SK13993A3 - Silage additive - Google Patents

Abstract

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

Description

Technical field

inventions

relates to a silage additive

C 'effect

M-3i silage production.

BACKGROUND OF THE INVENTION

Silage is produced by fermentation of crops such as grasses; cereals such as corn, wheat, barley and sorghum; leguminoses such as clover, peas and lantern; and rice. Fermentation, in its simplest form, is a natural fermentation caused by the natural lactic acid-producing bacteria present in the crop at the time of harvest. However, the fermentation may be better accomplished by adding silage ingredients to the crop, which silage ingredients may be chemical or bacterial, i.e. containing selected, lactic acid-producing bacteria. The use of these silage additives causes better preservation and increased stability of the silage product, with reduced strength losses, and. better performance of animals fed with this product. Silage additives may be added to the harvested crop either in the form of liquid suspensions applied using a suitable applicator or in the form of solid compositions containing the active ingredient which are mixed with carriers.

During silage, plant proteins are extensively hydrolyzed to peptides and amino acids by plant proteases and subsequently deaminated by microbial activity. The nitrogen components of silage are composed predominantly of amino acids, short peptides and ammonia, as opposed to green matter, which contains on average 750-850 g of true protein nitrogen per kg of total nitrogen.

As silage nitrogen is not well used as a substrate for protein synthesis by rumen microbes, it is necessary to supplement silage with expensive protein-rich feeds such as soy meal and fish meal.

-2There is little knowledge about the mechanism by which plant proteins hydrolyze during silage.

However, it is desirable to improve the nutritional value of silage by reducing or eliminating the proteolysis that occurs during silage.

SUMMARY OF THE INVENTION

According to the invention, the silage additive is characterized in that it comprises an active ingredient for silaging the crop and at least one protease enzyme inhibitor selected from the group consisting of cysteine protease inhibitors and aspartic protease inhibitors, the inhibitor or inhibitors being of biological origin.

Furthermore, according to the invention, the method of making silage is characterized in that the crop is silage in the presence of at least one of said inhibitors.

Cysteine and aspartic proteases can be identified by the method recommended by Storey and Wagner, Phytochemistry, 25, no. 12, 2701-2709 (1986), which identify protease properties in accordance with IUPAC-IUB Recommendation.

The use of an inhibitor of cysteine protease enzymes is preferred.

The silage additive according to the invention may contain any suitable active ingredient. It may be a chemical component, such as an acid, for example formic or sulfuric acid, mixtures thereof, or a component of bacterial origin. Lactobacilius, Streptococcus strains, Pediococcus acidilactici and Pediococcus pentosaceus, and in particular lactic acid producing Lactobacilius plantarum strains are very suitable bacterial components. Particularly useful for the silage of many materials is the Lactobacilius plantarum MTD1 strain, whose culture has been deposited in the National Collections of Industrial and Marine Bacteria (NCIMB).

Aberdeen AB2 1RY, UK, and was

Bacteria), 23 St Machar Drive, assigned NCIMB number 4002 ”. Deposits were performed on November 12, 1936 and were recognized as filings for patent purposes on July 20, 1933; international document according to

The Budapest Agreement was issued on July 26, 1933. Other suitable strains are strains of Lactobacillus amyiophilus, Lactobacillus caseii, Lactobacillus plantarum, Lactobacillus curvatus, Lactobacillus brevis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis, , Streptococcus thermophilus, and in particular Lactobacillus amyiophilus strain no. NCIMB 11546, Streptococcus lactis strain no. NCIMB 6681 and Streptococcus thermophiius strain no. NCIMB 8510. These strains are publicly available.

Preferred cysteine protease inhibitors are cystatins, antipain and especially peptide epoxides such as trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane (E-64). According to a preferred embodiment of the invention, the cross-linked crop is treated with an aspartic protease inhibitor in addition to a cysteine protease inhibitor, in order to obtain an extended inhibitory effect.

Suitable aspartic protease inhibitors include pepstatin A.

E-64 can be prepared according to an article by Hanada et al., Agric. Biol. Chem., 42 (3), 529-536 (1978).

