HUT68374A - Silage additive - Google Patents

Description

Announcement Days 1991 · 08 «27 *

Priorities 1990 · 08 * 31 (9019024 * 0)

The lAXv '^ - GeA-ÍGGYx kL} <Jí _Ύ Sagy Britain

OM ivia? Í7 / j-Aó {(Qi / 0-4 \ jj Q (7j 2. / 0 ς cy <,

The present invention relates to a silage additive. The invention further relates to a method of sliding.

Silage is produced by fermentation of haeson plants, such as wildflowers, cereal seeds (such as corn, bush, barley and sorghum), legumes (such as clover, alfalfa and peas) and riss. In its simplest form, fermentation takes place against naturally occurring lactic acid-producing bacteria on beta-shelled legumes. · However, to improve fermentation, silage additives may also be added to silage additives. These additives may be chemical or bacterial additives. well-selected lactic acid-producing bacteria · Use of such skiing additives improves the storage stability and stability of the silo · reduces the renal silage · and improves the utilization of your horse's feed. The silage additives may be applied to the harvested crop by means of a suitable device or in the form of a solid mixture containing the active ingredient and the carrier.

During silage, plant proteins are hydrolyzed by plant proteases to peptides and amino acids and then dehydrated by microorganisms present. Accordingly, the silage contains nitrogen compounds primarily in the form of amino acids, low-number peptides and ammonia, in contrast to the parent plants, which contain about 750-650 g of protein-bound nitrogen per kg of total nitrogen ·

The nitrogen content of silage is only marginally utilized as a sub-substrate during microbial protein synthesis in the rumen. Therefore, silage fodder must be supplemented with nutrients rich in high protein, such as soy meal or fish meal.

The mechanism of hydrolysis of plant proteins in grasses to silage stones is currently little known · However, there is a need to improve the nutritional value of silage by eliminating or reducing proteolysis during silage.

FIELD OF THE INVENTION The present invention relates to a silage additive comprising a crop scissor promoting agent and an enzyme inhibitor of the systeine and / or asparagineav protease.

The invention further relates to a silage process wherein the crop is treated with a cysteine and / or aspartic protease enzyme inhibitor.

Identification and characteristics of cysteine and asparaginea proteases are described in Storey and Wagner Phytochemistry, 25 (12) 2701-2709 (1986) | the authors characterize and classify the protégés according to the recommendations of IUPAJ-IUB.

In accordance with the present invention, it is preferred to use inhibitors of the oysteine protease enzyme for silageing.

The silage additive of the present invention may contain any known crop silage promoting agent. The silage promoting agent may be a chemical substance such as an acid such as formic acid or sulfuric acid or a mixture thereof, however, bacterial substances may be used as silage promoting agents. Lactic acid producing strains of Laotobaoillus and Strsptooocoue are particularly preferred as bacterial agents, and Laotobaoillus plantarum strains are particularly preferred amongst Pediocooous acidilactici and Pedioooccus pentosacaus strains. The silage of a number of materials is highly advantageous for the Betioné Collostlons of Xndustrial and Marine Bacteria (NJIMB), 23 St Machar Drlve, Aberdeen AB2 IR1, Great Britain, filed November 12, 1986 under SKJIMB 40027 and patented under the Budapest Treaty. Laotobaoillus plantarum & ITD1 strain, accepted as deposit on July 20, 1988 and published on July 25, 1988. Also preferably used are Laotobaoillus amylophllus, Laotobaoillus caaeii.

- 4 Laotobaoollus plantarum »Laotobaoillus acidophllus» Lactobaoillus ourvatus. Lactobacillus brevis · dtreptooocoue laotis · tolerances for streptooocoue thermophilum, Streptoooooue faeclum, Streptocooous fascalis, Lactobacillus arnylophlluB »Btreptooocoua laotis and Jtreptocooeua thermophilus species · front · line of Laotobaolllus amylophilua NGIMB 11546» the Streptococous laotis MGIMB 6681 and Otreptoooocus thermophilus NGIMB 8510 strain · These tolerances are available to the public

Any suitable oysteine protease inhibitor may be used in the silage additive of the invention. Inhibitors of biological origin are generally preferred, including but not limited to autostain and peptide epoxides. guanidino) butane (e-64). In a preferred embodiment, in order to increase the inhibitory effect, the crop to be silaged may be treated with an asparaginic protease inhibitor in addition to a cystlenase protease inhibitor.

