GB2046567A - Ensilaging vegetable matter - Google Patents

Abstract

A process for the preservation of vegetable material by lactic acidification which comprises admixing the vegetable material prior to ensilaging the same, with bacteria capable of initiating lactic fermentation, and enzymes and/or bacteria adapted to degrade higher glucides into fermentable sugars used by the bacteria initiating the lactic fermentation. The enzymes comprise a defined cellulolytic complex of fungal origin. The bacteria comprise Gram -ve bacteria belonging to the family of Enterobacteriaceae type Erwinia or Pectobacterium, group Herbicola, of the new species Enterobacter agglomerons, which breaks down starch, but not maltose. This composition with an enzyme and/or bacteria base, is incorporated on a cereal support.

Description

SPECIFICATION Process and product for preserving fresh vegetables and moist byproducts of the Agro-alimentary industry try The present invention is related to an improved process for preserving and valorizing fresh vegetables so as to allow the same to be consumed in an appetizing form in successive amounts, as the need arises, after harvesting said vegetables. The invention also relates to a composition or product for carrying out said process On account of the importance of the problems raised by the preservation of fresh vegetables, considerable research work has been conducted with a view to resolving the same by devising an appropriate technique; this research work has encountered a certain number of obstacles difficult to overcome.

The technique of ensilaging is based on the principle of preserving fresh vegetables during a more or less extended period of time within a closed, tight enclosure in an acid environment or medium with a view to preventing the proliferation of putrifying alkalizing and gas-producing germs; these germs will not develop in an acid medium. A satisfactory ensilaging method is difficult to devise, since a great number of obstacles are encountered when endeavouring to put such method to practical use, the most difficult problems to be overcome being the formation of fermentation products which are dangerous to the health of animals orthe health of man when the meat and dairy products are consumed by the latter.

The mechanism to be devised is as follows: allowing the lactic bacteria contained in the medium to produce lactic acid from available soluble sugars, in such a manner that the pH of the medium may be thus lowered to a value of about 4. However in many cases the vegetables to be ensilaged do not contain a sufficient amount of fermentable sugars, and the proliferation only of the lactic bacteria, which are contained in the medium is insufficient, the pH thus not being lowered rapidly enough to the required value 4, while butyric and anaerobic bacteria develop and convert the residual sugars into butyric and acetic acid, carbon dioxide and hydrogen, the proteins being degraded into ammoniacal and other metabolic phases.

Various approaches have been devised with a view to overcoming these drawbacks. More particularly, according to one known process, a chemical acidification is effected by adding various acids.

However this known method may be dangerous under certain conditions. According to another known method, a biological acidification is effected by adding highly glucidolytic bacteria. However this biological acidification process has been found to be difficult to carry out in practice.

According to other known methods, a raw material having a high contentofglucide, such as molasses, is added to the ensilaged material. The results obtained when carrying out such methods in practice are not consistent, and the process involves considerable expense.

French Patent specification No. 2 361 828 discloses a process of preserving and valorizing fresh vegetables by lactic acidification, which comprises adding to the vegetables, prior to ensilaging the same, bacteria capable of initiating lactic fermentation, and adding a degradation agent for degrading higher glucides into fermentable sugars which are adapted to be used by the bacteria that initiate the lactic fermentation. This degradation agent is constituted by gram+ bacteria which initiate the fermentation of starch, glucose, mannitol (mannite), rahmnose, saccharose, amygdaline, arabinose, while not initiating the fermentation of maltose, inositol and sorbitol; the bacteria areVP+, oxydase+, catalase+, nitrates, urea-, indol- and H2S- bacteria.Said degradation agent may also comprise one or more enzymes capable of splitting polysaccharides, especially starches, pentosames and other complex polysaccharides, so as to produce fermentable sugars. More particularly, according to the abovementioned patent specification, a hemicellulolytic complex of fungic origin the main activity of which is a galactomannanasic activity and which has secondary xylanase, amylase and pectylase activities, as well as an amylase may be added to the fodder with a view to obtaining the formation of the maltose necessary for the production of lactic acid by the bacteria.

