LU82150A1 - PROCESS AND PRODUCT FOR THE PRESERVATION OF GREEN PLANTS AND WET BY-PRODUCTS OF THE FOOD INDUSTRIES - Google Patents

Description

γ- * BL-2818 / M / m

£ I Y * y GRAND-DUCHY OF LUXEMBOURG

jarevei in- ............................................... .....

of the "1Z." £ & V.11! & G £ ... 18ÂQ .......... Minister, ÂxwJb of the National Economy and the Middle Classes

Book title: ........................................ „. ,, „„,,. ,,

Industrial Property Service

LUXEMBOURG

Patent Application ...........................

I. Request ......... IA ........ vmJkamiæx + ........ 8..220.Q ... leuilly-surzSeAne.t _______ Exxmae .________ (i). .... lM. * Ams ^ eïL ^ Î ^^ - <a> du .... CimeM.èmt ......... JMméaweg., ........ açissan ± .. ..in ... quality ... of ... man & xbatvea ................................... .....................

drop off ........ this o.rize .... fèvr £ .er ... miZ .... neu.f .... csnt ... quatT> e .vingt ..... .................................................. ...... (3) at .... XiL .................. hours, at the Ministry of National Economy and the Middle Classes, in Luxembourg: * 1. the present request for obtaining a patent for an invention concerning:,! SrD.aêM .... et.produit ... pour..la..ùomemotiûn ... âe..M & g & rates .. .en..rer.t ... and ... de..soii8 -........... (4) VX & MàsûmMmJtesL ^ ___________________________________________________________________________________________ declares, assuming responsibility for this declaration, that ( es) inventors) is (are): IksdRJiSSSBB * ..... 8Û..MUemie..3aimicnà.Æaùæxv? Âr ... 25-C: ie ... Va2? £ &.-________ Seanee _________________________________________ (5) 2. the delegation of power, dated d --------- le & ... £ 4VJ% MZ .... XB.8.Q ................ .....

3. description in language .. jÎ £ 3BÇ! Zl-.8 £ !. ........................................ of the invention in two copies; 4 ......... “________________ drawing boards, in two copies; 5. the receipt of the fees paid to the Luxembourg Registration Office, on February 8, nineteen hundred and eighty______________________________________________________________________________________________________________________________ claims for the above patent application the priority of one (s) request (s) from (6) filed (7) ... France_____________________________________________________________________________________________________________ le Dueuz & .... fétiziex ... m £ l .... neuf .... ccnt ... sazxarJ; e .. £ ix .. neuf sous ~ Ze-N & 03489. ...........- and— (¾ 1.β..ΏΜ ^^ ~ 0 ^ τΐΗη ^ ΜΪη .... ^ 1 .... ηε ^ ... οεηί ... 8αίχακ ± & Αζχ .... ΐΐ ^^ .... εΌΐί & .Λβ. · Ϋ́ο .. ^ 3 ··· 15Β8.? · _____________________ * on behalf of ....... Za.Æp.o.sani. e .__________________________________________________________________________________________________________________________________________________________________________________________________________________ O) elect domicile for him / her and, if designated, for his / her representative, in Luxembourg __ _________________________________ iâ ....... rue.du..C.ime ± ièr.e.3 ________ Luxembourg .___________________________________________________________________________________________________________________________________ uo) requests the grant of a patent for the subject described and represented in the aforementioned appendices, - with postponement of this issue to ....................................... month.

iw άψ mandgtaizee ..........................

y * S Π. Deposit Minutes

The aforementioned patent application has been filed with the Ministry of National Economy and the Middle Classes, Industrial Property Service in Luxembourg, on: fCPp • yr '- y; <v £ Pr. Minister # de l'Économie Nationale es i € f Middle Classes, II cr fM jh A 68007 \ * 3i. ^ /) ....... - —..... 'V * tf. "» Rn ^ v1 * /,' / - .......

»I» Μ

'BL-2818 / EM / BK

Claim of the priority of two requests for v, „certificate of addition filed in France HP on 12.2.1979 under No 79 03499 and J- on 21.6.1979 under No 79 15887.

! " \ * not a word

PATENT

(Addition to patent 77.975 of August 16, 1977 ü? I Process and product for the conservation of green plants and wet by-products of the agro-food industries • -ί 4 "·

NOT

V

ti Î! p CEVA S.A.