Ε-64 can be prepared by the method of Canada et al., Agrlc. Biol. Jhem. 42/3 / · 529 * 536 (1978). The purification and identification of the substance was reported by Possum et al., Arch. Bioohem. Biophys. 125, 367 (1968) (1963). Antipals are described by Suda et al. (J. Antlbiotios 25. 263 (1972)) and Umezawa et al. (J. Antiblotlos 25. 267 (1972)) Pepstatin is described by Umezawa et al. (J. Antibiotics 23. 259 (1970)). disclosed.

In the process of the invention, the oysteine protease inhibitor is usually applied as one of the components of the silage additive, together with the crop silage-promoting agent * ®. In many cases, it is preferable not to apply the ciestein protease inhibitor to the freshly cut ear edible prior to silage. · If both cysteine and aspartic protease inhibitors are used in the process of the invention, the 10-130 [mu] m Oysteine Protease Inhibitor Application · The inhibitor is preferably applied to the crop at a pH of 3 to 7.5. When used together with an aspartic protease inhibitor, the two substances are preferably applied simultaneously, however, they can be applied separately in any order. For the treatment of 1 kg of the crop, preferably 1-60 mg of the aspartic protease inhibitor is used. Preferably, the silage is accomplished by limiting the oxygen source or substantially excluding the oxygen.

The present invention may be applied to the crop in the form of a liquid or particulate solid. When the silage additive is applied in the form of a liquid, the carrier will usually be water, optionally in combination with another liquid carrier such as a lower alcohol such as propan-1-ol. The silage additives to be incorporated as solids contain a solid carrier. The silage additives to be incorporated as a solid are usually marketed in the form of a vehicle containing a carrier, while the liquid silage additives may be marketed as a mixture of substances to be suspended in water prior to use.

• ·

The silage additives to be incorporated into the silage material may optionally contain a solid carrier such as cereals, such as ground barley, ground wheat, ground corn cobs, and other solids such as limestone, clay, chalk, magnesite.

Silage additives to be incorporated in the form of solids usually contain additives other than the carrier, the silage-promoting agent (s) and the protease inhibitor (s), which are also suitable for various purposes. * Examples of such other additives are as follows! nutrients (such as sugars such as sucrose or lactose) # growth factors needed for the growth of certain bacteria (such as yeast extract, corn jam, vitamins and amino acids) * additives used to maintain bacterial viability! antioxidants and oxygen uptake agents # and other additives that reduce dust or increase adhesion of oils and additives *

The ratio of carrier to active ingredient in silage additives varies depending on the type and activity of the additive *

When the silage additive according to the invention is applied as a solid, 0.1 to 5 kg, preferably 0.2 * 1 kg of silage additive is usually added to 1 tonne of feed to be treated. , 5 to 5 11 volumes of liquid formulation containing 1 to 100 grams of active ingredient per liter of water * Any suitable liquid or solid formulation can be applied.

- 7 devices can be used ·

The silage additive of the present invention can be used to silage any type of silage, including the previously listed crop plants. The silage additive of the present invention can be used particularly advantageously for silage grasses.

The invention will be further described without limiting the scope of the invention in the following examples.

P.ÜM .....

The perjugivomtocate was purified by sieve precipitation with ammonium sulfate, gel blank and ion chromatography (sixty). In our experience, the enzymatic activity of as-purified extracts was heterogeneous, but the enzymatic activity of as was similarly inhibited by the protease inhibitors listed in Table 1 However, no inhibition was observed when using phenylmethylsulfonyl fluoride (sarin protease inhibitor). Based on the observed results, we concluded that the strain of oyster is a major protease component.