French Patent specification No. 2 390 908 discloses an improvement to the process according to French specification No 2 361 828. In the latter specification the enzyme is exoamylase of fungic origin which does not completely degrade starch, whereas the amylase used in accordance with French Specification No. 2,390,908 is an amylase of bacterial origin.

This enzyme (endoamyalase) acts on the liquefied starches and produces mainly alpha-D maltose and a small amountofalpha-D glucose said amylase being active in the pH range comprised between 5 and 8.

French specification No 2 390 908 also discloses the use of amyloglucosidase which is substantially more active with respect to the amylose and amylopectin chains for producing alpha-D glucose. The enzymatic composition according to said specification also comprises a hemicellulolytic complex of fungic origin which acts on the polysaccaric constituents of the cellular membranes and the dextrins in the pH range comprised between 5.5 and 8.

Patent Application No: 34183/77 (Serial No the contents of which are incorporated herein by reference, claims priority from the French Applications from which the aforementioned French Patent Specifications derive and describes and claims processes and compositions as hereinbefore discussed.

The present invention is aimed at providing an improved process and an improved product for ensilaging vegetables, the improvement residing inter alia in the provision of a novel enzymatic composition and in the provision of a novel bacterial composition. The enzymatic composition contains in addition to the enzymes indicated hereinabove a cellulolytic or cellulase complex capable of splitting the beta links (1--3) and (1-4) of the polysaccharides which are not attacked by the prior enzymatic com position.

The above-mentioned complex is a cellulolytic complex of fungic origin which degrades the natural cellulose and the other polysaccharides contained in the cellular membranes.

This enzyme is active in the pH range comprised between 2 and 5. A synergy phenomenon exists between the cellulolytic complex and the hemicellulolytic complex, as regards the degradation of the vegetable structures.

On account of the environment of the cellulose in the vegetable its hydrolysis requires a plurality of enzymes, or one single enzyme having a plurality of functions, to wit: - hydrolysis of the amorphous zones, - swelling of the crystalline regions and disrup ture of the hydrogen links so as to produce linear' molecules, - hydrolysis of cellobiose to produce glucose, - transglucosylation of disaccharides and oligosaccharides, as well as disrupture of the irregular links of cellulose and its primary hydrolysis products.

The cellulolytic complex has been chosen on the basis of the above criteria after a great number of tests had been performed on various vegetables.

A cellulase is defined by its activity as expressed in International Units per gramme of product, on International Unit (I.U.) corresponding to the amount of enzyme which sets free, in the form of reducing sugar, the equivalent of one micromole of glucose per minute on a given cellulosic substrate.

The activity varies depending on the nature of the substrate used: - when the activity is tested on "Whatman" (Registered Trade Mark) paper powder CC31, the result is C, negative, - when the activity is tested on carboxymethylcel lulose, the result is Cx activity.

The complex used in accordance with the present invention has a very high cellulolytic activity, as well as secondary activities of the hemicellulolytic type.

The high cellulolytic power is indicated by extremely high Ct and Cx activity values, and bycellobiase (beta-glucosidase) activity.

The activity of the C1 enzymatic complex, defined in accordance with current hypotheses as being a compound having Cx activity to which an unknown factor is added, expresses in particular a power of solubilisation of cellulose.

C, is capable of degrading the native cellulose (crystalline cellulose having a polymerization rate (DP) higher than 3,500 glucose units) into amorphous cellulose.

The activity of the Cx enzymatic complex expresses in particular the reducing of power endoglucanase and exoglucanase. These enzymes mainly act on the cellulose fibres in the zones of reduced crystallinity and splitthe long glucose chains into smaller units.

The activity of beta-glucodiase expresses an action on the units obtained as a result of the action of Cx.

These different activities cannot be considered separately. There exists a synergistic interaction between C1 and Cx as far as the degradation of these homopolymers is concerned. The enzymatic mechanisms C, and Cx lead to the production of cellobiase; the latter is then submitted to hydrolysis by a beta-glucosidase, thus producing two glucose units.

