SILAGE ADDITIVE

Abstract: Silage is made in the presence of a cysteine and/or aspartic protease inhibitors.

Description: 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 we provide a 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. 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 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, 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. 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, SUBSTITUTE SHEET antipain and particularly Peptide epoxides such as trans-epoxy- succinyl-L-leucyl-amido (4-guanidino) butane (E-64). 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 (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). In the method 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 μmol/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 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. Generally additives to be applied as solids will be supplied 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 5 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 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 SUBSTITUTE SHEET earlier in this specification. It is however particularly useful for ensiling grasses. The invention is illustrated by the following Examples:- EXAMPLE 1 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 Abbreviations in Table 1. PCMB : Para ercuribenzoate, IODAA: Iodoacetic Acid, PMSF : Phenylmethylsuphonyl 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 autolysis was less marked than that observed with the semi- SUBST1TUTE SHEET purified preparations in Example 1. However inhibition was again only detected using E-64 (a specific cysteine protease inhibitor) and pepstatin. TABLE 2 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 (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. 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 5. 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 SHE 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 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. 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 SUBSTITUTE SHEET TABLE 4 Abbreviations in Table 4 SE : Standard Error of Mean for 6 observations, Lac: Lactate, Ac : Acetate, Et : Ethanol, WSC: Water Soluble Carbohydrate. SUBSTITUTE.SHEET TABLE Abbreviations in Table 5 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 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. SUBSTITUTE SHEET Treatment Concentration μg/g Fresh Grass μm/g Fresh Grass Water (Control) E-64 11.25 0.03 22.5 0.06 45.0 0.12 Pepstatin 30.0 0.04 (Pepstatin) (E-64) 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/1) 'ECOSYL' & E-64 'ECOSYL' applied at above rate E-64 applied at 22.5 μg/g grass '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. All treatments were in triplicate. The silos were opened after 50 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 Eth means Ethanol. Lactate and Acetate are expressed as the free acids. Investigation of the products of ensilage showed that the SUBSTITUTE SHEET 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 - The Effect of Increasing Concentrations of E-64 on Nitrogenous Fractions of Silage SE, standard error for three observations Non-protein N does not include ammonia-N. For silage treated with E-64 at 11.25 μg/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 μg/g grass 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 μg/g grass was reduced to 70% of that of the control samples ammonia N. TABLE - The Effects of Increasing Concentrations of E-64 on Silage Compositions 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; 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 - The Effect of E-64 and Pepstatin on the Silage Composition 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. For silages treated with pepstatin and E-64 together, the soluble N content was reduced to 68% of control soluble N. SUBSTITUTE SHEET TABLE - The Effects of E-64 and Pepstatin on Silage Composition TABLE - The Effects of Cysteine Protease Inhibitors on the Nitrogenous Fractions of Silage 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 TABLE - The Effects of Cysteine Proteiase Inhibitors on Silage Composition 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 N content was 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. TABLE - The Effects of E-64 and 'ECOSYL' on Silage Composition 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 inhibitor or cysteine protease inhibitor applied with 'ECOSYL' appeared to reduce proteolysis in the silo to a greater extent than treatments imposed alone. SUBSTITUTE SHEET

Claims: l b CLAIMS 1 A silage additive which comprises an active ingredient for the ensilage of a crop and an inhibitor for cysteine or aspartic protease enzymes. 2 A process for the production of silage in which a crop is treated with an inhibitor for cysteine or aspartic proteases. 3 A process or additive according to Claim 1 or 2 in which the active ingredient is an acid or an ingredient of bacterial origin. 4 An additive or process as claimed in Claim 3 in which the bacterial ingredient is a lactic acid producing strain of Lactobacillus and/or Streptococcus and/or Pediococcus. 5 An additive or process according to any preceding claim in which the inhibitor is cystatin, antipain or a peptide epoxide. 6 An additive or process according to Claim 5 in which the peptide epoxide is trans-epoxy-succinγl-1-leucyl-amido (4-guanidino) butane. 7 An additive or process according to any preceding claim in which the inhibitor is a cysteine protease enzyme inhibitor. 8 An additive or process as claimed in any preceding claim in which both a cysteine and an aspartic protease enzyme inhibitor is present. 9 A process as claimed in any of Claims 2 to 8 in which with cysteine protease inhibitor is applied to newly cut grass before it is ensiled. 10 A process as claimed in any of Claims 2 to 9 in which the cysteine protease inhibitor is applied to the crop to be ensiled in a quantity in the range 10 to 130 um/kg of the crop. 11 A process as claimed in any of Claims 2 to 10 in which an aspartic protease inhibitor is applied in the range 1 to 60 mg/kg of crop. 12 A process as claimed in any of Claims 2 to 11 in which the pH is in the range to 3 to 7.5 when the inhibitor is applied to the crop. 13 A silage additive as claimed in any of ' Claims 1 to 8 which comprises a cysteine or aspartic protease inhibitor in a liquid carrier which comprises water. SUBSTITUTE SHEET . 14 A process as claimed in any of Claims 2 to 12 in which a liquid additive which comprises the protease inhibitor in an amount of 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. 15 A process as claimed in any of Claims 2 to 12 or 14 in which the silage is produced by the fermentation of grass, cereal grain, a legume or rice. 16 An additive as claimed in Claim 1 substantially as described in any of the examples. 17 A process of producing silage as claimed in Claim 2 whenever carried out substantially as described in any of the examples. 18 Silage prepared by a process as claimed in any of Claims 2 to 12, 14 or 15. SUBSTITUTE SHEET

Inventors: ROOKE JOHN ANDREW, WETHERALL JANE ANN, ROOKE, JOHN, ANDREW, WETHERALL, JANE, ANN
Applicants: ICI PLC, IMPERIAL CHEMICAL INDUSTRIES PLC
Application year: 1991
Main classification: A23K3
IPC codes: A23K1, A23K3