The results of As are reported in Table 1 · The abbreviations used in Table As 1 · have the following meaning: PUjB »p-mercuric acid ZODAAt iodoacetic acid PXSPt phenyl-ethylsulfonyl fluoride

inhibitor «Targeted ** enzyme Concentration Inhibition *% PBMCs oiszteiá 1 mmol 100 IOJMLA cysts 1 mmol 10 PW ezerin 1 mmol 2 Pepaztatin aszparaglnsav 5 _Z Bg / <a 17

2 ·

Example 1 was used to fill the forces of the puff with the extracts of the extracts. The extracellular unions were incubated at various pHs to prevent the fermentation during the incubation, and mixtures of antibiotics were added to the mixture. Inhibitors specific for various proteases, primarily of microbial origin, were used as protease inhibitors.

however, a lower degree of autolysis inhibition was observed in the semi-purified formulations tested in Example 1, but in this case only B-64 (a specific stein protease inhibitor) and pepstatin were inhibited.

The results are reported in Table 2. Abbreviations used in Table 2 have the following meaning: a-64t trans-epoxy-azuccinyl-L-leucyl-1-amido (4-guanidino) butane t-p-amidinophenyl-methyl-11-sulfonyl fluoride

TIUi anti-thrips unit

inhibitor Targeted enzyme Koncentráoió pH Inhibition,% • £ 64 cieztein 10 µmol 4 13 pepstatin aszparaglneav 5 / ug / ml 5 47 aprotinin sserin 5 »6 TIU 6 0 A-PMSF szerln 130 µmol 6 0

Silage is overtaken per yen (dry matter content / S'Lk / t

279 g / kg | water soluble carbohydrate / VOSV «113 g / g S2A> total nitrogen content * 26.5 g B / kg SKAj soluble nitrogen content: 433 g / kg B) pH of the silage 6.25» and 5 volts * in the latter case silage was performed in the presence of 1.5 g / kg plant formic acid. 6-6 treatments were performed on the pll values tested! the treatment conditions are listed in Table 3 ·

3. Push

management Tool Concentration Water ·· £ -64 10 yumol pepstatin 5 µg / ml £ -64 and pepstatin as above Horgonyakivonat undiluted extract formalin 3 g / kg plant

As shown in Table 3, four different protease inhibitors were used, namely? -64 (cysteine protease inhibitor), pepcututin (aspartic protease inhibitor), water.

- 10 potato extracts (containing serum ciaztein protease inhibitors) and formaldehyde (this substance inhibits proteolysis by forming reversible cerea bonds between proteins) · a total of 36 scissors! experiment * repeated three times under each test condition · 100 mL Volume measuring cylinders with Bunsen valves were used as silo tanks containing 70-70 g of plants to be silenced.

Silo tanks were opened after 82 days · All samples were well-silenced * no visible damage was observed · Analytical data for ^silo samples are given in Table 4 · θ ^Ώ 5 · »

The abbreviations used in Table 4 have the following meanings

Jiii standard error (based on average of 6 tests)

Laci lactate

Λα: acetate travel ethanol

Mo: dry matter

VOS: water soluble carbohydrate · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 11 Table 4

Keeelée SZU g / kg pH toast Stock g / kg AA Ac et í £ 14fefta control 249 3.75 30.1 120 24.3 5.37 E-64 244 3.76 30.6 115 22.7 3.15 Pepaztatin 239 3.75 30.2 126 22.2 9.26 E-64 + pepstatin 247 3.76 30.1 111 22.5 2.56 Sürgönyakivonat 244 3.72 30.0 124 23.1 7.22 Formaldehyde 254 3.76 29.6 115 20.8 2.67 Biloza oh at 5 control 250 3.69 30.2 124 22.1 0.5 L-64 249 3.72 30.2 115 19.6 0.92 pepstatin 251 3.72 29.9 115 21.0 0.5 E-64 + pepaztatin 244 3.80 30.9 102 17.5 2.25 potato Extract 247 3.74 30.8 110 24.2 13.7 Formaldehyde 252 4.49 29.7 51 19.3 18.5 NEITHER 0.5 0.086 0.60 7.0 1.16 1.06

Treatment Össsee N Soluble H Ammonia N g / kg AA g E / kg Π g N / kg « .Bfl ,, 6.1, ¾ M6W control 26.9 535 88.5 E-64 26.3 424 68.3 Pepaatatin 27.1 547 79.6 E-64 ♦ pepaztatin 26.8 454 54.5 Burgonyaklvonat 26.2 524 86.9 Formaldehyde 24.4 528 85.6

