CH643988A5 - PROCESS AND COMPOSITION FOR THE PRESERVATION OF GREEN PLANTS AND WET BY-PRODUCTS OF THE FOOD INDUSTRIES. - Google Patents

Description

The invention relates to an improved process for the conservation and enhancement of fresh plants, in order to allow their consumption in an appetizing form as and when required, after their harvest date; it also relates to a composition for carrying out the process.

Given the importance of the problem posed by the conservation of green plants, numerous research works have been devoted to the realization of this technique which presents a certain number of difficulties.

The silage rests on the principle of the conservation of fresh plants, for a more or less long period, in a closed and tight enclosure, in acid medium, in order to prevent the development of rotten, alkalizing and gas-forming, these germs do not develop in an acid medium. Achieving good silage is difficult and faces many obstacles, the main one being the formation of fermentation products dangerous to the health of animals and even humans who consume meat and dairy products.

The mechanism sought is as follows: allow the lactic acid bacteria contained in the medium to produce lactic acid from available soluble sugars, so that the medium can drop to a pH close to 4. But it very often happens 40 that, the silage plants not containing enough fermentable sugars, the proliferation of only lactic acid bacteria contained in the medium is insufficient, the pH does not drop quickly enough to 4, and the butyric and anaerobic bacteria develop, transforming the residual sugars butyric and acetic acids, carbon dioxide and hydrogen; proteins are degraded to the stage of ammonia and other metabolites.

Various solutions have been made to these drawbacks. In particular, it is known to carry out chemical acidification by adding various acids. But these processes are not without danger. It is also known to carry out biological acidification by the addition of very glucidolytic bacteria. But this biological acidification is difficult in its realization.

Processes have also been described in which a raw material rich in carbohydrates, for example me-55 lasses, is added to the silo. The results obtained are irregular and the process expensive.

In French patent No. 2361828, a process for the conservation and enhancement of green plants by lactic acidification is described, which comprises the addition to these plants, before ensilage, of bacteria capable of causing lactic fermentation, as well so that the addition of a factor of degradation of higher carbohydrates in fermentable sugars, usable by the bacteria producing lactic fermentation. This degradation factor is constituted by Gram + bacteria which ferment starch, glucose, mannitol, rhamnose, sucrose, amygdalin, arabinose and do not close maltose, inositol, sorbitol; these bacteria are VP +; oxidase +; catalase +; nitrates +; urea -; citrates -; indole -; H2S -. This degradation factor can also include one or more enzymes capable of cleaving Polysaccharides,

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in particular starches, pentosans and other complex polysaccharides in fermentable sugars. In particular, according to this patent, a hemicellulolytic complex of fungal origin can be added to the fodder having, as main activity, galactomannanasic activity and secondary activities as xy-lanases, amylases, pectinases and an amylase to ensure the formation of maltose. necessary for the production of lactic acid by bacteria.

French patent No. 2390908 constitutes an improvement to patent No. 2361828. While the amylase used in French patent No. 2361828 is an exoamylase of fungal origin which does not completely degrade the starch, the amylase used in the Patent No. 2390908 is an amylase of bacterial origin. This enzyme (endoamylase) acts on liquefied starches and produces mainly a-D maltose and a little a-D glucose, and acts in a pH range between 5 and 8. French patent N ° 2390908 describes also the use of amyloglucosidase, which has a much more extensive action with respect to the amylose and amylopectin chains for obtaining a-D glucose. The enzymatic composition according to this patent also comprises a hemicellulolytic complex of fungal origin which acts on the polysaccharide constituents of cell membranes and dextrins, in zones of pH between 5.5 and 2.

The present invention relates to an improved process and composition for plant silage, the improvement relating both to the enzymatic composition and to the bacterial composition. The enzymatic composition is characterized in that it contains, in addition to the enzymes previously used, a cellulolytic complex or cellulase capable of splitting the ß (1 - »3) and (1 -► 4) bonds of the Polysaccharides not attacked by the composition previous enzyme.