Apparently the hydrolysis reactions of cellulose comprise the stages schematically indicated herein-below: C1 Native cellulose ---------------------------------------- cellulose hydrolytic Cx Reactive cellulose ------------------------------------------ cellobiose Hydrolytic beta-glucosidase Cellobiose ---------------------------------------- molecules of glucose The C, comples solubilizesthe crystalline cellulose (polymerization degree DP higher than 3,500 glucose monomers) and produces reactive cellulose (amorphous cellulose).

Under the action of the enzymatic Cx complex (endoglucanase and exoglucanase) the chains of reactive cellulose are split either inside the chain and randomly, in the case of endoglucanase, or starting from the non-reducing chain ends, in the case of exoglucanase.

These reactions, taken as a whole, produce cellobiose units.

These cellobiose units are hydrolysed by betaglucosidase to produce two glucose molecules.

In the case of fungic cellulases according to the invention the enzymatic C1 and Cx complex is capable of hydrolyzing the insoluble native cellulose, so as to produce glucose.

The cellulases according to the invention are chosen not only on the basis of the above-indicated general criteria, but also on the basis of considerations regarding the particular ensilaging medium envisaged, taking into account the po lysaccha ro lytic behaviourofthese cellulases.

This polysaccharolytic behaviour has been determined on two types of substrates: a) - pure substrates; b) - dehydrated substrates.

CASE OF THE PURE SUBSTRATES: The activity of cellulases A, B, C, D, E, and F has been determined on various substrates at a given concentration in a 10-'M M acetate buffer medium.

The activity has been measured in accordance with the conventional standards, to wit, at pH 4.8 and 50"C, after hydrolysis during 20 minutes. The reducing power has been determined by the method using hexaferrocyanide.

TABLEI

SUBSTRATE "Whatman" C.M.C. Pectin Xylane Arabino Starch Locus (concen- paper galactane bean ration) 1% 1% 0.25% 0.25% 0.10% 0.25% 0.25 ENZYMES Ulig Ullg U Ullg Ullg Ulig Ulig (activity) (C, ) (Cx) A 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 CASE OF THE DEHYDRATED SUBSTRATES: The activity of enzymes A, B, C, D, E and F, has also been determined on various dehydrated vegetable substrates.

The activity has been tested at pH values of 3.8,4.8 and 5.8 et 30"C after hydrolysis during 24 hours. The reducing power has been determined by means of the method using dinitrosalicyclic acid.

TABLE II

Tested substrates Lucerne Ray-grass Maize Beetroot Straw Tested + (Rye-grass) cellulases pH ACTIVITY (tug) 3,8 2810 2280 1560 1810 1560 A 4,8 2440 2560 2080 1530 1690 5,8 I 2560 2220 1690 1500 1530 3,8 1560 1670 ' 1530 1220 1390 B 4,8 1280 2060 1670 1220 1500 5,8 890 1680 1940 890 1380 3,8 1280 1530 1390 1110 1110 C 4,8 1280 1750 1530 1110 1290 5,8 940 1560 1170 720 1110 3,8 1390 1440 1470 1278 1167 D 4,8 1190 2060 1390 1111 1306 5,8 890 1720 1530 972 1222 3,8 611 1190 1111 556 694 E 4,8 972 1528 1250 0 694 5,8 0 1167 1278 0 694 3,8 1306 500 694 556 F 4,8 889 972 1222 610 444 5,8 750 1000 560 550 333 The analysis of these various behaviours allowed the of the cellulolytic complex to be used in accordance with the invention to be used for ensilaging vegetables:: It was thus found that the cellulase or the cellulolytic complex to be added to the fresh vegetables when carrying out the process according to the invention must correspond to a C1 activity of 50 to 0.005 l.U. and a Cx activity of 500 to 0.05 l.U. per 100 grammes vegetable, these activities being determined at pH 4.8 at a temperature of 50"C after hydrolysis for 20 minutes.

Furthermore, the selected cellulase must retain a considerable activity even after a long dwelling time in the silo.

It has been shown experimentallythat under conditions close to those prevailing when vegetables are ensilaged the cellulolytic complex must retain at least 30% of its initial activity after 10 days. TABLE 111

Preservation 0 5 10 15 | 20 time (days) pH4,8 100% 97% 61% 26% 3% pH5,8 100% 85% 58% 27% 0% The velocity, or rate, of enzymatic degradation of the vegetable structure is a function of glucose elimination; this enzymatic unbalance is obtained by using bacteria having a high fermentation power; the acidification of the medium occurs very rapidly.

