HU185784B - Method for preserving vegetables for foddering purposes - Google Patents

Description

The invention relates to a process for preserving plants, characterized in that these crops are added to a formulation prior to ensilage, which is a higher fermentable glucidokat glucidokká _g transforms that can be used by the bacteria responsible for the lactic acid fermentation. This preparation is an enzyme and bacterial complex.

The enzyme-containing composition contains a fungal cellulolytic complex associated with a known enzymatic complex. The bacterial complex contains the Gram-negative bacterium of the Enterobacteriaceae family, which is already associated with known or non-Gram-positive bacterial bacteria that ferment maize starch, but not maltose. These enzyme-based and bacterial formulations available on cereal carriers provide particularly interesting results when plants are _2θ poor in glucides.

The invention relates to an improved process for adding value and the preservation of fresh plants in order to make it possible to reduce consumption of natural form, according to the needs, after the time of harvest, and for the process ²⁵ is required composition.

The ensilage is based on the preservation of fresh plant, so that the plants are closed for a longer or shorter period and impermeable at acid medium stored with a view to Preventing ³⁰ governs the alkalizing, gas generator, putrefactive germs development that are not in an acidic medium evolving.

Implementation of good silage is difficult and numerous obstacles, most of which are in common with that dangerous to their health-products of fermentation space ³⁵ formed, which is harmful to human health, those who consumed the meat and dairy products.

The method investigated is that the lactic fermentation bacteria should be able to produce lactic acid from the ⁴⁰ soluble sugars available by adjusting the pH to about 4. Often, however, the lack of sufficient silózandó plants fermentable sugars, the medium, the growth of bacteria is insufficient, and a pH of ⁴⁵ does not drop rapidly enough to 4; therefore, anaerobic and butyric acid bacteria multiply, converting the remaining sugar into butyric acid, acetic acid, carbonic acid, and hydrogen. Proteins are broken down into ammonia and other metabolites. 50

Various solutions have been found to overcome these difficulties. In particular, chemical acidification is known with the addition of various acids. However, these procedures are not harmless. Similarly, biological acidification with the addition of highly 55 glucidolytic bacteria is known. However, this biological acidification is difficult to accomplish.

Methods of adding silage-rich raw material such as molasses to silage have also been described. However, the results obtained are not always the same and the procedure is expensive.

French Patent No. 2,361,828 also discloses a method for preserving and adding value to plants

-2million with lactic acidification, which involves adding lactic fermentation bacteria to the plants before silage, and a digestion aid that converts higher-value glucides into fermentable sugars. This degradation enhancer contains Gram-positive bacteria Lactobacillus E and F, which ferment starch, glucose, mannitol, rhamnose, sucrose, coldalin, arabinose, but maltose, inositol and sorbitol no; the behavior of the bacteria in the reactions listed is as follows: VP positive (acetylmethylcarbinol reaction, Voges-Proskauer) oxidase positive; catalase negative, positive; nitrates negative, positive; urea negative; citrates are negative; indole negative; H ₂ S negative. This degradation enhancer may also contain one or more enzymes capable of degrading polysaccharides, such that starches, pentosans, and other polysaccharides can be transformed into sugars.

According to this patent, in particular, a fungal hemicellulolytic complex can be added to the feed, the main effect of which is galactomannase activity and its secondary effect is xylanase, anrlase, pectinase and an amylase activity, which provides the necessary maltose formation for bacterial lactic acid production.

The solution of French Patent No. 2,390,908 is an improvement on the solution described in French Patent No. 2,361,828. Amylase used in French Patent No. 2,361,828 also contains fungal exoamylase which does not completely degrade starch, whereas amylase used in Patent No. 2,390,908 is Gram positive Enterococcus B and C (also known as Streptococcus faecalis). This enzyme (endoamylase) acts on germinated starch, producing mainly α-D-maltose and a small amount of α-D-glucose at pH 5-8. French Patent No. 2,390,908 also discloses the use of amyloglucosidase, which has a much stronger effect on the amylose and amylopectin chains, thereby obtaining α-D-glucose. The enzymatic composition of this patent also contains a fungal hemicellulolytic complex which acts on polysaccharides and dextrins which form cell membranes at pH 5.5-2.