14 rue Garnier 92200 Neuilly-sur-Seine

France ί ’i

i: K

-1- "ft The invention relates to an improved process for the conservation and j (lt jsation of fresh plants, in order to allow their consumption under a _ ..... palatable as and when required, subsequently to their date f (i | r "* *.., -jolte: it also concerns a composition for the realization of a? 1 -ié 5 prv '*

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

Silage is based on the principle of conserving fresh plants, for a more or less long period, in a closed and tight enclosure, in an acid medium, with the aim of preventing the development of putative alealinizing and gasogenic germs, these germs not developing in an acid medium.

Achieving good silage is difficult and faces many obstacles, the main ones 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.

20 But it very often happens 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 butyric and anaerobic bacteria develop. , transforming residual sugars into butyric and acetic acids, carbon dioxide and hydrogen; the proteins are degraded in the ammonia and other metabolite stages.

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: 30 biological acidification is difficult in its realization.

Also described are processes in which a raw material rich in carbohydrates, for example molasses, is added to the silo. The results obtained are irregular and the process expensive.

In French patent 2,361,828, there is described a process for the conservation and enhancement of green plants by lactic acidification, which comprises the addition to these plants, before ensilage, of bacteria capable of causing lactic fermentation, as well that the addition of a factor of degradation of higher carbohydrates into fermentable sugars, usable by the bacteria producing lactic fermentation. This degradation factor is made up of 40 | gram + bacteria which ferment starch, glucose, mannitol, rahmnose, le! J. '' • sucrose, amygdalin, arabinose and do not ferment maltose, inositol! sorbitol; these bacteria are VP +; oxidase +; catalase +; nitrates +; ! urea -; citrates -; indole-; H ^ S -. This degradation factor can also include one or more enzymes capable of splitting polysaccharides, H 5 in particular starches, pentosans and other complex polysaccharides into I ’fermentable sugars. In particular, according to this patent, one can add to! fodder a hemicellulolytic complex of fungal origin having as main activity I galactomannanasic activity and secondary activities as being xylanases, amylases, pectinases and an amylase to ensure the formation of maltose necessary for the production of lactic acid by the bacteria.

French patent 2,390,908 constitutes an improvement to patent i 2,361,828. While the amylase used in French patent 2,361,828 is j. an exoamylase of fungal origin which does not completely degrade starch, amylase | used in patent 2,390,908 is an amylase of bacterial origin. This ! 15 enzyme (endoamylase) acts on liquefied starches and produces mainly K '! 1 yD maltose, and a little ot-D glucose, and acts in a pH range between 5 and 8. French patent 2,390,908 also describes the use of amyloglucosidase, which has a much more push against the amylose and f> amylopectin chains to obtain o (-D glucose. The enzymatic composition according to * 'y 20 this patent also includes a hemicellulolytic complex of fungal origin ί which acts on polysaccharide constituents of cell membranes and ni K dextrins, in pH zones between 5.5 and 2.

The present invention relates to an improved process and composition for the ensilage of plants, the improvement relating both to the enzymatic composition and to the bacterial composition. The enzymatic composition is $ j characterized in that it contains, in addition to the enzymes previously used, I a cellulolytic complex or cellulase capable of splitting the beta bonds I (1—> 3) and (1 -> 4) of non-polysaccharides attacked by the enzyme composition:! former.

î; '30 This is a cellulolytic complex of fungal origin which degrades the natural cellulose and other polysaccharides contained in the membranes! cell phones.

i!

This enzyme acts in a pH zone between 2 and 5. There is a phenomenon of synergy between the cellulolytic complex and the complex; 35 hemicellulolytic to degrade plant structures.

I Pu makes the environment of cellulose in the plant, its hydrolysis *. requires several enzymes or a single one fulfilling several functions which are: & - hydrolysis of amorphous zones,; - the swelling of the crystal regions and the rupture of the Hydrogen bonds to? î 40 I give linear molecules, ’t •,’ I '-3- * \; - the hydrolysis of cellobiose into glucose, - the transglucosylation of di and oligosaccharides as well as the rupture of the irregular bonds of cellulose and its primary products of hydrolysis.

The cellulolytic complex was chosen according to the above criteria p 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 one micromole of glucose per minute, on a given cellulosic substrate. .

