 26.9 E-64 26.3 SPepstatin 27.1 E-64 Pepstatin 26.8 Potato 26.2 Formaldehyde 24.4 Ensiled at pH SControl 22.1 E-64 27.4 Pepstatin 25.3 E-64 Pepstatin 26.5 Potato 27.1 Formaldehyde 26.7 SSE 0.49 Soluble N (gN/kg N) Ammonia N I (gN/kg) N) 1 88.5 68.3 79.6 54.5 86.9 85.6 520 357 597 414 522 518 2.4 85.3 58.1 75.8 49.6 68.2 100.8 0.48 Abbreviations in Table SE Standard Error of Mean for 6 observations.

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 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 content 487 g/kg of nitrogen. The r 'ining nitrogen being present substantially as protein. The 2L of water or solution contained where indicated sufficient of tl,- enzyme inhibitor and/or bacterial preparation indicated to give the quantities in micrograms per gram of fresh grass shown in the following table.

SUBSTITUTE

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PCT/-B 014 38 12 June 1992 1 tI Ub 62 TABLE 6 Treatment Concentration I g/g Fresh Grass pm/g Fresh Grass Water (Control) E-64 11.25 0.03 S 22.5 0.06 45.0 0.12 I Pepstatin 30.0 0.04 I(Pepstatin) (E-64) I Pepstatin E-64 30.0 22.5 0.04 0.06 Antipain 32.0 0.04 Cystatin 1.2 0.9 'ECOSYL' 3 1/t (2.85 g/l) 'ECOSYL' E-64 'ECOSYL' applied at above rate E-64 applied at 22.5 pg/g grass 'ECOSYL' is a commercially available silage additive for aqueous suspension made by Imperial Chemical Industries PLC. 'ECOSYL' is Trade Mark of Imperial Chemical Industries PLC.

The product comprises as its major active constituent freeze dried L.plantarum strain NCIMB 48027 together with nutrients.

All treatments were in triplicate. The silos were opened after days and all appeared well ensiled with no visible spoilage.

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. For the following tables non protein nitrogen (soluble nitrogen) does not include ammonia.

SE means standard error and in each table is for three observations; La means Lactate; Ac means Acetate and Et means Ethanol. Lactate and Acetate are expressed as the free acids.

Investigation of the products of ensilage showed that the T '-'dom Pc.tont Office SUBSTITUTE SHEET i i :,al Application UBST UTE SH PCTGB 9 1 0 143 8 28 August 1992 11 protease inhibitors had little effect on silage fermentation and the production of lactic acid was not affected. In the following tables DM means dry matter.

TABLE 7 The Effect of Increasing Concentrations of E-64 on Nitrogenous Fractions of Silage JE-64 Concentration Total N Non-protein N Ammonia N pg/g grass gN/kg DM gN/kgN I gN/kgN 0 407 643 72.4 11.25 360 558 61.1 22.5 366 586 70.8 45.0 359 481 51.0 S SE 0.89 2.22 1 0.71 SE, standard error for three observations Non-protein N does not include ammonia-N.

For silage treated with E-64 at 11.25 tg/g grass the soluble N (non-protein N) content was reduced to 87% of that of the control sample N. For silages treated with E-64 at 45.0 pg/g grass the soluble content was reduced to 75% of control sample's so',uble N. The ammonia N content of silages treated with E-64 at pg/g grass was reduced to 70% of that of the control samples ammonia N.

TABLE 8 The Effects of Increasing Concentrations of E-64 on Silage Compositions IE-64 Application Silage DM WSC La Ac Eth Rate pH S 0 3.48 156 20.6 184 22.5 16.0 11.25 3.41 158 24.5 202 19.3 38.9 22.5 3.48 163 24.2 188 22.2 33.3 45.0 3.40 164 31.3 175 18.4 35.5 SE 0.02 0.15 1.25 13.5 2.94 5.57 7 UrTitTld Kingdom Patent Office SUITHE EET -POT n.e:nulonal Application -A2 ft

Y

12 June 19' 12 12 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; All expressed as grams per kg of dry matter.

By dry matter is meant the residue after dehydration of the grass or silage as the case may be.