Cystatin has been described by Fossum et al., Ar. Biochem. Biophys. 125, ·

367, 1968. It was further purified and characterized by Sen et al.,

Arch. Biochem. and Biophysics 158, 623 (1973). Antipain has been described by Suda et al., J. Antibiotics. 25,263 (19732) and Umezawa et al., J. Antibiotics 25,267 (1972). Pepstatin described

Umezawa et al., J. Antibiotics 23 259 (1970).

In the method of the invention, the cysteine protease inhibitor is generally applied to the crop as part of the silage additive together with the active ingredient for ensiling the crop, although the cysteine protease inhibitor may also be applied separately, either before or after the active ingredient. In many cases, it is preferable to apply the cysteine protease inhibitor to fresh cut grass before silage. When both cysteine and aspartic protease inhibitors are used, it is preferably applied at the same time.

The cysteine protease inhibitor is preferably applied to the crop to be ensiled in an amount ranging from 10 to 130 / mol / kg of crop. Preferably, the inhibitor is administered at a pH in the range of 3 to 7.5. When an aspartic protease inhibitor is also administered with this inhibitor, it is most advantageous to administer the two agents together, although they may also be administered separately in any order. The aspartic protease inhibitor is preferably applied in an amount ranging from 1 to 60 mg / kg of crop. may be carried out under conditions where oxygen supply is limited or oxygen is substantially eliminated.

The silage additive according to the invention can be applied as a liquid or as a particulate solid. If it is to be applied as a liquid, the carrier comprises water, and optionally e.g. a lower alcohol such as propan-1-ol, while the solid material is the carrier when it is to be applied as a solid. Generally, the ingredients to be applied as solids are supplied to the user as a complete mixture including carriers, but when the ingredient is applied as a liquid, the active ingredient is generally provided to the user and suspended in an appropriate volume of water before use.

If the additive according to the invention is to be applied as a solid, any suitable material can be used as the carrier of the silage additive according to the invention. Examples of suitable carriers are cereals such as crushed corn sticks, barley and wheat meal and other materials such as limestone, clay, chalk, magnesite and talc.

The crosslinking additives generally contain, in addition to carriers, active ingredients and protease inhibitors, other substances added for various reasons. A further substance may also be added as a component in the active ingredient of the invention. Such other substances include nutrients (which may be sugars such as sucrose, lactose, etc.); growth factors that are required for the growth of some bacteria (including yeast extract, corn extract, vitamins and amino acids); substances added to protect the viability of bacteria; antioxidants; substances that aid the uptake of oxygen and oil, and other substances that reduce the propensity of additives to dust or improve adhesion to crops.

The relative proportions of the carrier and the active ingredients present in the silage additive depend on the additive and its action.

When the additive according to the invention is applied in the form of a solid, it is suitable to supply it in green feed in an amount of 0.1 to 5 kg / tonne of treated green feed, preferably in the range of 0.1 to 1 kg / tonne. When the additive is applied as a liquid, it is desirable to supply in an amount ranging from 0.5 liters to 5 liters per tonne of treated fodder, the active ingredient being added to the water in an amount of i to 100 g per liter. Any suitable applicator may be used to apply the additive of the invention, either as a solid or a liquid.

The silage additive according to the invention can be used to make silage from any suitable material, including the aforementioned crops, but is particularly suitable for silage grassing.

The following Examples illustrate the invention.

EXAMPLES

Example 1 (diagnostic)

The extracts of perennial butter are purified 60 times by ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The activities of these purified enzymes were found to be heterogeneous but nevertheless share the same model of inhibition by different protease inhibitors (Table 1). Substantial inhibition was only found with p-chlorometuribibenzoate, which is a cysteine protease inhibitor. Inhibition was also found with pepstatin, which is an aspartic protease inhibitor, but to a much lesser extent. No inhibition was found with phenylmethylsulfonyl-1-fluoride, a serine protease inhibitor. From these data it was concluded that the major group of proteases present in the perennial butter are cysteine proteases.

Table 1

inhibitor objective enzyme the end entrace inhibition PCMB cysteine and mM 100 IODA cysteine and mM 10 PMSF serine 1 mM 2 pepstatin asparágrový 5 g / ml 17

Legend to Table i:

PCMB: parachlormerkuribenzoate, IODAÄ: iodoacetic acid,

PMSF: phenylmethylsulfonyl-fluoride.