The activities vary depending on the substrate used: - if the activity is tested on Whatman CC 31 paper powder, the activity is obtained; . - if we test the activity on carboxymethylellulose we obtain the 0χ activity.

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

The activity of the enzyme complex C ^, defined according to the assumptions, corme being composed of an activity C to which is added an unknown factor,

X

- · more particularly reflects a power to dissolve the cellulose.

is capable of degrading native cellulose (crystalline cellulose of degree of polymerization DP ^. 3,500 units of glucose) into amorphous cellulose.

The activity of enzyme complex C more particularly reflects the

X

reducing power of endo and exoglucanases. These enzymes act mainly; on the cellulose fibers in the zones of low crystallinity and cut the long sequences of glucose into smaller units, j The activity of beta-glucosidase translates an action on the units of cellobiose j obtained after the action of 0χ.

These different activities cannot be taken separately. There is a synergistic mechanism between C ^ and Cx for the degradation of these homopolymers. The enzymatic mechanisms C. and C lead to the production of cellobiose; I Λ this is then hydrolyzed by a beta-glucosidase to give two units of glucose.

Cellulose hydrolysis reactions seem to break down depending on the; following diagram: C1

Cellulose Native -y Reactive Cellulose C Hydrolytic

X

i I Reactive cellulose - >> Cellobiose 't / • "S’ -4- beta-glucosidase i hydrolytic; {

Cellobiose -——--> 2 glucose molecules

The complex solubilizes crystalline cellulose with a degree of polymerization ((DP) greater than 3,500 glucose monomers) and produces reactive cellulose 5 (amorphous cellulose).

:not a word . Under the action of the enzymatic complex (endo and exoglucanases) the reactive cellulose chains are cut, either inside and at random in the case of endo-glucanases, 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 beta-glucosidase to give two glucose molecules.

In the case of fungal cellulases according to the invention, the complex: (enzynatic and Cx is capable of hydrolyzing native insoluble cellulose, in gluccse.

The cellulases according to the invention were chosen, not only as a function of the general criteria indicated above, but also taking into account the silage medium which is particular, as a function of the polysaccharoly-I behavior; tick of these cellulases.

This polysaccharolytic behavior was determined on two types of I substrates: I a) - pure substrates; ί b) - dehydrated substrates.

! CASE OF PURE SUBSTRATES: (25 The activity of cellulases A, B, C, D, E, F, was determined on various substrates at a given concentration in an acetate buffer medium 10-¼.

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

j 30 ^ 35 \ î · \ i, i! 40 y '' · ^ λ -5- \ SuBSTRAT paper T CM cTI Pectin Xylanc Arâhino Starch I LoCU XfcQncen- Walh galaetane bean X ^ ation] __________ N. 1% 1% 0.25% 0.25% 0.10% 0.25% 0.25 5 ENZYMES -----: --- (activity), IU / s} "/ *" / g ül / g Ul / g Ul / g Ul / (cO (Cx) ______ A 400 6444 356 5Π 22 394 544 10 "

B 211 5660 322 611 33 100 30C

μμ · ι «μμ · * μ C 266 1999 411 311 0 244 389 15 D 52 6440 255 511 0 120 132

Ε 3? 2 8500 305 605 0 0 C

20

F 960 7666 1030 1200 0 0 14C

ν ν., 25 30

’I

Γ \ uo u -6- <it I - CASE OF DEHYDRATED SUBSTRATES: I We have also determined the activity of enzymes A, B, C, D, E and F on i -. · Various dehydrated plant substrates.

Ί,,, jlj The activity was tested at pH 3.8 - 4.8 - 5.8, at 30 ° C after 24 hours hydrolysis. The reducing power was determined using the dinitro-salicylic acid technique.

Ix. Substrate ”_.