TABLE 9 The Effect of E-64 and Pepstatin on the Silage Composition Treatment Total N Non-protein N Ammonia N gN/kg DM gN/kgN gN/kgN Control 407 643 72.4 E-64 366 556 70.8 22.5 g/g grass Pepstatin 435 521 60.6 g/g grass E-64 (30 pg/g grass) 428 438 62.9 and Pepstatin (22.5 pg/g grassf) SE I 1.51 0.63 0.54 Pe:,statin 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. For silages treated with pepstatin and E-64 together, the soluble N content was reduced to 68% of control soluble N.

oi dI- om P..ent Office SUBSTITUTE SHEET CT .:..ional Appiication PCT/GBJ' 10 14 3, 12 Juns 199 12 06 92 13 TABLE 10 The Effects of E-64 and Pepstatin on Silage Composition Treatment Silage DM WSC La Ac Eth pH IContro. 3.48 156 20.6 184 22.5 16.0 JE-64 22.5 .g/g 3.48 163 24.2 188 22.2 33.3 1 grass !Pepstatin 3.61 164 17.5 168 16.1 36.0 1 30 i'g/g grass jE-64 30 tg/g grass 3.56 163 11.1 171 23.9 38.9 land Pepstatin 122.5 Vg/g grass I SE 0.02 0.12 0.66 8.48 5.39 8.68 TARLE 11 The Effects of Cysteine Protease Inhibitors on the Nitrogenous Fractions of Silage I Treatment ITotal N Non-protein N Ammonia N gN/kg DM I gN/kgN IgN/kgN IControl I 407 I 643 I 72.4 IE-64 I 366 556 I 70.8 22.5 V-g/g grass ICystatin I 406 571 I 75.7 1.2 I.g/g r'rass IAntipain I 411 I 524 1 69.1 32 pVg/g grass I SE I 1.02 I 2.40 I 0.76 The soluble N content of cystatin treated silages was reduced to control soluble N. The soluble N content of silage treated with antipain was reduced to 81% of control soluble N.

rt~A ffce SUBSTITUTE SHEE .:,onal Application 12 June 1992 14 12 06 92 TABLE 12 The Effects of Cysteine Protease Inhibitors on Silage Composition

I

Treatment Silage DM WSC La Ac Eth pH lControl 3.48 156 20.5 184 22.5 10.0 IE-64 22.5 pg/g 3.48 163 24.2 190 22.2 33.3 1 grass lAntipain 3.55 161 24.1 163 28.8 4.31 1 32 jg/g grass ICystatin 3.65 160 17.2 183 29.4 4.55 1.2 ig/g grass SE 0.03 3.09 0.93 19.2 8.57 5.07 TABLE 13 The Effects of E-64 and 'ECOSYL' on Nitrogenous Fractions of Silage I Treatment Total N Non-protein N Ammonia N I gN/kg DM gN/kgN gN/kgN

I

Control 407 643 72.4 E-64 366 586 70.8 22.5 pg/g grass I'ECOSYL' 378 533 63.2 8.55 Vg/g grass E-64 22.5 'g/g grassl 355 421 53.3 and 'ECOSYL' 8.55g/i

I

SE 0.79 1.51 0.79 For 'ECOSYL' treated silage the quantity of soluble N was reduced to 82% of control soluble N. For silage treated with E-64 and 'ECOSYL' a greater reduction in soluble M content was (Iqom Pvtent Office SUBSTITUTE SHE T 1, .:onal Application PCT/GB 1 01458.

12 June 1992 12 06 92 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.

TABLE 14 The Effects of E-64 and 'ECOSYL' on Silage Composition Treatment Silage DM WSC La Ac Eth Treatment Silage DM WSC La Ac Eth I IControl 3.48 156 20.6 184 22.5 16.0 JE-64 22.5 ig/g 3.48 163 24.1 188 22.2 33.3 grass 'ECOSYL' 3.63 154 16.0 149 42.6 11.2 8.55 ig/g grass

I'ECOSYL'

8.55 Pg/g grass 3.45 164 28.9 132 42.0 30.2 and E-64 S(22.5 g/g grass) SE 0.03 0.11 0.92 13.9 8.53 5.45

CONCLUSIONS

The results show that cysteine and aspartic proteases are active during ensilage. The addition of specific cysteine and aspartic protease inhibitors of biological origin reduced the extent of proteolysis in the silo, without adversely affecting the production of lactic acid in the silo.