BACKGROUND OF THE INVENTION The present invention relates to an improved process for silage plants, an improved process for enzymatic and bacterial methods. Prior to silage, a higher enzyme composition consisting of an enzyme complex and a bacterial complex is converted to fermentable glucids, which are utilized by lactic fermenting bacteria, to yield high glucides. The enzyme complex is characterized by containing a cellulolytic complex or cellulase or a mixture thereof which, in addition to the enzymes already used, is capable of breaking down the β - (- + 3) and (14) bonds of the polysaccharides which have not been attacked by the known enzymatic preparation. a.

It is a fungal cellulolytic complex that degrades natural cellulose in the cell walls and other polysaccharides.

This enzyme is effective over a pH range of 2-5. In the breakdown of plant structures, cellulolytic c-

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and the hemicellulolytic complex is synergistic.

More enzymes necessary to hydrolyze cellulose contained in the plant, or an order to perform several functions, which are as follows: ⁵

- hydrolysis of amorphous zones,

- swelling of crystalline regions and hydrogen bonding to obtain linear molecules,

- hydrolysis of cellobiose to glucose, ¹⁰

- transglycosylation of di- and oligosaccharides and cleavage of irregular bonds in the products of cellulose and primary hydrolysis.

The cellulolytic complex is selected so as to meet the above criteria basis of a large number of extra test ¹⁵ with various plants.

Cellulase activity is defined as the activity per 1 g of product expressed in International Units (IU), 1 IU is the amount of enzyme which liberates 1 micromole of glucose from a particular cellulose-containing substrate per minute from ²⁰ reducing sugars.

The activity varies with the substrates used: ²⁵

if the activity is assayed on Wathman CC 31 paper powder, the activity is C 1;

- if the activity is assayed on carboxymethylcellulose then C _{x is} the activity.

Both the cellulolytic activity and the hemicellulolytic secondary activity of the complex of the invention are very high. The strong cellulolytic effect is expressed in the very strong C and C _x as well as in the cellobiase (β-glucosidase) activity. .

The effect of C enzyme complex, which consists of ^35, according to the hypotheses and C _x activity at an unknown factor, as determined by the ability of cellulose.

C, is capable of breaking down native cellulose (crystalline cellulose with a degree of polymerization of PF> 3500 glucose units) to amorphous cellulose.

The effect of the C _x enzymatic complex is primarily due to its ability to reduce endo- and exoglucanases. These enzymes mainly act on cellulose fibrils in the weakly crystalline zones and cleave the long glucose chains into smaller units.

The β-glucosidase activity after _Cx effect manifests _5Q it in the impact on cellobiose units.

The different effects cannot be assessed separately. There is synergism between C and C _x in the degradation of homopolymers. The enzymatic mechanisms of C 1 and C _x lead to the production of cellobiose; this is followed by ₅₅ pairs of β-glucosidase hydrolysis, which results in two units of glucose.

Cellulose hydrolysis reactions proceed as follows:

C native cellulose reactive cellulose 60, ",, C" hydrolytic, ", reactive cellulose->cellobiose,", hydrolytic β-glucosidase, ",. , cellobiose - ^E - ^s -> 2 molecules of glucose.

The Cj complex solubilizes crystalline cellulose (degree of polymerisation (PF) of more than 3,500 glucose monomers) and produces reactive cellulose (amorphous cellulose).

The C _x enzymatic complex (endo- and exoglucanases) cleaves the reactive cellulose chains, either internally and randomly in the case of endoglucanases or starting from the non-reducing sites in the case of exoglucanases.

All reactions together result in cellobiose units.

These cellobiose units are hydrolyzed by a β-glucosidase to form two molecules of glucose.

In the present invention, for fungal cellulases, the C and C _x enzymatic complexes are capable of hydrolyzing insoluble native cellulose to glucose.

The cellulases according to the invention are selected not only on the basis of the above general criteria, but also, taking into account the particular medium of silage, on the basis of the polysaccharide-degrading effect of the cellulases.

This polysaccharide degrading effect is determined on two types of substrate (plant):

a) - on pure substrate;

b) - on a dehydrated substrate.

EXAMINATION OF THE PURE SUBSTRATE:

The activity of A, B, C, D, E, F cellulases on various substrates at a given concentration in 10 to ⁴ molar acetate buffer medium is determined.