When the vegetable mass has been stabilized the bacterial proliferation and the formation of lactic acid are discontinued, contrary to the cellulolytic activity of cellulase, which continues for more than 10 days.

This activity of producing and accumulating the glucose formed within the ensilaged material increases the nutritive value of the ensilaged vegetables.

The enzymatic composition used in accordance with the present invention thus comprises a cellulase or cellulolytic complex having the aboveindicated characteristics, in association with the enzymes described in French Patent specification No 2390 908, to wit, a fungic amylase, a bacterial amylase, an amyloglucosidase and a hemicellulolytic complex.

These enzymes present in the composition thus exhibit a complementary activity on account of their effect on the glucides-which may be extremely simple glucides or extremely complex ones - in pH ranges comprised between 7 and 2. The respective maximum activities of the various enzymes occur in relays as the pH decreases (which latter does not reach values lowerthan 3.5 in the silo), and within temperature intervals varying from 10 to 30"C which are compatible with the conditions prevailing in the silo.

The bacterial composition used in accordance with the present invention is characterized by the utilisation of novel bacterial strains adapted to be used when ensilaging vegetables, said strains being adapted to be used separately or in admixture with the bacterial strains andlorthe enzymes disclosed in the prior art and in the present description. This novel composition leads to considerably improved ensilaging results. The novel bacterial strains according to the invention are gram - bacteria; they belong to the Enterobacteriaceae family, the Erwinia or Pectobacterium type and the Herbiola group; they are called Enterobacter agglomerans (classification according to Bergey's Manual of Bacteriology, eighth edition).

The properties of such bacteria are listed in the appended TABLE IV.

TABLE IV EnterobacterAgglomerans Adonitol Gram - Inositol Utilisation malonate + Sorbitol Glucose + Arabinose + Phenylalamine desaminase - Maltose ONPG + Trehalose + Indole - Xylose H2S - Starch + LDC - Oxydase ODC - Gelatine + Urease - Amygdaline + Saccharose + Melibiose Argininedihydrolase - V.P. + Salicine - Rhamnose The utilisation of these novel bacterial strains, separately or in combination with other bacterial strains and/or enzymes, for ensilaging vegetables is effected in accordance with the operating conditions described in the prior publications. The bacteria may be grown on a nutritive support comprising cereal wheats. The composition to be introduced into the silo comprises a mixture of the required bacteria deposited on a soluble or insoluble support, with the enzymes defined in the present disclosure.

In a preferred embodiment of the present invention, the enzymes and the bacteria are placed on a cereal support (corn, barley, germinated barley), preferably in a finely ground condition.

In another preferred embodiment of the invention the bacteria and the enzymes are placed onto a support of a type constituted by pre-gelated starch, maltohexose, etc..., which support is highly soluble in cold water.

The starch and other polysaccharides contained in the supports combine with the glucides of the vegetables and thus increase the nutritive value of the ensilaged matter. The composition comprising the mixture of bacteria on their support with the enzymatic complex, deposited or not on a cereal support, may include various proportions of each of one of these constituents. The dry mixture comprises an amount of about 100,000 to 1 million cocci and 100,000 to 1 million bacilli pergramme.

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

The amount of cellulase to be added to the ensilaged fresh vegetables must correspond to a Cl activity of 50 to 0.005 I.U. per 100 g. vegetable; it must thus be equal to 2 to 2.10-4#by weight of fresh vegetables when a cellulase having a 250 I.U./gramme titre is used.The proportions of the other enzymes are comprised between 0.05 and 0.2/ bacterial amylases having a 250 units/gramme titre (by weight of vegetables), 0.10 to 0.20%o fungic amylases having a 50,000 units AG/gramme titre (by weight of vegetables), 0.10 to 0.70 /Oo amylglucosidase having a 200 units AG/gramme titre (by weight of vegetables), 0.05 to 0.20%o hemicellulolytic complex having a 35,000 I.U./gramme titre (by weight of vegetables).