• ···· 4 · ♦ ··· * · · · · · · · · · · ·

Total N Soluble in M ama ^ Knia g g K / kg S2A g H / kg H g S / kg 11 scissors at pH 5.Q control 22.1 520 85 »3 £ -64 27.4 357 58.1 pepstatin 25 »3 597 75 »8 £ -64 + pepstatin 26.5 414 49 ye Curtain akiv onat 27 »! 522 68.2 Porínaldehid 26.7 518 100.8 0.49 2.4 0.48

Α Tables 4 and 5 · The following conclusions can be drawn from your data

Decreasing the pH of the silage with formic acid only slightly changed the composition of the silage. However, due to the formic acid treatment, the lactic acid, acetic acid and ethanol content of the formaldehyde-treated silage changed significantly.

Protease inhibitors also had little effect on silage fermentation. Compared to formaldehyde-treated silage, the protein degradation in the silage was unchanged when the potato extract was added (see Table 5). The quality of the silage did not change with the addition of pepstatin. However, the addition of £ -64 (oisstein * protease inhibitor) significantly reduced (p <0.001) the amount of soluble nitrogen (0.80 times control) and ammoniacal nitrogen (0.73 times control).

The results of the studies show that cleate proteases are primarily involved in the degradation of the protein in the silage, and the addition of specific microbial inhibitors of cysteine protease can reduce the proteolysis during the silage without adversely affecting lactic acid production ·

The edges of the edges were cut into 2 cm long filaments and mixed with 20 ml of water or equal volumes of solutions of the composition described below · The mixture was compressed, placed in a silo container, sealed to prevent air from entering, and the container closed. · The dry matter content of the test plant was 190 g / kg, the water soluble carbohydrate content was 241 g / kg dry matter, the total nitrogen content was 42.5 g / kg dry matter and the soluble nitrogen content

It was 487 g / kg nitrogen * and the remaining prescription contained essentially protein nitrogen.

The quantity and quality of the enzyme inhibitor and / or bacterial material in the aqueous solutions used for the treatment are given in Table 6, the quantitative data in Table 6 being the amount of enzyme inhibitor and / or bacterial material applied to 1 g of fresh plant in 20 ml solution. how much is burned in ^ ug and yumol units.

Ecosyl ”(trademark) in Table 6 is a commercially available silage additive for use as an aqueous suspension for silage treatment by Imperial Chemical Inaustries? LC · This material contains freeze-dried Lactobaoillus plantarium SC1MB 48027 as a major component with nutrients · · · ···

- 14 *

Table 6

Treatment Concentration / Ug / g fresh plant / umol / g fresh plant Vis (control) • Μ » V-64 11 * 25 0 * 03 E-64 22.5 0, O6 E-64 45 »0 0 * 12 2epsstatin 30.0 0 * 04 Machine Satin + b-64 30 * v + 22.5 o, Q4 * 0, ü6 Antlpain 32.0 0 * 04 uisztatln 1.2 0 * 9 vcosyl 3V, (2.85g / L) ucoeyl + E-64 the above amount of Ecosyl + 22.5 / ug / g E-64

Each experiment was repeated three times. Sill containers were opened after $ 0 days · All samples were well-silenced * no visual damage was observed · pepstatin aspartic protease inhibitor * antlpain olstein and azerine protease inhibitor * oystatin ozothiazine protease inhibitor is linked to proteases.

Analytical data for the resulting silage samples are shown in Figures 7-14. in Table. The amount of non-protein nitrogen (soluble nitrogen) specified in the tables does not contain ammonia nitrogen. The abbreviations and symbols in your tables have the following meanings

SE: standard error (based on average of three tests), La: lactate (expressed as free acid), · ♦ 9 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 15 A <m of steel (expressed as free acid),

8ths ethanol,

SC: dry matter!