The activity is assayed according to standard protocols, i.e. at pH 4.8, 50 ° C, after 20 minutes of hydrolysis. The reducing capacity was determined by the hexacyano ferrate (II) method.

Szjbszt ATUM ^r \ (con- \ c3ntrá- \ tion) Wathman paper Carboxymethyl -cellulose Pectin xylan arabino -galaktán Starch Locus bean 1% 1% 0.25% 0.25% 0.10% 0.25% 0.25% Enzyme (activity) IU / g (C) NE / g (C _x ) IU / g IU / g IU / g IU / g IU / g THE 400 6444 356 511 22 394 544 B 211 5660 322 611 33 100 300 C 266 1999 411 311 0 244 389 D 52 6440 255 511 0 120 132 E 372 8500 305 605 0 0 0 F 960 7666 1030 1200 0 0 140

EXAMINATION OF THE DEHYDRATED SUBSTRATE:

The activity of enzymes A, B, C, D, E, F was determined on various dehydrated feeds.

The activity was assayed at pH 3.8-4.8-5.8 at 30 ° C after 24 hours of hydrolysis. The reducing capacity is determined by the dinitrosalicylic acid method.

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Based on the evaluation of the table, the activity of the cellulolytic complex can be determined so that the _3θ process of the invention can be used for silage:

we find that the process of the invention, these crop to be ensiled cellulase or cellulolytic complex _C of activity of from 50 to 0.05 shine, the _Cx activity of 500 to 0.05 Nene ^J corresponds to 100 g of the plant; these activities are pH

Determined at 4.8 and 50 ° C after 20 minutes of hydrolysis.

The cellulase of choice should be highly active _4Q , even after prolonged storage in the silo.

Experiments show that under conditions similar to silage, the cellulolytic complex must retain at least 30% of its initial activity even after 10 days.

Preservation time in days 0 5 10 15 20 pH 4.8 100% 97% 61% 26% 3% pH 5.8 100% 85% 58% 27% 0% 50

The rate of enzymatic degradation of plant structure depends on the elimination of glucose; this shift in enzymatic equilibrium is achieved by employing bacteria with a high fermentative capacity, the medium becoming very acidic. Once the weight of the plant has stabilized, bacterial growth and hence lactic acid production stops, in contrast to the cellulolytic activity of cellulase, which lasts for more than 10 days. 60

This activity, i.e. the production and accumulation of glucose during silage, increases the nutritional value of the silage plant.

Thus, the enzymatic composition used in the process of the invention comprises a cellulase or a cellulolytic complex having the above characteristics and may be accompanied by the enzymes described in French Patent No. 2,390,908, i.e., a fungal exoamylase, an amyloglucosidase and a hemicellulolytic agent. complex, as well as Gram-positive Lactobacillus E and F enzymes and Enterococcus B and C amylase.

The enzymes in these formulations also have an additive effect on glucid activity, from the most complex to the simplest, at pH 7-2. The maximum activity of each enzyme is related to a decrease in pH that does not fall below pH 3-5 in the silo and occurs at a temperature range of 10-30 ° C, which is consistent with the conditions of the silo.

The bacterial composition used in the process of the invention is characterized in that it contains a new bacterial strain which can be used for silage of the plant; these strains are used in combination with known bacterial strains and enzymes. The compositions used in the process of the invention lead to much better silage results. The novel bacterial strain of the invention is Gram-negative bacteria; belonging to the family Enterobacteriaceae, genus Erwinia or Pectobacterium, herbicide known as Enterobacter agglomerans (for classification see Bergey's Manual of Bacteriology, 8th edition).

The following table lists the characteristics of this bacterium:

/. spreadsheet

Enterobacter agglomerans

Gram - sorbitol - Utilization of malonate + arabinose - glucose + maltose - phenylalanine deaminase - trehalose + ONGP + xylose - indole - starch + H ₂ S - oxidase - LDC gelatine + ODC - amygdalin - urease - melibiose - saccharose - Voges-Proskauer + arginine dehydrolase - rhamnose - salicin - ribitol - inositol -

New bacterial strains used for silageing plants may be used alone or in combination with other bacterial strains and enzymes known in the art. Bacteria can be grown on medium containing cereal flour. The formulation to be introduced into the silo is used in combination with a soluble or insoluble carrier containing a mixture of the required bacteria, and the enzyme mixture of the invention.