It is a cellulolytic complex of fungal origin which degrades natural cellulose and the other Polysaccharides contained in cell membranes.

This enzyme acts in a pH zone between 2 and 5. There is a phenomenon of synergy between the cellulolytic complex and the hemicellulolytic complex to degrade plant structures.

Due to the environment of cellulose in the plant, its hydrolysis requires several enzymes or only one fulfilling several functions which are:

- hydrolysis of amorphous zones,

- the swelling of the crystalline regions and the rupture of the hydrogen bonds to give linear molecules,

- the hydrolysis of cellobiose into glucose,

- the transglucosylation of the di- and Oligosaccharides, as well as the rupture of the irregular bonds of the cellulose and its primary products of hydrolysis.

The cellulolytic complex was chosen according to the above criteria and after a large number of tests on various plants.

A cellulase is defined by its activity expressed in international units per gram of product, an international unit (IU) being the quantity of enzyme which releases in reducing sugar the equivalent of 1 nmol of glucose per minute, on a given cellulosic substrate.

The activities vary according to the substrate used:

- if the activity is tested on Whatman CC 31 paper powder, activity Q is obtained;

- if the activity on carboxymethylcellulose is tested, the activity Cx is obtained.

The complex used according to the invention has a very high cellulolytic activity as well as secondary activities of the hemicellulolytic type. The high cellulolytic power results in very strong Q and Cx activities and in cellobiase (ß-glucosidase) activity.

The activity of the enzymatic complex Q, defined according to the hypotheses as being composed of an activity Cx to which an unknown factor is added, more particularly reflects a power of solubilization of the cellulose.

C, is capable of degrading native cellulose (crystalline cellulose with a degree of polymerization DP> 3500 glucose units) into amorphous cellulose.

The activity of the Cx enzyme complex more specifically reflects the reducing power of endo- and exoglucanases. These enzymes act mainly on cellulose fibers in areas of low crystallinity and cut the long sequences of glucose into smaller units.

The activity of ß-glucosidase reflects an action on the cellobiose units obtained after the action of Cx.

These different activities cannot be taken separately. There is a synergy mechanism between C! and Cx for the degradation of these homopolymers. The Ct and Cx enzymatic mechanisms result in the production of cellobiose; this is then hydro-lysed by a ß-glucosidase to give two units of glucose.

Cellulose hydrolysis reactions seem to break down according to the following scheme:

native cellulose £ 2 reactive cellulose reactive cellulose hydrolytic Cx ^ cellobiose ß-glucosidase hydrolytic cellobiose ^ two glucose molecules

The Ci complex solubilizes crystalline cellulose [degree of polymerization (DP) greater than 3500 glucose monomers] and produces reactive cellulose (amorphous cellulose).

Under the action of the enzymatic complex Cx (endo- and exoglucanases) the reactive cellulose chains are cut either inside and at random in the case of endoglucanases, or from non-reducing ends in the case of exoglucanases.

All of these reactions give units of cellobiose.

These cellobiose units are hydrolyzed by a ß-glucosidase to give two glucose molecules.

In the case of fungal cellulases according to the invention, the enzyme complex C, and Cx is capable of hydrolyzing native cellulose insoluble in glucose.

The cells according to the invention were chosen not only according to the general criteria indicated above, but also taking into account the silage medium which is particular, according to the polysaccharolytic behavior of these cellulases.

This polysaccharolytic behavior has been determined on two types of substrates:

a) pure substrates;

b) dehydrated substrates.

PURE SUBSTRA TS:

The activity of cellulases A, B, C, D, E, F, was determined on various substrates at a given concentration in an acetate buffer medium 10 ~ 'M.

The activity is tested according to the usual standards, that is to say at pH 4.8, at 50 ° C., after 20 min of hydrolysis. The reducing power is determined using the hexaferrocyanide technique.

DEHYDRATED SUBSTRA TS:

The activity of enzymes A, B, C, D, E and F was also determined on various dehydrated plant substrates.