N., Pulpe * * j 1Q * Luierne Rty> gran Mail de Paille

CellulaseVv 'beet »pH ACTIVITY (ül / g) i ι I ——i —— 1 1" i -j 1e 3.8 2810 2280 1560 1810 15ô0 A 4.8 2440 2560 2080 1530 1690 i 5.8 1560 2220 1690 1500 1530 i 't »3.8 1560 1670 1530 1220 1390? N B 4 · 8 1 2 8 0 2 060 1 67 0 1 22 0 1 5 00 d (5.8 890 1680 1940 890 1280 1 3. 8 1280 1530 1390 1110 Π10 i C 4, 8 1280 1750 1530 1110 1290 J ^ 5.8 940 1560 1170 720 l * lu μ! 25 $ -----: -—, ------- I ') 3 , 8 1390 1440 1470 1278 li 0 '/ | D 4.8 II90 2060 1390 1111, 306 i 5.8 890 1720 1530 972 122; P 30 ------- j ----- [3.8 611 1190 1111 556 694 IE 4, 8 972 1528 1250 0 694 t 5.8 o 1167 1278 0 094 35 3, 8 1306 1306 500 694 5it s F 4.8 889 972 1222 610 44-! 5.8 750 1000 560 550 332!: / 1 * ____ _. I -τι'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. lay an activity of 50 to 0.005 IU, and an activity of 500 to 0.05 IU, per 100 g of plant, these activities being determined at pH 4.8, at 50 ° C, after 20 minutes of hydrolysis.

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

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

15 Storage time 0 5 10 15 20 in days pH 4 8 100% 97% 61% 26% 3% pH 5.8 100% 85% 58% 27% 0% 20

The rate of enzymatic degradation of the plant structure is a function of the elimination of glucose; this shift in enzymatic equilibrium is obtained by using bacteria with high fermentative 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 days.

This activity by the production and the accumulation of glucose which it generates within the silage, will increase the nutritive value of the ensiled plant.

The enzymatic composition used in the process according to the invention therefore comprises a cellulase or a cellulolytic complex having the above characteristics, associated with the enzymes described in BF 2,390,908, that is to say a fungal amylase, an amylase bacterial, an amyloglueosidase and a hemicellulolytic complex.

35 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 activity maxima for each enzyme take turns as the pH decreases, which does not drop below pH 3.5 in the silo, and in temperature intervals varying from s ^ 0 J ^ IQ to 30 ° C compatible with the silo conditions.

at 8-

U

fi \ i The bacterial composition according to the invention is characterized by the use of new bacterial strains usable in the silage of plants, [t jü these strains can be used alone or in mixture with the bacterial strains fi and / or the enzymes described in the prior art and in this present invention.

T

^ 5 This new composition leads to very improved silage results. The i j. new bacterial strains according to the present invention are gram- bacteria; they belong to the family of Enterobacteriaceae, to the Erwinia or Pectobacterium group, to the Herbicola group; they are called Enterobacter agglomerans (classification Bergey's Manual of Bacteriology, eight edition).

The table below gives the characteristics of such bacteria:

TABLE I

‘I Enterobacter Agglomerans! Gram - Arabinose + i <j Use malonate + Maltose 15 Glucose + Trehalose +

Phenylalamine desaminase - Xylose ONPG + Starch +

Indole - Oxydase ij H_S - Gelatin + H 2 I20 LDC - Amygdalin + ODC - Melibiosis

Urease - V.P. +

Sucrose + Rhamnose -

Arginine dihydrolase 25 Salicin

Adonitol Isositol Sorbitol

The use of these new strains of bacteria for silage of plants, alone or in combination with other bacterial strains and / or enzymes, i, is carried out according to the methods described in the prior art. The bacteria can be grown 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 bac-;! _ tériées on a support of cereals (wheat, barley, germinated barley) preferably in finely ground form.

Another preferred form of the invention consists in placing the bacteria and μ 40 f the enzymes on a support of the pregelatinized starch, maltohexose, etc. type, support 9 which is easily soluble in cold water.

The starch and other polysaccharides contained in the carriers add 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 an amount of the order of 100,000 to 1 million cocci and 100,000 to 1 million bacilli / g.

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

The quantity of cellulase to be added to the fresh ensiled plant must correspond to an activity C. from 50 to 0.005 IU per 100 g of plant; it must therefore be from v -4 Ί 2 to 2.10% s by weight of the fresh vegetable in the case of a cellulase grading 250 IU / g 15 The proportions of the other enzymes are between 0.05 and 0.20% o by weight vegetable, bacterial amylases grading 250 units / g; between 0.10 and 0.20% e by weight of the plant, of fungal amylases grading 50,000 PS 50 units / g; between 0.10 and 0.70% o by weight of the plant, of amyloglucosidase titrating 200 AG units / g; between 0.05 and 0.20 'by weight of the plant, of hemicellulolytic complex assaying 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 advantage of a cellulolytic complex, and bacteria of the Enterobacteriaceae family, tests were carried out in micro-silos and in mini-silos. The different conservation parameters are studied as well as those reflecting the plant structure.