In a preferred embodiment of the process of the invention, the enzymes and bacteria are applied to cereal carriers (wheat, barley, germinated barley), preferably finely ground flour.

In another preferred method of the invention, the bacteria and enzymes are applied, for example, to a pre-latinized starch maltohexose-type carrier that is readily soluble in cold water.

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Starch and other polysaccharides in the carrier are added to the plant's glucides and thus increase the nutritional value of the silage material. The composition, which is both a bacterial mixture on a carrier and an enzymatic complex on a cereal carrier, may contain different proportions of each ingredient. The dry mixture may contain from about 100,000 to 1,000,000 Enteracoccus and 100,000 to 1,000,000 Lactobacillus per gram of dry mixture.

The amount of cellulase, hemicellulolytic complex, amylase and amyloglucosidase may vary; these are calculated depending on the type of feed to be treated.

The amount of cellulase to be added to the fresh silage plant should correspond to 50-0.005 IU activity / 100 g plant; so the fresh plant

2-2.10 ^-4 weight relay if the cellulase factor is 250 IU / g. The ratio of other enzymes to bacterial amylase is 0.05-0.20% by weight of the plant when the factor is 250 units / g; for fungal amylase, the plant is 0.10-43.20 wt.% if its factor is 50,000 polysaccharide units / g; for an amyloglucosidase, the plant is 0.10-0.70% w / w if the factor is 200 amyloglucosidase E / g; for the hemicellulolytic complex, the plant is 0.05-0.20% w / w at a factor of 35,000 IU / g.

When the enzymes are applied to the cereal carrier according to the preferred method of the invention, the product to be loaded into the silo may contain 1 kg of enzyme per approximately 9 kg of carrier. In the case of a soluble vehicle, the ratio of enzyme concentration to vehicle will vary and may vary from 1: 1.5 to 1: 4.

The incorporation of cellulase into the carrier increases the amount of fermentable sugars available, and the formation of lactic acid is more significant and faster. The plant protein is better preserved. In addition, the nutrient value of the medium increases.

To demonstrate the importance of a cellulolytic complex and a bacterium of the Enterobacteriaceae family, experiments were performed in microsilos and minisilos. In doing so, we study the various parameters that affect preservation and the structure of plants.

1. Experiments in a micro silo

The first series of laboratory experiments were performed in microsilos.

These experiments were conducted in May with alfalfa: the capacity of the microsilos was 1 kg. The alfalfa is finely chopped and placed in a silo according to the following experimental protocol:

A) The microsilicates are treated with a preservative according to the following general procedure:

grain carrier 90% premix 10%

The composition of the premix:

- a bacterial complex (on 5 Lactobacillus E and F lactose supports)

- an enzymatic complex consisting of:

• fungal amylase, • enterococcus B and C amylase, • amyloglucosidase, • fungal hemicellulolytic complex whose main activity is of the galactomannase type;

- an energy-rich carrier; finely ground barley + lactose serum and enzymatic and bacterial complexes, the premix contains 1-4% of each.

2% of the preservative additive is added to alfalfa (so the added amount of premix is 0.2%) and 1% of barley is added.

B) The microsilicates were treated with a preservative as described in A, but fungal cellulase (derived from Trichoderma viridis) having a Cj activity of 250 U / g and a C _x activity of 2500 U / g (pH 4) was added. At 8, 50 'Con for a reaction time of 20 minutes, 2 g / t of this cellulase (i.e. 2.10 &^gt; ⁴ % by weight) relative to the fresh plant (Experiment B) and 200 g / t, i.e. 0.02% relative to the fresh plant (experiment B ').

C) Control test in microsilicate

- microsilution of lucerne without preservative T = 0

After 100 days of silage, the microsilos were opened and the following parameters were tested: pH, lactic acid, NH ₃ / total N. The results are shown in the following table:

Amount of preservative T = 0 pH lactic acid g / kg dry matter NH, / all N 5.66 26g 25.15 Lucerne + 0.2% premix (A) Lucerne + 0.2% premix + Fungal cellulase 2 g / t 4.13 143.68 16.33 (B) Lucerne + 0.2% premix + fungal cellulase 4.32 144.75 16,20 200 g / t .B ') 3.73 169.47 15.02

The use of a formulation which contains a cellulase in addition to the bacterial enzyme complex disclosed in U.S. Patent No. 2,390,908 results in a better result with the preservative.