When the enzymes are deposited on a cereal support in accordance with a preferred embodiment of the present invention, the product to be incorporated in the ensilaged matter may contain about 1 kg enzymes per 9 kg support. When a soluble support is used, the ratios of enzyme concentration to support vary and may be comprised between 1/1.5 and 1/4.

Due to this incorporation of cellulase in the support, the amount of available fermentable sugars is increased, whereby a larger and more rapid production of lactic acid is obtained. The vegetable protein wil thus be more efficiently preserved. Furthermore, the nutritive value of the medium will be increased.

With a view to showing the advantages brought about by using a cellulolytic complex and bacteria of the Enterobacteriaceae family, tests were performed in micro-silos and mini-silos. The various preservation parameters as well as the vegetable structure parameters were studied.

1. TESTS PERFORMED IN MICRO-SILOS A first laboratory test series was performed in micro-silos. These tests were carried out in May on lucerne (alfafa). The micro-silos had a capacity of 1 kg. The lucerne was finely chopped and then ensil aged in accordance with the following operating mode: A- One series of micro-silos were treated by means of a preserving additive having the following general formula: cereal support 90% pre-mixture 10%, the pre-mixture being constituted by: - a bacterial complex (5 lactic bacteria on lactose support): - an enzymatic complex comprising:: an amylase of fungic origin; an amylase of bacterial origin; an amyloglucosidase; a a hemicellulolytic-complex of fungic origin, the main activity of which was a galactomannasic activity; - a high-energy support: finely ground barley + lactoserum, the bacterial and enzymatic comp lexes representing each 1% of the pre-mixture composition.

The preservation additive was admixed with the lucerne in a proportion of 2% (the rate of incorporated pre-mixture thus being 0.2%), and 1% barley was added.

B - A series of micro-silos were treated with the same preservation additive as that used according to A, however, a cellulase of fungic origin (stemming from Trichoderma viride) was added, said cellulase presenting a C, activity of 250 units/gramme and a Cx activity of 2,500 units/gramme, as determined at pH.

4.8 at a temperature of 50 C, for a reaction time of 20 minutes, the proportion of incorporation of this cellulase being 2 g/T (i.e.2.10-4% by weight) as referred to the fresh vegetables (Test B) and 200 g/T (i.e.

0.02%) by weight as referred to the fresh vegetables (Test B').

C - A series of micro-silo reference tests: - a series of micro-silo tests with lucerne contain ing no preservation product : T = 0.

After 100 days duration of ensilaging the microsilos were opened, the following parameters were recorded: pH, lactic acid, NH$N (total). The results are listed in TABLE V overleaf.

It can be seen that the admixing of a cellulase to a bacteria + enzyme complex according to patent specification no 2 390 908 leads to an improvement of the performances of the preservation additive. A more satisfactory preservation of the vegetables is obtained, as illustrated by the comparison of A and B', as follows: - a decrease of the pH value from 4.13 to 3.73 (decrease of 10%); TABLE V

Lactic acid pH Total NH3/N (g/kg MS) Référence: T = 0 5.66 26g 25.15 Premixed lucerne 4.13 143.68 16.33 + 0,2%(A).

. Premixed lucerne + 0,2% + cellulase of fungic 4.32 144.75 16.20 origin 2 g/T (B)- Premixed luceme + 0,2% + cellulase of fungic 3.73 169.47 15.02 origin 200 g/T (B') - a 15% decrease of the lactic acid production; - a 8% decrease of the total NHJN ratio; this shows an improvement of the preser vation of the vegetable protein which results from the rapid acidification of the medium.

2. TESTS PERFORMED IN MINI-SILOS These tests were carried out in June with lucerne in mini-silos having a capacity of 150 kg.

The lucerne was finely chopped, then ensilaged in accordance with the operation conditions indicated herein-above.

A. -A series of mini-silos was treated by means of the same preservation additive as that used in the micro-silo tests described under A, the proportion of incorporation being 0.2% pre-mixture.

B. -A series of mini-silos was treated in accor dance with the formula and the dose indicated under A, to which the same cellulase of fungic origin was added in a proportion of 2 grammes/T.