1- Tests carried out in micro-silos

A first series of experiments was carried out in the laboratory in 35 micro-silos.

These experiments were carried out in May, on alfalfa: the micro-silos have a capacity of 1 kg. The alfalfa was finely chopped and then ensiled by following the experimental protocol below: A - A series of micro-silos was treated using the conservation additive of which 40 / the general formula is as follows: 4 -10- * cereal support 90% I premix 10%; the premix consisting of: - a bacterial complex (5 lactic acid bacteria on a lactose support); 5 - 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; and the bacterial and enzymatic complexes each entering for 1% in the composition of the premix.

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

B - A series of micro-silos was treated with the same preservative additive as in A, but by adding a cellulase of fungal origin (from Trichoderma viride) with an activity of 250 units / g and an activity of 2,500 20 units / g (activities determined at pH 4.8, at 50 ° C, for a reaction time of 20 minutes) the incorporation rate of this cellulase was 2g / T (i.e. 2.10 ”^% by weight) per compared to fresh vegetables (test B) and 200g / T (ie 0.02%) compared to fresh vegetables (test B ').

C - A series of control micro-silos: 25 - a series of alfalfa micro-silos containing no preservatives: T = 0.

After 100 days of ensilage, the micro-silos were open, the following parameters were followed: pH, lactic acid, total NH 2 / N. The results are given in the following table: 30 pH Lactic acid NH_ / N total in g / kg DM ^ Control: T = 0 5.66 26 g 25.15 d Alfalfa + 0.2% premix (A) 4, 13 143.68 16.33 ^ Alfalfa + 0.2% oeïSSVoriglne ^ 32 ™. 75 16.20 fungal 2 g / T (B)

Alfalfa + 0.2% premix + - „,.

I 40, Orisine cellulase 3'73 15.02 i Fungal Ij 200g / T (B *) -11-

It is found that the addition of a cellulase to the bacteria + enzymes complex according to patent 2,390,908 results in an improvement in the performance of the preservative additive. A better conservation of the plant is obtained which, if A and B 'are compared by: 5 - a decrease in pH: 4.13 3.73 (decrease of 10% - an increase in the production of lactic acid by 15% - an 8% decrease in the total NH ^ / N ratio; this fact translates into better conservation of the vegetable protein / consequence of rapid acidification of the medium.

10 2 - Tests carried out in mini-silos

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

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

15 A - A series of mini-silos was treated using the same preservation additive as for the micro-silos in A, the incorporation dose being 0.2% of premix.

B - A series of mini-silos was treated using the formula and the dose indicated in A to which the same cellulase of fungal origin was added at a dose of 2 g / T.

After 100 days of ensilage, the mini-silos are open, the analysis of the silages led to the following results:

A B

25 0.2% premix silos 0.2% premix silos + cellulase (2 g / T)

Dry matter (DM)% 23.67 27.27 E.N.A. (g / kg DM) 51.25 48.97 30 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 35 N total g / kg DM 21.70 23.12 N ammonia 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 2,390,908.

40 H This addition produces: -12- \ - a lower pH 3.94 against 4.20 (minus 6%) | - a greater amount of lactic acid (plus 29.53%) - the protein part of the silage is much better preserved when supplemented with a cellulase; this is confirmed by the analysis. In this case, we have:. - a lower total NH ^ / N ratio (minus 27.6%) - a higher protein level (plus 6%) - a lower ammoniacal nitrogen level (minus 20.1%)

This higher rate of acidification can be explained by the fact that the bacteria will dispose more quickly of a large mass of fermen tescible sugars easily transformable into lactic acid. In this availability, we find the effect of the cellulase introduced into the medium. s In the examples which follow, the mixture on a support according to the process of the present invention consists of the complex described in French patent 2,361,828 and / or patent 2,390,908, the enzymatic complex described in the present invention, to which gram-bacilli are added, from the Enterobacteriacea family! genus Erwinia or Pectobacterium, Herbicola group, the rate of incorporation of these * bacilli into the mixture corresponds to 103 to 10 bacilli / g.