The activity was tested at pH 3.8, 4.8, 5.8, at 30 ° C, after 24 h hydrolysis. The reducing power was determined using the dinitrosalicylic acid technique.

(Tables at the top of the next page)

Analysis of these different behaviors made it possible to determine the activity of the cellulolytic complex to be used according to the invention in silage.

It has thus been found that the cellulase or the cellulolytic complex to be added in the process according to the invention to the fresh ensiled plant must correspond to an activity C! 50 to 0.005 IU and Cx activity

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SUBSTRATE (concentration)

Wathman paper

CMC

Pectin

Xylane

Arabino-galactin

Starch

Bean locus

ENZYMES

1%

1%

0.25%

0.25%

0.10%

0.25%

0.25%

(activity)

U.I./g (Ct)

U.I./g (Cx)

U.I./g

U.I./g

U.I./g

U.I./g u.i./g

A B C D E F

400 211 266 52 372 960

6444 5660 1999 6440 8500 7666

356,322,411 256,305 1,030

511 611 311 511 605 1200

22 33 0 0 0 0

394 100 244 120 0 0

544 300 389 132 0 140

Substrates tested

Cellulases tested

Alfalfa

Ryegrass

But

Beet pulp

Straw

pH

ACTIVITY (U.I./g)

AT

3.8 4.8 5.8

2810 2440 1560

2280 2560 2220

1560 2080 1690

1810 1530 1500

1560 1690 1530

B

3.8 4.8 5.8

1560 1280 890

1670 2060 1680

1530 1670 1940

1220 1220 890

1390 1500 1280

VS

3.8 4.8 5.8

1280 1280 940

1530 1750 1560

1390 1530 1170

1110 1110 720

1110 1290 1110

D

3.8 4.8 5.8

1390 1190 890

1440 2060 1720

1470 1390 1530

1278 1111 972

1160 1306 1223

E

3.8 4.8 5.8

611 972 0

1190 1528 1167

1111 1250 1278

556 0 0

694 694 694

F

3.8 4.8 5.8

1306 889 750

1306 972 1000

500 1222 560

694,610,550

556 444 333

from 500 to 0.05 IU per 100 g of plant, these activities being determined at pH 4.8, at 50 ° C, after 20 min of hydrolysis.

In addition, the cellulase chosen must retain significant activity even after a long stay in the silo.

It has been experimentally demonstrated that, under conditions approaching those of silage, the cellulolytic complex must retain, after 10 d, at least 30% of its initial activity.

Time

conservation (d)

0

5

10

15

20

pH 4.8

100%

97%

61%

26%

3%

pH 5.8

100%

85%

58%

27%

0%

The rate of enzymatic degradation of the plant structure is a function of the elimination of glucose; this enzymatic equilibrium shift is obtained by using bacteria with a high fermentation power; the acidification of the medium is very rapid. When the plant mass is stabilized, the bacterial proliferation as well as the production of lactic acid are stopped, contrary to the cellulolytic activity of the cellulase which is prolonged more than 10 d.

This activity, through the production and accumulation of glucose that it generates within the silage, will increase the nutritional value of the ensiled plant.

The enzymatic composition used in the process according to the in-45 vention therefore comprises a cellulase or a cellulolytic complex having the above characteristics, associated with the enzymes described in French patent No. 2390908, that is to say an amylase fungal, a bacterial amylase, an amyloglucosidase and a hemicellulolytic complex.

50 These enzymes present in the composition thus have a complementary action by their activity on carbohydrates, from the most complex to the simplest, in pH zones which could range from 7 to 2. The maximum activity for each enzyme take turns as the pH decreases, which does not drop below 3.5 in the silo, and in temperature ranges varying from 10 to 30 ° C compatible with the conditions from the silo.

The bacterial composition according to the invention is characterized by the use of new bacterial strains which can be used in plant silage, these strains can be used alone or as a mixture with the bacterial strains and / or the enzymes described in the prior art and in this present invention. This new composition leads to very improved silage results. The new bacterial strains according to the present invention are Gram - bacteria; they belong to the family of Enterobacte-65 riaceae, in the genus Erwinia or Pectobacterium, to the group Herbicola 'they are called Enterobacter agglomerates (classification "Bergey's Manual of Bacteriology", 8th ed.), and were deposited on February 11, 1980 'Institut Pasteur under N ° 1-114.