After 100 days ensilaging the mini-silos were opened, the analysis of the ensilaged materials gave the results shown in TABLE VI overleaf.

The results listed listed herein-above clearly show the advantages of adding a cellulase to the bacterial + enzyme complex described in Patent specification no 2 390 908.

This addition results in: - a decreased pH :3.94 as compared to 4.20 (less 6%); TABLE Vl

A B silos 0.2% premixture | silos 0.2% premixture + cellulase (2 gfT) . Dry matter (MS)% ....... 23.67 27.27 . E.N.A. (g/kg MS) ....... 51.25 48.97 . pH ....... 4.20 3.94 - Lactic acid (glkg MS) ....... | 80.62 | 114.40 . Acetic acid (g/kg MS) ....... 17.42 20.04 . Total NH3/N ....... 18.40 13.31 . Total N (g/kg MS) ....... 21.70 23.12 . Ammoniacal N(g/kg MS) ....... 3.87 3.09 - an increased proportion of lactic acid (plus 29.53%); - a more satisfactory preservation of the proteinic part of the ensilaged matter, when a cellulase is added; this is confirmed by analysis.

The following results are obtained in this case: - a decreased total NH/N ratio (less 27.6%); - an increased proteic rate (plus 6%); - a decreased ammoniacal nitrogen rate (less 20.1%).

This increased acidification velocity may be explained by the fact that the bacteria are provided more rapidly with an important amount of fermentable sugars which are easily converted into lactic acid.

The effect of the cellulase introduced into the medium can be seen here again.

In the examples given herein-below, the mixture applied on the support in accordance with the invention was constituted by the complex described in French Patent Specification no 2 361 828 and/or in French Patent Specification no 2 390 908, the enzymatic complex disclosed in the present description, to which gram - bacilli of the Enterobacteriaceae family, of the Erwinia or Pectobacterium type, Herbicola group, were added, the proportion of incorporation of these bacilli in the mixture corresponding to 105 to 106 bacilli per gramme EXAMPLES Tests were carried out in silos having a capacity of 4 m3 with Dactile Lucifer harvested in June, at the starting stage of heading.The finely chopped fodder was stocked under four different conditions: A. ensilaging without treatment; B. ensilaging with 4 litres formic acid/ton in accordance with known methods; C. ensilaging with the pre-mixture, the composi tionofwhich is described in French Patent specifications no 2 361 828 and no 2 390 908: ensilaging the matterto be treated with an addition of 7 kg of the strain of 7 bacteria on support, the properties of which are indicated in French Patent Specification no 2 361 828 page 7, no 1 to 7 and enzymes; proportion of incorporation: 10 kg/ton; D. ensilaging with the pre-mixture according to C, which bacilli according to the invention were added, to wit: gram - bacteria, bacteria of the Enterobacteriaceae family, Erwinia or Pec tobacterium type, Herbicola group, named Enterobacter agglomerans.The proportion of the incorporation of the complex was 10 kg/T.

After 90 to 100 days the different silos were opened, and the vegetables were analysed. The results thus obtained were listed herein-after in TABLE VII.

The results show that the mixture according to the French Patent specifications no 2361 828 and no 2 390 906 led already to a considerable improvement, as regards the preservation of the ensilaged matter, which is illustrated by: TABLE Vll

Parameters N-Nk3 N soluble Organic acids (g/kg M.S.) pH o/ Total N Total N lactic propionic acetic butyric Treatments A: not treated. 5.28 17.76 63.80 23 1.38 29.46 ' traces B : treatment with 4.05 7.10 45.43 41.36 traces 14.95 0 formic acid 4%.

C : treatment according 4.02 14 48.10 90 0 14.20 0 French Patent 2.390.908.

D: novel treatment 3.70 8 44 112 0 18.28 0 (C=bactericid straties according to the invention).

- a decrease of pH value, which was lowered from 5.28 to 4.02; - an increased lactic acid content of 90 g/kg MS as compared to 23 g/kg MS; - an improved proteinic protection which is illus N-NH3 trated by decrease of the ---- ratio dropping total N soluble N from 17.76 to 14 and of the ratio dropping total N from 63.80 to 48.10.