Example 2

Example i is repeated using the purified extracts

- 7 perennials. The extracts are incubated at different pH values in the presence of a combination of antibiotics that are used to prevent fermentation during incubation. The protease inhibitors used were specific for the various classes of proteases and were predominantly of microbial origin. The results are summarized in Table 2. Inhibition of autolysis was less pronounced than that observed with the partially purified preparations of Example 1. However, inhibition was again only observed using E-64 (specific cysteine protease inhibitor) and pepstatin.

Table 2

inhibitor objective enzyme concentration pH inhibition E-64 cysteine 10 µM 4 13 pepstatin aspartic 5 µg / ml 5 47 aprotinin serine 5,6 TIU 6 0 A-PMSF serine 130 µM 6 0

Legend to Table 2:

E-64: trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane,

A-PMSF: (p-amidinophenyl) methylsulfonyl fluoride,

TIU: trypsin inhibition unit.

Example 3

Silage is perennial (dry matter) (DM = 279 g / kg, water-soluble carbohydrates 113 g / kg dry matter, total nitrogen 26.5 g N / kg dry matter, soluble nitrogen 433 g / kg N).

The grass is ensiled, either treated or untreated with formic acid (.1.6 g / kg green matter) to silage the grass at pH 6.25 or pH 5. Six grass treatments (listed in Table 3) are performed at each pH. Four different protease inhibitors are used: E-64 (cysteine protease inhibitor) pepsatine (aspartic protease inhibitor), aqueous potato extract containing both serine and cysteine protease inhibitors, and formaldehyde, which reduces proteolysis by creating reversible, saturated white bonds between white proteins . A total of 36 forces are fulfilled, as each treatment is repeated in three forces. The silos consist of 100-ml graduated cylinders equipped with Bunsen valves, and 70 g of grass is harvested at each force.

The silos open after 82 days. In all the silage looks well preserved without visible damage. The results of the silage analyzes are shown in Tables 4 and 5.

Lowering the pH of the ensiled grass with formic acid had little effect on the composition of the silage. However, treatment with formic acid had a significant effect on the concentration of lactic acid, acetic acid and ethanol, these differences being due to poor fermentation quality of the silage preserved after treatment with both formic acid and formaldehyde. The reasons for this phenomenon are unclear.

Protease inhibitors also had little effect on silage fermentation. Compared to non-formaldehyde-treated silage, the addition of potato extract did not affect the breakdown of protein in the silo (Table 5). Nor did the use of pepstatinutin affect the quality of silage. However, E-64, which is a cysteine protease inhibitor, significantly (P < 0.001) reduced the amount of soluble nitrogen (0.30 soluble control nitrogen) and ammonia nitrogen (0.73 ammonia nitrogen control) produced during silage.

The results of these studies show that cysteine proteases are a major type of protease involved in the breakdown of proteins in strength, and that the addition of specific cysteine protease inhibitors, of microbial origin, can reduce the extent of proteolysis occurring in the strength without adversely affecting lactic acid production.

Table 3

treatment

concentration

Water

E-64

pepstatin

E-64 and Pepsatin Potato Extract

Formalin as undiluted 3 g / µM 5 _of µg / ml above extract kg grass

Table 4

Treatment Dry matter pH WSC Lac AC ET (g / kg) (g / kg dry matter)

Silage at pH 6.2

Control 249 3.75 30.1 120 24, 3 5.37 E-64 244 3.76 30.6 115 22.7. 3.15 Pepsatin 239 3.75 3 0, 126 22.2 9.26 E-64 + Pepsatin 247 3.76 30.1 111 22.5 2.56 extract from potatoes 3.72 30.0 124 23.1 7.22 Formaldehyde 254 3.76 29.6 115 20.8 2.67

Table 4 continuation:

Treatment Dry matter PH (G / kg) WSC Lac Ac (g / kg DM) et Silage at pH 5.0 inspection 250 3.69 30.2 124 22.1 0.5 E-64 249 3.72 30.2 115 19.6 0.92 pepstatin 251 3.72 29.9 115 21.0 0.5 E-64 + Pepsatin 244 3.80 30.9 102 17.5 2.25 extract from potatoes 247 3.74 30.3 110 24.2 13.7 formaldehyde 252 4.49 29.7 51 19.3 18.5 SE 0.5 0,036 0.60 7.0 1.16 1.06

Legend to Table 4:

SE: Standard deviation of mean of 6 observations.