VvbZi is a water soluble carbohydrate

Examination of silage products showed that protease inhibitors have little effect on silage fermentation · and lactic acid production remains unchanged ·

Increasing Quantity! Effect of 8-64 on nitrogen-containing fractions of silage *

i-64 concentration yUg / g plant Total Weight g / kg 88A Non-protein N g il / kg 1 Ammonia N g N / kg N 0 407 643 72.4 11.25 360 558 61.1 22.5 366 586 70.8 45.0 359 481 51.0 SB 0.89 2.22 0.71

The amount of soluble nitrogen (non-protein nitrogen) in the silage treated with S-64 at 11.25 µg / g was 87 times the control value · The amount of soluble nitrogen in the silo treated with 45.0 / ug / g Β-64 75% of control value, while ammonia nitrogen decreased to 70 &lt; control value ·

9 9 • 999 • · • ·>

Concentration of £ -64 / µg / g plant PH · of the silo 3ZA Section 8 / * plant VOSZ La g / kg wt Λ0 dth 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 24.2 183 22.2 33.3 45.0 3.40 164 31.3 175 18.4 35.5 0.02 0.15 1.25 13.5 2.94 5:57

"dry matter" means the amount of residue obtained after drying the plant or silage ·

Table 9: Effect of pound and pepstatin on silage composition

Treatment Total M g Wkg wd Non-protein N g N / kg N Ammonia S g N / kg H control 407 643 72.4 £ -64l 22.5 / g / g plant 366 556 70.8 Papastatin 30 yg / g plant 435 521 60.6 £ • 64 ♦ pepstatin 22.5 * 30 / g / g plant 428 438 62.9 • NEITHER 1:51 0.63 0.54

As a result of pepstatin treatment, soluble nitrogen (non-protein nitrogen) content of the silo was reduced to a greater value (68 s) of control when using 81 pts · pepstatin and p-64 together. inhibitor is applied to potassium-keel * Table

Effect of E-64 and pepstatin on silage composition

Treatment

The silo J? 2A VOSZ La Ac uth p »-: ® SaXj ³⁸⁴

control 3.43 156 20.6 184 22.5 16.0 22.5 zug 3-64 / g plant 3.48 163 24.2 188 22.2 33.3 30, ug of pep- tatin / g plant 3.61 164 17.5 168 16.1 36.0 22.5 zug E-64 ♦ 30 ug _z pepszta- tin / g plant 3.56 163 11.1 171 23.9 38.9

JU u, O2 0.12 0.66 8.48 5.39 8.68 th jtMáaai effect of leptin-pro tea lJAlbltor.Qk .a. silo Mtrogen content Treatment Total N g ii / kg AA Kern protein g S / kg K Ammonia H g M / kg S control 407 643 72.4 22.5 / ug S-64 / g plant 366 556 70.8 _Z olsztatin 1.2 .mu.g / g of plant ' 406 571 75.7 32 / ug antipain / g plant 411 524 69.1 NEITHER 1.02 2.40 0.76

A. Silage is soluble in nitrogen when treated with oystatin

Its content was 88% of the control value, while the anti-opine treatment was l

caused the soluble nitrogen content of the silo to decrease to a% of the control value ·

Effect of olefin protease inhibitors on allele composition

Treatment PH of the silo oZA g / kg plant voaz ta g / kg AA Ao £ th control 3.48 156 20.5 184 22.5 10.0 22.5 / Ug Κ-64 / g 163 plant 3.48 24.2 190 22.2 33.3 32 yug antlpain / 161 g 'plant 3:55 24.1 163 28.8 4.31 1.2 ₇ ug cystatin / g plant 3.65 160 17.2 183 29.4 4.55 NEITHER 0.03 3.09 0.93 19.2 8.57 5.07

$ Ut..Mb.láza

Effect of E-64 and Ecoayl on Nitrogen-containing Fractions of Silo

Treatment Total g N / kg AA Non-protein N g a / kg a Ammonia H g R / kg N control 407 643 72.4 22.5 / ug £ -64 / g plant 366 586 70.8 8.55 / ug of plantyl / g plant 378 533 63.2 22.5 / ug K-64 ♦ 8.55 / ug Eoosyl / g plant 355 421 53.3 0.79 1.51 0.79

Treatment with Eoosyl resulted in a reduction of the soluble nitrogen content of the control to 82 'of the control. Treatment with Ζ-64 and ozosyl resulted in a greater decrease (65 / o) of the soluble nitrogen content of the silo

- 19 than if the two materials were applied separately.