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Table I gives the characteristics of such bacteria: Table I

Enterobacter agglomerates

Gram

-

Arabinose

-

Malonate use

+

Maltose

-

Glucose

+

Trehalose

+

Phenylanine deaminase

-

Xylose

-

ONPG

+

Starch

+

Indole

-

Oxidase

-

H2S

-

Gelatin

+

LDC

-

Amygdalin

+

ODC

-

Melibiose

-

Urease

-

VP

+

Sucrose

-1-

Rhamnose

-

Argininedihydrolase

-

Salicin

-

Adonitol

-

Inositol

-

Sorbitol

-

The use of these new strains of bacteria for plant silage, alone or in combination with other bacterial strains and / or enzymes, is carried out according to the methods described in the prior art. The bacteria can be cultivated on a nutritious support comprising cereal flours. The composition to be introduced into the silo comprises a mixture of the necessary bacteria deposited on a soluble or non-soluble support, with the mixture of the enzymes recommended in the present invention.

A preferred form of the invention consists in putting the enzymes and the bacteria on a cereal support (wheat, barley, germinated barley), preferably in finely ground form.

Another preferred form of the invention consists in putting the bacteria and the enzymes on a support of the pregelatinized starch, maltohexose, etc. type, support which is easily soluble in cold water.

The starch and other Polysaccharides contained in the supports are added to the carbohydrates of plants and thus increase the nutritional value of the silage. The composition comprising the mixture of supported bacteria and the enzymatic complex deposited or not on a cereal support can comprise different proportions of each of these constituents; this dry mixture contains a quantity of the order of 100,000 to 1,000,000 Cocci and 100,000 to 1,000,000 bacilli / g.

The amounts of cellulase, hemicellulolytic complex, amylase and amyloglucosidase can be variable; they are calculated according to the nature of the fodder to be treated.

The amount of cellulase to add to the fresh ensiled vegetable must correspond to an activity C! from 50 to 0.005 IU per 100 g of vegetable; it must therefore be 2 to 2 • 10_4% o by weight of the fresh vegetable in the case of a cellulase grading 250 IU / g. The proportions of the other enzymes are between 0.05 and 0.20% by weight of the plant of bacterial amylases grading 250 units / g; between 0.10 and 0.20%> by weight of the plant with fungal amylases grading 50,000 PS 50 units / g; between 0.10 and 0.70% o by weight of the plant amyloglucosidase grading 200 AG units / g; between 0.05 and 0.20% o by weight of the plant of hemicellulolytic complex grading 35,000 IU / g.

When the enzymes are deposited on a cereal support according to the preferred form of the invention, the product to be incorporated into the silage may contain 1 kg of enzymes for approximately 9 kg of support. In the case of a soluble support, the concentration ratios of the enzyme relative to the support change and can vary from 1 / 1.5 to 1/4.

This incorporation of cellulase into the support will increase the quantity of fermentable sugars available, which will induce a greater and faster production of lactic acid. The vegetable protein will thus be better preserved. On the other hand, the nutritional value of the environment will only be increased.

In order to illustrate the interest of a cellulolytic complex, and bacteria of the Enterobacteriaceae family, tests were carried out in microsilos and in minisilos. The different conservation parameters are studied as well as those reflecting the plant structure.

1. Tests carried out in microsilos

A first series of experiments was carried out in the laboratory in microsilos.