Lac: Lactic acid

Ac: Acetic acid

ET: Ethanol

WSC: Water-soluble carbohydrates

Table 5

treatment

Total N (gN / kg dry matter)

Soluble N (gN / kg N)

Ammoniacal N (gN / kg N)

Silage at pH 6.2

inspection

26.9

535

33.5

Table 5 continuation:

treatment Total N Soluble N Ammoniacal N gN / kg dry matter) gN / kg M) gN / kg N)

E-64 26,3 424 68.3 pepstatin 27.1 547 79.6 E-64 + · Pepstatin 26.8 454 54, 5 extract from potatoes 26.2 524 86.9 formaldehyde 24.4 523 35.6

Silage at pH 5.0

inspection 22.1 520 35.3 E-64 -27.4 357 58.1 pepstatin 25.3 597 75.8 E-64 + Pepstatin 26.5 414 49.6 extract from potatoes 27.1 522 63.2 formaldehyde 26.7 513 100.8 SE 0.49 2.4 0.48

Legend to Table 5:

SE: Standard deviation of mean from 6 observations.

Example 4

The perennial butter is chopped to a length of approximately 2 cm and 20 ml of water or a lower aqueous solution described below are added per 70 grams of grass. The mixture was compressed and placed in a non-airtight closure 12 stored at room temperature for the duration of the experiment. The perennial butter consisted of a dry matter of 190 g / kg, a water-soluble carbohydrate of 241 g / kg of dry matter, a total nitrogen of 42.5 g / kg of dry matter and a soluble nitrogen of 4S7 g / kg of nitrogen. The remaining nitrogen is present essentially as a protein. 20 ml of water or solution contained, where indicated, a sufficient amount of enzyme inhibitor and / or said bacterial preparation to obtain an amount in micrograms per gram of fresh grass listed in the following table.

Table 6

treatment concentration µg / g fresh grass / µm / g fresh grass

Water (control) E-64 11.25 22.5 45.0 0.03 0.06 0.12 pepstatin 30.0 0.04 (Pepstatin) (E-64) Pepstatin and E-64 30,0 22,5 0 04 0,06 antipain 32.0 0.04 cystatin 1.2 0.9 'ECOSYĽ 3 l / t (2.65 g / l) 'ecosyľ ECOSYL applications aný in the above dose E-64 applied 22,5 / µg / g grass

ECOSYL is. a commercial available silage additive for aqueous suspensions manufactured by Imperial Chemical Industries PLC. ecOSYL 'is a trademark of Imperial Chemical Industries PLC. This product contains, as its main active ingredient, kmert L. plantarum NCIMB 48027 freeze-dried, together with wild game. All treatments were performed in triplicate. The silos were opened after 50 days and the silage in all silos looked well preserved without visible deterioration. 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. In the following tables, non-protein nitrogen (soluble nitrogen) does not include ammonia .SE means the standard deviation that is in all tables for three observations; La denotes lactate; Ac represents acetate and Et represents ethanol. Lactate and acetate are expressed as free acids. Examination of the ensilage products showed that protease inhibitors had little effect on silage fermentation and that lactic acid production was not affected. In the following tables DM means dry matter.

Table 7

Effect of increasing E-64 concentration on nitrogen silage irrigation

Concentrations E-64 Total N Nebielkovinný N Ammoniacal N zug / g grass gN / kg DM GN / kg N GN / kg N 0 407 643 72.4 11.25 360 553 61.1 22.5 366 586 70.8 45.0 359 481 51.0 SE 0.89 Cj / 2 Art 0.71

SE: Standard deviation in three observations Non-protein N does not include ammonia.

For silages treated with E-64 at a dose of 11.25 µg / g grass, the soluble nitrogen (non-protein nitrogen) content was reduced to 37% compared to the nitrogen content of the control. For silage treated with E-64 at a dose of 45.0 _of µg / g grass, the soluble nitrogen content was reduced to 75% compared to the soluble nitrogen content of the control. The ammonia nitrogen content of silages treated with E-64 at 45 µg / g grass was reduced to 70% compared to the ammonia nitrogen content of the control.