Better preservation of the plant is achieved by comparing the results of A and B ':

- pH drop: from 4.13 to 3.73 (10% decrease),

- 15% increase in lactic acid production,

- an 8% reduction in the NH ₃ / total N ratio; as a result, the plant protein is better preserved and the medium becomes more acidic when mixed.

2. Experiments in minisilos

These experiments are conducted in June with alfalfa in mini-silos with a capacity of 150 kg.

The alfalfa is finely chopped and then placed in a silo according to the following experimental protocol:

A - The mini-silos were treated with the same preservative additive as in Experiment 1 A, the microsilos were added at 0.2%.

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Β - The mini silos are treated with the additive of the dosage and composition according to claim 1A to which the same fungal cellulose is added at a dose of 2 g / t.

After 100 days of silage, the minisilos are opened and analyzed for their _g content to give the following results:

Solids %-the THE Silos containing 0.2% premix B Silos containing 0.2% premix + cellulase (2 g / t) 10 23.67 27.27 E. N. A. g / kg dry 51.25 48.97 material 1R PH 4.20 3.94 I □ Lactic acid g / kg dry 80.62 114.40 material Acetic acid g / kg dry 17.42 20.04 material NHj / total N 18.40 13.31 you total N g / kg dry 21.70 23.12 zu material Ammonia N) g / kg 3.87 3.09 dry substance

The above results clearly demonstrate the importance of a formulation of the type which, in addition to the bacterial-enzyme complex described in French Patent No. 2,390,908, also contains cellulose. The effect of the additive is as follows:

- lower pH 3.94, compared to pH 30 4.20 (less than 6% reduction), more lactic acid (29.53%), protein content of silage plants is better preserved when cellulose supplementing; this is confirmed by the analysis. In this case: ₃₅

- NH ₃ / total N ratio is 27.6% lower, protein fraction is 6% higher, ammonia nitrogen is 20.1% lower.

Larger acidification can be explained by the fact that ₄₀ bacteria process faster higher levels of fermentable sugar, which is easily converted into lactic acid. This ease of processing is due to the cellulose introduced into the medium.

In the following examples, the carrier mixture of the present invention is a complex as described in French Patent Nos. 2,361,828 and 2,390,908, for which the enzyme complex of the invention and the Gram species of the genus Erwinia or Pectobacterium belonging to the genus Enterbacteriaceae adding a negative bacterium; of which the mixture contains 10 ^s- 10® bacteria / gram.

Examples

An experiment is conducted in silos with a capacity of 4 m ³ , with a knotweed ewe harvested in June, at the onset of the cereal decay. Finely chopped feed is stored under four conditions:

Á: silage without treatment,

B: 4 liters of formic acid / ton, known per method,

C: silageing with a premix known from French Patent Nos. 2,361,828 and 2,390,908; 7 kg of 7 bacterial cultures are added per well, the characteristics of which are known from French Patent No. 2,361,828 (page 7, Nos. 1-7) and also contain enzymes; the dosage is 10 kg / t.

D: silencing is carried out using the premix C, to which the bacterium of the invention is added; and Gram-negative bacteria belonging to the family Enterobacter agglomerans of the genus Enterobacteriaceae, genus Erwinia or Pectobacterium, group Herbicola. The dosage of the complex is 10 kg / t.

After 90-100 days, the silos are opened and the plants analyzed. The results are shown in Table 2.

Parameters N-NR Soluble N Organic acids g / kg dry matter treatments pH all N% all N lactic acid propionic acetic acid butyric traces- THE: untreated 5.28 17.76 63.80 23 1.38 29.46 in B: 4% formic acid treatment 4.05 7.10 45.43 41.36 traces 14.95 0 C: No. 2,390,908. treatment according to French patent specification 4.02 14 48.10 90 0 14.20 0 D: treatment according to the invention (C + is the bacterial strain according to the invention 3.70 8 44 112 0 18.28 0

Evaluation of the results conclude that the mixture of the 2,361,828 and No. 2,390,908 French Patent ⁵⁵ already improves the preservation of the ensiled material, and this manifests itself as follows:

- decrease in pH from 5.28 to 4.02,

- increase in lactic acid content from 23 to 90 g / kg in 60 dry matter,

better protection of proteins by

N-NH

The reduction of the -π-rr ratio from 17.76 to 14 shows total N, and the lubricated N ¹ lubrication of 65 soluble N, - = - rj - 'from 63.80 to 48.10.