These experiments were carried out in May, on alfalfa: the microsilos have a capacity of 1 kg. The alfalfa was finely chopped, then ensiled by following the experimental protocol below:

A. A series of microsilos was treated using the preservation additive, the general formula of which is as follows:

cereal support: 90%

premix: 10%

the premix consisting of:

- a bacterial complex (5 lactic acid bacteria on a lactose support);

- an enzymatic complex comprising:

an amylase of fungal origin;

an amylase of bacterial origin;

an amyloglucosidase;

a hemicellulolytic complex of fungal origin having as main activity a galactomannanasic activity;

- a medium rich in energy: finely ground barley + whey;

the bacterial and enzymatic complexes each entering for 1%

in the composition of the premix.

The conservation additive was added to the alfalfa at the rate of 2% (the rate of incorporation of the premix being therefore 0.2%) and 1% of barley was also added.

B. A series of microsilos was treated with the same preservative additive as in A, but by adding to it a cellulase of fungal origin (originating from Trichoderma viride) having a C activity, of 250 units / g and a Cx activity of 2500 units / g (activities determined at pH 4.8, at 50 ° C, for a reaction time of 20 min); the incorporation rate of this cellulase was 2 g / t (i.e. 2-10 ~ 4% o by weight) relative to the fresh plant (test B) and 200 g / t (i.e. 0.02%) compared with fresh vegetables (test B ') -

C. A series of control microsilos: a series of alfalfa microsilos containing no preservatives: t = 0.

After 100 d of silage, the microsilos were opened; the following parameters were followed: pH, lactic acid, total NH3 / N. The results are given in the following table:

Control: t = 0

pH

Lactic acid

(g / kg dry matter)

NH3 / N total

5.66

26g

25.15

Alfalfa + 0.2% premix

(AT)

4.13

143.68

16.33

Alfalfa + 0.2% premix + cellulase of fungal origin 2 g / t (B)

4.32

144.75

16.20

Alfalfa + 0.2% premix + cellulase of fungal origin 200 g / t (B ')

3.73

169.47

15.02

It has been observed that the addition of a cellulase to the bacteria + enzymes complex according to patent No. 2390908 results in an am5

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improved performance of the preservative additive. We obtain a better conservation of the plant which results, if we compare A and B ', by:

- a decrease in pH: from 4.13 to 3.73 (decrease of 10%),

- a 15% increase in lactic acid production, 5

- a decrease of 8% in the total NH3 / N ratio; this fact reflects a better conservation of the vegetable protein, consequence of a rapid acidification of the medium.

2. Tests carried out in 1C mini-silos

These tests were carried out during June on alfalfa in minisilos with a capacity of 150 kg.

The alfalfa was finely chopped, then subjected to silage following the experimental protocol below:

A. A series of minisilos was treated using the same preservative additive as for the microsilos at A, the incorporation dose being 0.2% of premix.

B. A series of minisilos was treated using the formula and the dose indicated in A, with the addition of the same cellulase of fungal origin at a dose of 2 g / t.

After 100 d of silage, the minisilos were opened; silage analysis led to the following results:

AT

B

0.2% silos

0.2% silos

premix

premix

+ cellulase (2 g / t)

Dry matter (DM) (%)

23.67

27.27

ENA (g / kg DM)

51.25

48.97

pH

4.20

3.94

Lactic acid (g / kg DM)

80.62

114.40

Acetic acid (g / kg DM)

17.42

20.04

NH3 / N total

18.40

13.31

Total N (g / kg DM)

21.70

23.12

N ammoniacal (g / kg DM)

3.87

3.09

The above results fully demonstrate the advantage of adding a cellulase to the bacteria + enzymes complex described in patent No. 2390908.

This addition produces:

- a lower pH: 3.94 against 4.20 (- 6%),

- a higher amount of lactic acid (+ 29.53%),

- ■ the protein part of the silage is much better preserved when supplemented with a cellulase; this is confirmed by analysis; we have in this case;

a lower total NH3 / N ratio (- 27.6%),

a higher protein level (+ 6%),

a lower ammoniacal nitrogen level (- 20.1%).

This higher rate of acidification can be explained by the fact that the bacteria will have more rapidly a large mass of fermentable sugars easily convertible into lactic acid. In this availability, we find the effect of the cellulase introduced into the medium.