Table 8

Effects of increasing E-64 concentration on silage composition

Application pH DM WSC la Ac et dose of E-64 silage 0 3.48 156 20.6 1S4 22.5 16.0 11.25 3.41 158 24.5 202 19.3 38.9 ci / 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

Legend to Table 8:

DM: means dry matter in grams per kilogram WSC: means water-soluble carbohydrates. La: means lactic acid. Ac: means acetic acid, Et: means ethanol,

and

-15 everything is expressed in grams per kilogram of dry matter.

By dry matter is meant the residue after dehydration of grass or silage.

Table 9

Effect of E-64 and pepstatin on silage composition

treatment Total N Nebielkovinný N Ammoniacal N gN / kg dry matter GN / kg N gN / kgN

Control 407 643 72.4 E-64, 366 556 70.8 22.5 / µg / g grass Pepstatín 435 521 60.6 30.0 µg / g grass E-64 (30.0 ug / g grass) and pepstatin 428 438 62.9 (22.5 zug / g grass) SE 1.51 0.63 0.54

Pepstatin reduced the amount of soluble nitrogen (81% of control nitrogen). The reduced amount of soluble nitrogen in the silages was higher when pepepsin and E-64 were co-administered compared to each inhibitor alone. For silages treated together with pepsatin and E-64, the soluble nitrogen content was reduced to 68% soluble nitrogen in the control.

Table 10

Effect of E-64 and pepstatin on silage composition

Treatment ⁵ pH silage DM WSC la Ac et inspection 3.48 156 20.6 184 22.5 16.0 E-64 3.43 163 24.2 138 22.2 33.3 (22.5 / µg / g grass) pepstatin 3.61 164 17.5 168 16.1 36.0 (30.0 / µg / g grass) E-64 (30.0 µg / g grass) and pepstatin 3.56 163 11.1 171 23.9 38.9 (22.5 yug / g grass) SE 0.02 0.12 0.66 8.48 5.39 8.68

Legend to Table 10

DM : means dry matter in grams per kilogram WSC : means water-soluble carbohydrates. la : means Lactic acid Ac : means acetic acid. et : means ethanol,

SE is expressed in grams per kilogram of dry matter, and means the standard deviation for the three observations.

Table 11

Effects of innostasis protease inhibitors on nitrogen fractions

silage treatment Total N Nebielkovinný N Ammoniacal N g N / kg DM gN / kg N GN / kg N

inspection 407 643 72.4 S-64 366 556 70.8 (22.5 zug / g grass) Cystatin 406 571 75.7 (1,2 _from ug / g grass) antipain 411 524 69, 1 (32.0 zug / g grass) SE 1.02 2.40 0.76

Legend to Table 11:

DM: means dry matter,

SE: standard deviation for three observations.

The soluble nitrogen content of cystatin-treated silages was reduced to 38 µs of soluble nitrogen control. The soluble nitrogen content of the antipain treated silage was reduced to 81% of the soluble nitrogen of the control.

Table 12

Effects of cysteine protease innibitors on silage composition

treatment are you pH Lase DM WSC la A c et inspection 3 48 156 20.5 134 22.5 10,0 E-64 3 48 163 ti * 4 / £ 190 22.2 33.3 (22.5 / µg / g grass) antipain 3 55 161 24, 1 163 28.8 4, 31 (32.0 zg / g grass) cystatin 3 65 160 17.2 183 29.4 4, 55 (1.2 µg / g grass) SE 0 03 3.09 0.93 19.2 8.57 5.07

Legend to Table 12:

DM

WSC

la

Ac

Et means means means means dry matter in grams per kilogram of water-soluble carbohydrates, lactic acid acetic acid ethanol,

SE all expressed in grams per kilogram of dry matter, and means the standard deviation for the three observations.

Table 13

Effects of E-64 and ECOSYL on nitrogenous silage fractions

treatment Total N Nebielkovinný N Ammoniacal N gN / kg DM g N / kg N gN / kgN

inspection 407 643 72.4 E-64 366 586 70.8 (22.5 x ug / g grass) 'ECOSYL' 378 533 63.2

(8.55 xug / g grass)

E-64 (22.5 xug / g grass) and 'ECOSYL' (8.55 g / ^z 1)

355 421 53.3 SE 0.79 1.51 0.79

Legend to Table 13:

DM: means dry matter,

SE: means the standard deviation for three observations.