14 .. «Table of Tables gr6A.ao-SgO8X.l.>

Treatment The pH of the phila SZA g / k§ plant V03Z La g / kg QSA AO i. fith control 3.48 156 20.6 184 22.5 16.0 22.5 / ug fi-64 / g plant 3.48 163 24.1 188 22.2 33.0 8.55 / ug ücosyl / g plant 3.63 154 16.0 149 42.6 11.2 8.55 µg Ecoeyl ♦ $ 22.5 ug κ-64 / g plant 3.45 164 28.9 132 42.0 30.2 0, u3 0.11 0.92 13.9 8.53 5.45

7-14. The following conclusions can be drawn from the data presented in Table 1, which shows that cysteine and aspartic proteases exert their proteolytic activity during shedding. specific cysteine of biological origin _? the addition of para-protease inhibitors can reduce the amount of proteolysis occurring at 311, but without adversely affecting the amount of milk produced during silage.

The amount of soluble nitrogen in the silage treated with Ecosyl is reduced compared to the control, which can be explained by the effect of the pH change on the activity of the proteolytic enzymes in its stomach (which operate optimally in the pH range of 5 to 7). taste In Hcosyl-treated silos, the pH decreases more rapidly in the initial phase of the silage than in the control sample.

Co-silylation with -cosyl and aspartic protease inhibitors or oysteine protease inhibitors results in a greater reduction in proteolysis than if these components were used separately.

Claims (17)

1 µl of an additive comprising an active ingredient that aids in the ailization of the crop and an enzyme inhibitor of cysteled / or aspartic protease. 2 · A method of silage digging, characterized in that the crop to be harvested is treated with a protease enzyme inhibitor of tysteine and / or aspartic acid. The silage additive or the lower silage according to claim 1 or 2, characterized in that the anti-emetic agent is an acid or a bacterial agent. A silage additive or process according to claim 3, characterized in that the bacterial agent is a lactic acid producing Lactobaoillus and / or octropoococcal and / or Pediocoaous strain. Silage additive or process according to any one of the preceding claims, characterized in that the inhibitor is cystatin, antipain or a peptide epoxide. 6. The silo additive or process of claim 5, wherein the peptide epoxide is tranazo-epoxy-succinyl-L-leuylamido (4-guanidino) butane. A silage additive or process according to any one of the preceding claims, wherein the inhibitor is an inhibitor of an enzyme protease. Silage additive or process according to any one of the preceding claims, characterized in that the cysteine aspartic protease enzyme inhibitor is co-present. 9. The process according to any one of claims 1 to 3, wherein the cysteine protease inhibitor is applied to freshly cut grass before silage. 10. The method of any of claims 2-9, wherein the cysteine protease inhibitor is 11. A process according to any one of claims 1 to 6, characterized in that the aspartic protease inhibitor is applied to the crop to be controlled in an amount of 1 to 60 mg / kg of crop. 12. A 2-11. A process according to any one of claims 1 to 3, wherein the inhibitor is applied to the crop to be silenced at pH values between 3 and 7.5. 13 «Figures 1-8. A silage additive according to any one of claims 1 to 5, characterized in that it comprises a cysteine or aspartic protease inhibitor together with an aqueous liquid carrier. 14 · A 2-12. A process according to any one of claims 1 to 5, characterized in that 0.5 to 5 liters of liquid silage additive is added to 1 tonne of feed to be treated, containing 1 to 100 g of protease inhibitor per liter of water-containing liquid carrier. 15 · A 2-12. The process according to any one of claims 1 to 14 and 14, characterized in that the silo is formed by fermentation of grasses, cereal seeds, legumes or rice. · The silo additive according to claim 1, characterized in that it is essentially as described in the examples. The method of claim 2, characterized in that it proceeds essentially as described in the examples. 16. _f »2-12. 14 and 15 · sec to any one of claims 23 • silo produced Rint procedure.