In the examples which follow, the mixture on a support according to the method of the present invention consists of the complex described in French patent No. 2361828 and / or patent No. 2390908, the enzyme complex described in the present invention, to which adds Gram bacilli, from the family of Enterobacteriaceae, genus Erwinia or Pectobacterium, Herbicola group, the rate of incorporation of these bacilli into the mixture corresponds to 105 to 106 bacilli / g.

Examples:

Tests have been carried out in silos with a capacity of 4 m3 on cocksfoot Lucifer harvested in June, at the beginning of the heading. Finely chopped fodder is subjected to storage under 4 conditions:

A. silage without treatment,

B. ensilage made with 41 formic acid / t, in the manner known from the prior art,

C. ensilage made with the premix whose composition is given in French patents Nos 2361828 and 2390908: ensilage

40 supplemented with 7 kg of the culture of 7 support bacteria, the characteristics of which are indicated in French Patent No. 2361828, p. 7, Nos 1 to 7, and of enzymes: incorporation dose 10 kg / t,

D. ensilage made with the premix according to C to which are added bacilli according to the invention: Gram bacteria, of the family

45 of Enterobacteriaceae of the genus Erwinia or Pectobacterium, of the Herbicola group called Enterobacter agglomerates. Dose of incorporation of the complex: 10 kg / t.

After 90 to 100 d, the various silages are opened, and the analyzes of the plants are carried out. They lead to results

50 given in Table II.

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Table II

Settings

PH treatments

N-NH, total N (%)

N soluble N total

Organic acids (g / kg DM)

lactic propionic acetic butyric

A: untreated

5.28

17.76

63.80

23

1.38

29.46

traces

B: treatment with formic acid 4%

4.05

7.10

45.43

41.36

traces

0

C: treatment according to French patent No 2390908

4.02

14

48.10

90

0

14.20

0

D: treatment according to the invention (C + bacterial strains according to the invention)

3.70

8

44

112

0

18.28

0

7

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The composition of the results shows that the mixture according to French patents Nos 2361828 and 2390908 already brought an improvement in the conservation of the silage, this resulting in:

- a lowering of the pH which went from 5.28 to 4.02,

- a higher lactic acid content which went from 23 to 90 g / kg DM,

- better protein protection which results in a reduction in

N — NH

report ? which went from 17.76 to 14, and from

N soluble ratio ^ which went from 63.80 to 48.10.

This mixture had the advantage of not giving rise to any formation of propionic acid and butyric acid.

The mixture according to the present invention provides an additional improvement resulting in:

- a lowering of the pH which goes from 4.02 to 3.70,

- a higher lactic acid content which goes from 90 to 112 g / kg DM,

- better protein protection which results in a decrease in the total N - NH3 / N ratio; this goes from 14 to 8, and from the soluble N / total N ratio; it goes from 48.10 to 44.

This mixture has the advantage of not giving rise to any fermentation of propionic acid and butyric acid.

The digestibility and the indigestion of these silages were measured on sheep. After the various treatments, it was found:

B. treatment with formic acid: 0.64 UF / kg,

C. treatment according to French patents Nos 2361828 and 2390908: 0.74 UF / kg,

D. treatment according to the invention: 0.82 UF / kg.

The nitrogen balance on growing animals leads to results showing the advantage of the treatments with silage products according to the present invention as can be seen in Table III.

Table III

Settings

Treatments

N ingested (g / d)

Fecal

N urinary

N retained

(g / d)

N ingested (%)

(g / d)

N ingested (%)

(g / d)

N ingested (%)

A: untreated

21

7.90

3.76

13

61.9

0.10

0.50

B: treatment with formic acid 0.4%

18.9

7.34

38.8

10.4

55

1.1

5.88

C: treatment according to main patent + 1st addition

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 nitrogen retention in the case of the present invention is more than doubled compared to that obtained after treatment with formic acid.