For ECOYSYL treated silage, the amount of dissolved nitrogen was reduced to 82% of the soluble nitrogen of the control. The silage treated with E-64 and ECOSYL showed a greater reduction in the soluble nitrogen content compared to separate application of each treatment. With the addition of ECOSYL and E-64, the soluble nitrogen content of the silage was reduced to 65% of the soluble nitrogen of the control.

Table 14

Effects of E-64 and ECOSYL on silage composition

treatment PH silage DM WSC la Ac et inspection 3.48 156 20.6 184 22.5 16.0 E-64 3.48 163 24, 1 188 tĹj čj f tí. 33.3 (22.5 / µg / g grass) 'ecosyľ 3.63 154 16.0 149 42.6 11.2 (8.55 / µg / g grass) 'ecosyľ (8.55 / µg / g grass) and E-64 3.45 164 28.9 132 42.0 30.2 (22.5 / µg / g grass) SE 0.03 0.11 0.92 13.9 8.53 5.45

Legend to Table 14:

DM : means dry matter in grams per kilogram, WSC : means water-soluble carbohydrates, la : means acid milk, Ac : means acid acetic acid, et : means ethanol,

SE is expressed in grams per kilogram of dry matter, and means the standard deviation for the three observations.

- 21 Conclusions:

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

The reduction in the soluble nitrogen content of ECOSYL-treated silages compared to that of control silages can be attributed to the effect of pH on perennial proteolytic enzymes (these exhibit the highest activity at pH 5-7); ECOSYL-treated silage is expected to reduce the pH more rapidly in the early silage stages compared to control silages.

It can be concluded that combinations of cysteine and aspartic protease inhibitors or cysteine protease inhibitors and ECOSYL u reduce proteolysis in strength to a greater extent than treatment with a single agent.

Claims (16)

PATENT 7 N .Á .R Silage additive, characterized in that it contains an active ingredient for crop silage and an inhibitor of cysteine or aspartic protease enzymes. 2. A method for producing silage, characterized in that the crop is treated with a cysteine or aspartic protease inhibitor. A method or additive according to claim 1 or 2, characterized in that the active ingredient is an acid or a component of bacterial origin. The additive or process according to claim 3, characterized in that the bacterial component is a strain of Lactobaccilus and / or Streptococcus and / or Pediococcus producing lactic acid. An additive or method according to any one of the preceding claims, wherein the inhibitor is a cystatin, an antipain, or a peptide epoxide. δ. An additive or process according to claim 5, wherein the peptide epoxide is trans-epoxy succin-L-leucyl-amido-4-guanidino) butane. An additive or method according to any one of the preceding claims wherein the inhibitor is a cysteine protease enzyme inhibitor. Additive or method according to any one of the preceding claims, characterized in that it is present both as an inhibitor of the enzyme cysteine protease and asparagus protease. Method according to any one of claims 2 to S, characterized in that it is a cysteine protease inhibitor II applies to freshly mowed grass before it is ensiled. The method of any one of claims 2 to 9, wherein the cysteine protease inhibitor is applied to the crop to be ensiled in an amount ranging from 10 to 130 µm / kg of crop. The method according to any one of claims 2 to 10, wherein the aspartic protease inhibitor is applied in the range of 1 to 60 mg / kg of crop. A method according to any one of claims 2 to 11, wherein during application of the inhibitor to the crop, the pH is in the range of 3 to 7.5. A crosslinking additive according to any one of claims 1 to 8, characterized in that it comprises a cysteine or aspartic protease inhibitor in a liquid carrier containing water. A method according to any one of claims 2 to 12, characterized in that a liquid additive comprising a protease inhibitor is applied in an amount of from 1 gram to 100 grams per liter in a water-containing carrier in a dose ranging from 0.5 to 5 liters. per ton of treated green fodder. A method according to any one of claims 2 to 12 or claim 14, characterized in that the silage is produced by fermentation of grasses, cereals, leguminoses or rice. The additive of claim 1 substantially as described in any of the examples. The method of making silage according to claim 2 whenever practiced essentially as described in any of the examples. A silage produced by the method of any one of claims 2 to 18 III 12, claim 14 or claim 15