This mixture had the advantage of leading to no formation of propionic and butyric acid.

The mixture according to the present invention resulted in a supplementary improvement which is illustrated by: - a decrease of the pH value (dropping from 4.02 to 3.70); an increased lactic acid content (112 g/kg MS as compared to 90 g/kg MS); - an improved proteic protection which is illus trated by a decrease of the N-NH3/total N ratio (dropping from 14 to 8 and of the soluble N/total N ratio (dropping from 48.10 to 44).

One advantage of this mixture resides in the fact that it leads to no fermentation of propionic and butyric acid.

The digestibility and ingestibility of these ensilaged matters were measured on mutton; after the various treatments the following results were found: B : treatment with formic acid : 0.64 UF/kg; C : treatment according to French Patent specifica tion no 2 361 828 and no 2 390 908: 0.74 UF/kg; D : treatment according to the present invention 0.82 UF/kg.

The nitrogen balance on animals in the growth period produced results which showed the advantages brought about by the treatment with ensilaging products according to the present invention, as illustrated in TABLE VIII.

The nitrogen retention obtained when applying the method according to the present invention is more than twice as large as that obtained by treatment with formic acid.

The invention is not limited by the foregoing description and the various embodiments illustrated therein. Various modifications may be envisaged by those skilled in the art without departing from the spirit and the scope of the invention as defined by the appended claims.

The EnterobacterAgglomerans bacteria employed in embodiments of the present invention has been lodged at the C.N.C.M., Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, Cedex 15, France on 11th February 1980, under accession number 1-114. TABLE Vlil

Parameters Ingested Fecal N Urinary N Retained N N Ingested Ingested Ingested Treatments < g/j 9/i % N g/j % N g/j % N A: nottreated. 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 + 17 7.07 41.6 8.12 47.7 1.8 10.50 1 st Addition.

D : treatment according to invention. 16.5 5.4 38.8 7.70 47 2.40 14.50

Claims (2)

1. A process of preserving and valorizing fresh vegetable matter by lactic acidification, comprising admixing to said vegetable matter prior to ensilaging the same, bacteria capable of initiating lactic fermentation, and adding degradation agent which is adapted to degrade higher glucides into fermentable glucides for use by the bacteria initiating said lactic fermentation, and which is constituted by enzymes and/or bacteria, wherein said enzymes, if present, comprise, optionally in combination with a fungic amylase, with an amyloglucosidase, or with a hemicellulolytic complex of fungic origin having primarily a galactomannanasic activity, a cellulase or cellulolytic complex of fungic origin capable of degrading the native cellulose into glucose and having a C, activity of 50 to 0.005. International Units and a Cx activity of 500 to 0.05 International Units per 100 grammes fresh vegetables and retaining under the prevailing ensilaging conditions at least 30% of said activity after 10 days, and said bacteria, if present, being gram-bacteria which initiate starch fermentation but do not initiate maltose fermentation and belonging to the family of Enterobacteriaceae, type Erwinia or Pectobacterium, group Herbicola, named Enterobacter agglomerans.

1. A process of preserving and valorizing fresh vegetable matter by lactic acidification, comprising admixing to said vegetable matter prior to ensilaging the same, bacteria capable of initiating lactic fermentation, and adding degradation agent which is adapted to degrade higher glucides into fermentable glucides for use by the bacteria initiating said lactic fermentation, and which is constituted by enzymes and/or bacteria, wherein said enzymes if present, comprise in combination with a fungic amylase, an amyloglucosidase, a hemicellulolytic complex of fungic origin having primarily a galactomannanasic activity, a native glucose cellulose having a C1 activity of 50 to 0.005 International Units and a Cx activity of 500 to 0.05 International Units per 100 grammes fresh vegetables and retaining under the prevailing ensilaging conditions at least 30% of said activity after 10 days, and said bacteria, if present, being gram-bacteria which initiate starch fermentation but do not initiate maltose fermentation, and belonging to the family of Enterobacteriaceae, type Erwinia or Pectobacterium, group Herbicola, named Enterobacter agglomerans.