R

Claims (10)

643,988 1. A method for preserving and enhancing green plants by lactic acidification which comprises adding to these plants, before silage, a factor for degrading higher carbohydrates into fermentable carbohydrates usable by the bacteria producing lactic fermentation, this factor consisting of an enzyme complex and / or a bacteria complex, characterized in that the enzyme complex comprises, associated or not with a fungal amylase, a bacterial amylase, an amyloglucosidase, a hemicellulolytic complex d fungal origin having as main activity a galactomannanasic activity, a cellulase or cellulolytic complex of fungal origin, capable of degrading native cellulose into glucose and having a Q activity of 50 to 0.005 international unit and a Cx activity of 500 to 0.05 international unit for 10 g of fresh plant and conserves, under the conditions of silage, at least 30% of this activity after 10 d, and in that the bacterial complex includes Gram bacteria which ferment starch but do not ferment maltose belonging to the family of Enterobacteriaceae, genus Erwinia or Pectobacterium, group Herbicola, called Enterobacter agglomerates. 2 • 10 ~ 4 to 2% o of cellulase grading 250 IU of activity Cj / g 0.05 to 0.20% o of bacterial amylase grading 250 units / g 0.10 to 0.20% o of fungal amylase grading 5000 PS 50 units / g 0.10 to 0.70% o of amyloglucosidase grading 200 AG units / g 0.05 to 0.20% o of hemicellulolytic complex grading 35,000 IU / g, the% o being calculated by weight compared to the ensiled plant. 2. Method according to claim 1, characterized in that the bacteria of the family of Enterobacteriaceae are used in association with bacteria known from the prior art capable of degrading higher carbohydrates into fermentable carbohydrates. 2 3. Method according to one of claims 1 or 2, characterized in that the bacteria of the family of Enterobacteriaceae are used in combination with Gram + bacteria which ferment starch, glucose, mannitol, rhamnose, sucrose, amygdalin, arabinose and do not ferment maltose, inositol, sorbitol. 4. Method according to claim 1, characterized in that the mixture of enzymes consists of: 5. Method according to one of claims 1 or 2, characterized in that the enzymes are incorporated on a cereal support at a rate of about 1 kg of enzymes per 9 kg of support. 6. Method according to claim 1, characterized in that the bacteria are cultivated on a support comprising cereal flours. 7. Method according to one of claims 1 or 2, characterized in that the bacteria and enzymes are incorporated on a support of the starch type of cereals, pregelatinized starch, maltohexose, soluble in cold water, in the proportion 1 kg of bacteria and enzymes for 1.5 to 4 kg of support. 8. Method according to one of claims 1 to 5, characterized in that the quantities of enzymes and bacterial culture with their support, in the dry state, added per tonne of plants to be ensiled, are from 10 to 15 kg , this additive containing 105 to 106 live germs per gram. 9. Composition for implementing the method according to claim 1 for the conservation and enhancement of green plants which comprises strains of lactic acid producing bacteria, and a factor for degrading higher carbohydrates into fermentable carbohydrates, consisting of an enzyme complex and / or a bacteria complex, characterized in that the enzymatic complex comprises, associated or not with a fungal amylase, a bacterial amylase, an amyloglucosidase, a hemicellulolytic complex of fungal origin having as main activity a galactomannanasic activity, a cellulase or cellulolytic complex of fungal origin, capable of degrading native cellulose into glucose and having a Q activity of 50 to 0.005 international unit and a Cx activity of 500 to 0.05 international unit per 100 g of fresh vegetable and preserves, under the conditions of silage, at least 30% of this activity after 10 d, and in that the complex b actérien includes Gram bacteria which ferment starch but do not ferment maltose, belonging to the family of Entero-5 bacteriaceae, genus Erwinia or Pectobacterium, group Herbicola, called Enterobacter agglomerates. 10. Composition according to claim 9, characterized in that the Gram— Enterobacteriaceae bacteria are associated with bacteria capable of degrading higher carbohydrates into fermented sugars-target, and in particular Gram + bacteria which ferment starch, glucose, mannitol, rhamnose, sucrose, and amygdalin, arabinose, and do not ferment maltose, inositol, sorbitol.