The advantage of this mixture is that it does not allow the formation of propionic acid and butyric acid.

The mixture of the invention also provides further improvement, which is manifested in the following:

- pH drop from 4.02 to 3.70,

- increase in lactic acid content from 92 to 112 g / kg of dry matter,

- better protection of proteins as it is

-6_ decrease 185,784 ίί-NH / _r ent of 14 to 8 with N muosszes tat and soluble N ^ _ar decrease from 44 ⁵ also indicates total N 48.10 message.

Another advantage of this blend is the absence of propionic acid and butyric acid fermentation.

The digestibility and ingestibility of these silage products are measured in sheep; after various treatments we find the following:

B: formic acid treatment 0.64 HE / kg,

C: treatment according to French Patent Nos. 2,361,828 and 2,390,908: 0.74 HE / kg,

D: Treatment according to the invention: 0.82 HE / kg.

Nitrogen balance results for growing animals confirm the efficacy of treating plants silenced with the method and composition of the invention; this is III. shown in the table.

IU. SPREADSHEET

Parameters N in fecal matter N in urine The retention N treatments The recorded N · g / day g / day % of N recorded g / day % of N recorded g / day. % of N recorded THE: untreated 21 7.90 3.76 13 61.9 0.10 0.50 B: 0.4% formic acid 18.9 7.34 38.8 10.4 55 U 5.88 C: basic patent treatment + 1. additives 17 7.07 41.6 8.12 47.7 1.8 10.5 D: treatment according to the invention 16.5 5.4 38.8 7.70 47 2.40 14.5

The retention of nitrogen in the process and composition of the invention is more than twice that obtained after formic acid treatment.

Claims (2)

CLAIMS 1. A method of preserving and adding value to plants by acidification of lactic acid comprising adding to the plants, prior to silage, an enzymatic composition which decomposes higher-value glucids into fermentable glucids which can be utilized by lactic fermenting bacteria, comprising an enzyme complex and a bacterial complex. comprising a fungal cellulolytic complex or cellulase or a mixture thereof capable of degrading native cellulose to glucose and having a C activity of 50-0.005 IU per 100 g fresh plant and a C _x activity of 500-0.05 IU per 100 g fresh plant and conditions of silage at least 30% of this activity is retained after 10 days, and optionally contains a fungal amylase, amyloglucosidase, a fungal hemicellulolytic complex having a major effect on galactomannase activity and containing a bacterial complex. we are that (a) Gram-positive bacteria Enterococcus B and C (Streptococcus faecalis) capable of digesting higher glucids to fermentable glucids, as well as fermenting starch, glucose, mannitol, rhamnose, sucrose, amygdalin and arabinose; but contains inositol and non-Serbian fermenting Lactobacillus E and F Gram-positive bacteria and contains between 100,000 and 1,000,000 grams of Enterococcus and Lactobacillus per gram of dry mixture, or b) the bacterial complex comprises negative bacteria in addition to bacteria according to a) of the Enterobacteriaceae family, Erwinia or Pectobacterium genus herbicola group belonging called Enterobacter agglomerans, starch fermentation, maltose non-fermenting Gram, the dry mixture grams of 10 ⁵ to 10 ⁶ . (Priority: June 21, 1979) 2. The method of claim 1, wherein the enzyme mixture is of the following composition: ^{2x10 4} -4% cellulase having C activity of 250 IU / g; 0.05-0.20% o bacterial amylase having an activity of 250 U / g; 0.1C-0.20% o fungal amylase having an activity of 5000 polysaccharide E / g; 0.1C-0.70% amyloglucosidase having an activity of 200 amyloglucose E / g; 0.05-0.20% o hemicellulolytic complex with activity of 35,000 IU / g, thousandths per weight of silage plants.