2. A process according to Claim 1, wherein the bacteria belonging to the family of Enterobacteriaceae are used in association with other bacteria capable of degrading higher glucides into fermentable glucides.

3. A process according to claim 2, wherein said other bacteria are selected from the bacteria capable of degrading higher carbohydrates which are disclosed in Patent Application No: 34183/77 (Serial No..

..............).

4. A process according to claim 1 or 2, wherein the bacteria belonging to the family of Enterobacteriaceae are used in association with gram + bacteria which ferment starch, glucose, mannitol, rahmnose, saccharose, amygdaline and arabinose and do not ferment maltose, inositol and sorbitol.

5. A process according to any preceding claim, wherein the enzymes are present as a mixture cons tituted by: -

2.10-4 to 20/or cellulose having a titre of 250 l.U.

C1/g, - 0.05 to 0.2/oo bacterial amylase having a titre of 250 units/g, - 0.10 to 0.2/oo fungic amylase having a titre of 5,000 units P.S. 50/9, - 0.10 to 0.70'/or amyloglucosidase having a titre of 200 unitsAG/g, - 0.05 to 0.200/, hemicellulolytic complex having a titre of 35,000 l.U./g, the percentage values being expressed by weight with reference to the ensilaged vegetables.

6. A process according to any one of the preceding claims, wherein the enzymes are present and are incorporated on a cereal support in a proportion of about 1 kg enzymes per 9 kg support.

7. A process according to any one of the preceding claims, wherein the bacteria are present and are cultivated on a support comprising cereal wheats.

8. A process according to any one of claims 1 to 5, wherein the bacteria and the enzymes are present and are incorporated on a support of the cereal starch type, pregelated starch type, malto-hexose type, which is soluble in cold water, in a proportion of 1 kg bacteria and enzymes per 1.5 to 4 kg support.

9. A process according to one of the preceding claims wherein the amount of enzymes and bacterial culture with their support, in the dry state, added per ton of vegetables to be ensilated is 10 to 15 kg, this additive containing 105 to 10off live germs per gramme.

10. A composition for preserving and valorizing fresh vegetables which comprises lactic acidproducing bacteria strains and a degradation agent capable of degrading higher glucides into fermentable glucides constituted by an enzymes complex and/or bacteria, wherein the enzymes if present comprise, possibly in association with fungic amylase, amyloglucosidase, a hemicellulolytic complex of fungic origin having primarily a galactomannanasic activity, a cellulase orcellulolytic complex of fungic origin, capable of degrading the native cellulose into glucose and having a C1 activity of 50 to 0.005 International Units and a Cx activity of 500 to 0.05 International Units for 100 grammes of fresh vegetables and retained under the prevailing ensilaging conditions at least 30% of said activity after 10 days, said bacteria if present, being grambacteria which initiate starch fermentation but do not ferment maltose, belonging to the family of Enterobacteriaceae, type Erwinia or Pectobacterium, group Herbicola, named EnterobacterAgglomerans.

11. A composition according to claim 9, wherein the Enterobacteriaceae gram-bacteria are present and are associated with bacteria capable of degrading the higher glucides into fermentable sugars, and especially gram + bacteria which ferment the starch, glucose, mannitol, rahmnose saccharose, and amygdaline, arabinose, and do not ferment maltose, inositol and sorbitol.

12. A process of preserving and valorizing fresh vegetable matter by lactic acidification, as claimed in claim 1 and substantially as hereinbefore described, with reference to any one of runs B and B' of Test 1, run B of Test 2, and run D of the Examples.

13. A composition for preserving valorizing fresh vegetable matter, as claimed in claim 10 and substantially as hereinbefore described with reference to any one of runs B and B' of Test 1, run B of Test 2 and run D of the Examples.

14. Vegetable matter when treated buy a process according to any of claims 1 to 9 and 12 or by means of a composition, according to any of claims 10,11 and 13.

15. An animal fed upon vegetable matter as claimed in claim 14.

16. The features hereinbefore disclosed, or their equivalents, in any novel selection.

New claims or amendments to claims filed on 28 July 1980.

Superseded claims 1.