\ EXAMPLES

i o

’20 Tests were carried out in silos with a capacity of 4 m on cocksfoot Lucifer harvested in June, at the beginning of heading. Finely chopped fodder S is subject to storage under 4 conditions:

AT

A: silage without treatment ί B: silage produced with 4 liters of formic acid / ton, in the manner known from the prior art! C: ensilage made with the premix whose composition is given in! French patents 2,361,828 and 2-390,908: silage added with: d 7 kg of the culture of 7 bacteria on a support, the characteristics of which! are indicated in French patent 2,361,828, page 7, Nos. 1 to 7, and 30 of enzymes: incorporation dose 10 kg / ton.

D: ensilage made with the premix according to C to which are added; bacilli according to the invention: gram- bacteria, of the family of

Enterobacteriaceae of the genus Erwinia or Pectobacterium, from the Herbic group called Enterobacter agglomerans. Dose of incorporation of the complex 35 10 kg / T.

| After 90 to 100 days, the different silages are opened and analyzed: plants are made. They lead to the results below (Table II).

! 40 -! w -13- 0 3 cr m

• H 4) O O O

C O

c >> Λ. 0 Pt,

• 3 P

CO X) • ς: ο -------

XJ

b3 0 ü; 3 vo m o oo cr cr> cm eu W Ή ·· ~ λ “-P CT” * = T - = T 00 10 C '0 eu “- T- r-

0 O

ra m. o ------ 3 CJ 0. -H 3 C cr ro ή m toi c oo ω t. o m o o -h ro

Q, · - t, O O

0 O P

15 0 c, XJ O.

H · I.I .. II · I —I - - - '- o <0

3 MD

cr oo H ·>

-P OO T- o CM

O CVI if CTi r- M ™ 20 3 Cü 0 ►J r — 1 ED jQ Γ-) O OO o “C 3 (0 00. = R r- t- <ri *)“ oo oo m œ zt 0 P MD. = R J3- ^ 3- 2 2 25 * co> po OOP t- S o 2 P f- ZT oo 2 2 * ~ * co m cm o 30 * £. | ^ o O f- iT »zr st oô I p I" fc5 · p rc 0 / · = τ c § -h 0 / OO,> 3 t- / PO) ri ^ -H 0 35 1 / “I“ a »CSSî

2 / S gE ΐ | «-g-S-SêS

fl »Ο g <2 ££ g.ï £ / p h 0„ 0 p · 000 ^ 0

/ C P P 0 4-J0C

/ 0 _ -p "o -h> g: -Hc + pc / E C CD-iri 00ΓΟ 0 · Η O-iri / 0 o c o and, · t, - U te -

/ PC Pro PJDCM H

Γ / Ή fi J ro ··. · ..

Uo sjw I £ C CQ o Q

-14-% ""

The composition of the results shows that the mixture according to French patents 2,361,828 and 2,390,908 already brought an improvement in the conservation of the silage, this resulting in: - a lowering of the pH which passed from 5.28 to 4.02; 5 - a higher lactic acid content which went from 23 to 90 g / kg of Mi | * - better protein protection which results in a reduction in the I N-NH3 ratio which went from 17.76 to 14 and in the total IN 1 soluble N ratio which went from 63.80 to 48.10 S 10 N total 1 Ce mixture had the advantage of not giving rise to any training! ! propionic acid and butyric acid.

I,,, g. The mixed according to the present invention provides additional improvement | resulting in:] 15 - a drop in pH from 4.02 to 3.70 | - a higher lactic acid content which goes from 90 g to 112 g / kg of f- i - better protein protection which results in a reduction in the total N-NH3 / N ratio. This goes from 14 to 8 and from the soluble N / total J ratio. This 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 indigestibility of these silages were measured on sheep; after the various treatments, it was found: B: treatment with formic acid: 0.64 UF / kg

: 25 C: treatment according to French patents 2,361,828 and 2,390,908: 0.74 IU

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 the table III.