The reduction of soluble N content of 'ECOSYL' treated silage compared to that for control silages could be attributed to the effect of pH on ryegrass proteolytic enzymes (shown to be optimally active at pH 5 to 7 a more rapid reduction in pH during the initial stages of ensilage would have been expected for 'ECOSYL' treated silage compared to control silages.

A combination of cysteine and aspartic protease inhibitors S or of cysteine protease inhibitor and 'ECOSYL' appeared to reduce proteolysis in the silo to a greater extent than single-agent treatments.

''ii I;T -od om P.:noie Office SUBSTTUTE S ET PCT IrlL ,ional Application

Claims (5)

13. A process as claimed in any one of claims 9 to 12 in which the pH is in the range to 3 to 7.5 when the inhibitor is applied to the crop.

14. A process as claimed in any one of claim 9 to 13 in which a liquid additive which comprises the protease inhibitor in an amount of 1 gram to 100 grams per litre in a carrier which comprises water is applied at a rate in the range 0.5 to 5 litres per tonne to the forage to be treated. A process as claimed in any one of claims 9 to 14 in which the silage is produced by the fermentation of grass, cereal grain, a legume or rice.

16. Siiage produced by a process as claimed in any one of claims 9 to 15 or by use of an additive as claimed in any one of claims 1 to 8.

17. A silage additive as claimed in claim 1 substantially as hereinbefore described with reference to any one of the examples.

18. A process as claimed in claim 9 substantially as hereinbefore described with reference to any one of the examples. DATED: 3 February, 1995 PHILLIPS ORMONDE FITZPATRICK Attorneys for: ZENECA LIMITED JOWLIPS r I c- -r fVN C INTERNATIONAL SEARCH REPORT International Application No PCT/GB 91/01438 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) b According to International Patent Classification (IPC) or to both National Classification and IPC A 23 K 3/00 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols A 23 K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched t Ill. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category a Citation of Document, It with Indication, where appropriate, of the relevant passages Relevant to Claim No. 13 Y Canadian Journal of Plant Science, volume 61, no. 1,2,16- 1, 1981 B.D. McKersle: "Proteinases and 18 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 A 7,12,13 Y EP,A,0144993 (GENETIC DIAGNOSTIC 1,2,16- CORPORATION) 19 June 1985, see the,whole document 18 A DE,C, 701217 FARBENINDUSTRIE) 1-3,15- 11 January 1941, see claim; page 1, lines 39-45 18 o Special categories of cited documents o T' later document published after the International filing date document defining the general state of the art which is not r prority date and not in conflict theory anppie ion but cited to understand the principle or theory underlying the considered to be of particular relevance invention T E earlier document but published on or after the international X' document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to 'I document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication dat of another Y' document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considtred to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled P' document published prior to the international filing date but in the art. later thai the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 05-11-1991 12. 91 Internaonal Searching Authority Signature of Authorized Officer EUROPEAN PATENT OFFICE Fonr PCT/ISA/210 Icond iseet) IJanuary 1985) International Application No Page 2 Ill. DOCUMENTS CONSIDERED 2!E RELEVANT (CONTINUED FROM THE SECOND SHEET) Category I I Citation of Document, with Indication, where appropriate, of the relevant passages Relevant to Claim NO. Phytochemistry, volume 25, no. 12, 1986 (Oxford, GB), RA. Storey et al.: "Plant proteases: A need for uniformity", pages 2701-2709, see page 2704, right-hand column, last paragraph page 2706 Form, PCrflSA/21O te~1ra shct( t0anuary 1955, ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. GB 9101438 SA 50878 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 03/12/91 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document cited in search report Publication date Patent family member(s) Publication date EP-A- 0144993 19-06-85 AU-A- 3638884 13-06-85 JP-A- 60155122 15-08-85 DE-C- 701217 None iE For more details about this annex see Official Journal of the European Patent Office, No. 12/82