ί f-! i; 35 40 * ”, -15- v *

TABLE III

j parameters N N Fecal N urinary 'N retained I

ingested -------—-- d 5 treatments g / d 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: formic acid treatment 18.9 7.34 38.8 10.4 55 1.1 5.88 0.4% i 10 -------- iC: treatment according to main patent + 17 7.07 41.6 8.12 47.7 1.8 10.5 1st fb addition: 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. 20 | Γ 25 30 1 40

Claims (9)

1. A method for preserving and enhancing green plants by lactic acidification which comprises adding g 'to these plants before silage a factor for degrading the above carbohydrates into fermentable carbohydrates which can be used by the bacteria producing the S lactic fermentation, this factor being made up of a complex! of enzymes and / or by a complex of bacteria characterized! in that the enzymatic complex comprises, associated or not I with a fungal amylase, with a bacterial amylase, with 1 an amyloglucosidase, with a hemicellulolytic complex I of fungal origin having as main activity j a galactomannanasic activity, a cellulase or îj cellulolytic plex of fungal origin, capable of degrading native cellulose into glucose and having activity | from 50 to 0.005 international units and a 0χ activity of î | 500 to 0.05 international units per 100 g of fresh plant 1 and preserves in silage conditions at least 30% of this activity after 10 days, and in that the bacterial complex includes gram bacteria which ferment starch but do not ferment maltose belonging to the family, Enterobacteriaceae, genus IErwinia or Pectobacterium, Herbicola group being called, Enterobacter agglomerans. 2. Method according to claim 1 characterized in that the bacteria of the Enterobacteriaceae family are used in combination with bacteria known from the prior art capable of degrading higher carbohydrates into fermentable carbohydrates. 3. Method according to one of claims 1 to 2, characterized j in that the bacteria of the family of Enterobacteriaceae are used in association with bacteria gram + j “which ferment starch, glucose, mannitol, rah - mnose, sucrose, amygdalin, arabinose and ne; \ do not ferment maltose, inositol, sorbitol. t- / - 17 - V V- <U - Process according to claim 1, characterized in that the mixture of enzymes consists of: 2.10 to 2% tf of cellulase titrating 250 IU of activity C ^ / g 0, 05 to 0.20% * of bacterial amylase grading 250 units / g 0.10 to 0.20 io <* of fungal amylase grading 5,000 PS units 50 / g 0.10 to 0.70% c of amyloglucosidase grading 200 AG units / g, 0.05 to 0.20% e of hemicellulolytic complex grading 35,000 IU / g, the% c being calculated by weight relative to the ensiled plant. 5. Method according to one of claims 1 to 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 to 2, characterized in that the bacteria and enzymes are incorporated on a support of the cereal starch, pregelatinized starch, malto-hexose type, soluble in cold water, in the proportion of 1 kg of bacteria and enzymes for 1.5 to 11 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 10 to 10 live germs per gram. 9. Composition for the conservation and enhancement of green plants which comprises strains of lactic acid producing bacteria, and a factor of degradation of higher carbohydrates into fermentable carbohydrates, consisting of an enzyme complex and / or a complex {of bacteria, characterized in that the enzymatic-1 complex which comprises or not associated with a fungal amylase, a bacterial jfr * amylase, an amyloglucosidase, a P - 18 - complex: i hemicellulolytic of fungal origin having main activity a galactomannanasic activity, a cellulase or cellulolytic complex of fungal origin, capable of degrading native cellulose into glucose and having an activity of 50 to 0.005 international units and an 0χ activity of 500 to 0.05 international units per 100 g of fresh vegetable and preserved in the silage conditions at least 30% of this activity after 10 days and in that the bacterial complex eom- * takes bacteria éries gram - yes ferment starch but 'do not ferment malt, belonging to the family Ides Enterobacteriaceae, genus Erwinia or Pectobacterium, group Herbicola, being called Enterobacteriobacter Agglomerants. 10. Composition according to claim 9 characterized in that the gram bacteria - Enterobacteriaceae are associated with bacteria capable of degrading higher carbohydrates into fermentable sugars, and in particular gram + bacteria which ferment starch, glucose, mannitoljle rahmnose , sucrose, and amygdalin, arabinose, and do not ferment maltose, inositol, sorbitol. ^ 4 i * / 'BL-2818 / EM / BM "* The strain" Enterobacter agglomerans "was deposited on eleventh February one thousand nine hundred and eighty at the Institut Pasteur, under No. 1-114. »M