WO2009016482A2

WO2009016482A2 - Method for the treatment of vegetal matter

Info

Publication number: WO2009016482A2
Application number: PCT/IB2008/001996
Authority: WO
Inventors: Leonardo Setti; Dario Zanichelli; Alessandro Filippini; Francesco Carloni
Original assignee: Alma Mater Studiorum - Universita' Di Bologna
Priority date: 2007-07-31
Filing date: 2008-07-31
Publication date: 2009-02-05
Also published as: ITBO20070542A1; WO2009016482A3

Abstract

Method for the treatment of vegetal matter, according to which the vegetal matters undergo to an hydrolysis induced by endo-enzymes and another following hydrolysis induced by exo-enzymes; systems of micro and ultra- filtrations characterized by specific recirculation mechanisms are provided.

Description

METHOD FOR THE TREATMENT OF VEGETAL MATTER

TECHNICAL FIELD The present invention relates to a method for the treatment of vegetal matter. BACKGROUND ART

In the cosmetic and food sectors, the need of new functional products able to promote human health and to confer "natural" hedonistic properties is increasing. The so called "fresh cosmetics", those that are presented alike a fresh food product (with the denomination of origin as well the expiring date) , are extremely topical as well as the so called "nutraceuticals products" , that have both nutritional and pharmaceutical properties such as yogurts, bakery products, juices and snack bars having anti- cholesterolemic, antioxidant, anti-age and so on properties, are gaining an interesting share of the market .

These functional products are technically known as "fortified" foods or cosmetics because of they are obtained by adding to commercial products

"functionalizing" additives such as vitamins (A, B, E), fibers, antioxidants (i.e. Co-enzyme Q, biophenols, carotenoids) , Pre/Pro biotics

(Bifidobacteria, inulin) . The "functionalizing" additives can be obtained via chemical synthesis or fermentation, otherwise can be directly extracted from natural sources throughout refining processes that are generally long, expensive and with an high environmental impact.

The majority of the "functionalizing" additives are phytocomponents i.e. chemical compounds, such as phytosterols or biophenols, exclusively present in vegetal matter. Known are sitosterols used as anti- cholesterolemics in yoghurt and bread, or biophenols, obtained from green tea, grape and olives very diffused in cosmetics, or also γ-oryzanol present in rice oil.

These Phytocomponents are obtained by more or less complicated extractive processes from vegetal matters (leaves, skins, seeds, etc.) that can be easily treated with water as well as more complicated hydro-alcoholic solution, organic solvents, more expensive steam extractions (such as in the production of flavors and essential oils) or CO₂ in supercritical phase (such as in the production of caffeine from coffee powder) . Moreover, technologies for the recovery of phytocomponents from aqueous systems based on sorbent resins are known, on which resins the components are firstly adsorbed and then recovered by de-adsorption using hydro-alcoholic or organic solvents . The described processes yield to products usually containing low amounts of phytocomponents and quite often with high content of impurity due to the solvents used. The yield of extraction of phytocomponents is mainly impaired by the fact that these molecules are often covalently linked to the solid fractions of the vegetal matter.

Regarding applications of vegetal matter in these fields, it should be stressed that the only relevant example is wheat bran, one of the by-products of cereal transformation, used as ingredient in bakery

"whole" products .

To overcome these downsides it was recently proposed to homogenize the vegetal matter (such as carrots, apple, etc.) in order to produce purees. However, with this technology the insoluble biomass is suspended and the phytocomponents remain prevalently linked to the macromolecular structures present in the suspension, still being not bio- available .

It was also proposed to disassembly the vegetal matter in their main fractions such as starch, celluloses, proteins, fat and phyto-components, and recover then the fractions of interest throughout separation or extraction techniques with solvents

(glycols, alcohols, etc.). In this way only a small - A -

part of the non covalently bound to the macromolecules phyto-components are recovered. DISCLOSURE OF INVENTION

It is an object of the present invention to provide a method for the treatment of vegetal matter designed to at least partly eliminate the drawbacks of the known art, and which, at the same time, is cheap and easy to implement .

According to the present invention, there are provided a method for the treatment of vegetal matter and organic molecules as claimed in the accompanying independent claims, and, preferentially, in any one of the claims depending directly or indirectly from the independent claims. Unless explicitly specified the opposite, in the present document the following terms have the hereinafter reported meanings .

Vegetal matter indicates organic matter of vegetal origin, in particular comprising mono and/or poly-saccharides . Vegetal matter can comprise untreated vegetal matter (cereals, beet, sugar cane etc.), or treated matter of vegetal origin (agro-food by-products such as, vinasse, olive oil milling waters, tomato skins, cereal bran, etc.) . Advantageously, the vegetal matter comprises, in particular consists of cereal transformation byproducts in particular wheat bran. Endo-lysis indicates a lysis (particularly an hydrolysis) occurring in a intermediate position of a molecule's backbone. Advantageously an endo-lysis can be obtained using endo-enzymes having at least one of the following activities: amylase, cellulase, protease, pectinase, xylanase .

Exo-lysis indicates a lysis (particularly an hydrolysis) occurring in a extremity position of a molecule's backbone. Advantageously, an exo-lysis can be obtained using exo-enzymes having at least one of the following activities: glucosidase, arylesterase, maltase, cellobiases.

Enzymatic preparation indicates a solution comprising one or more enzymes identical or different among themselves. According to some advantageous embodiments, the enzymatic preparation can comprise several different enzymes.

According to further advantageous embodiments, the enzymatic preparation can comprise a single enzyme .

In the present document the porosity of a membrane is defined as the maximum dimension of the particle that can pass through the membrane itself .

BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, in which: figures 1-4 schematically show some embodiments of a plant designed to implement a method according to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

According to a first aspect of the present invention, a method for treating a vegetal

(vegetable) matter comprising organic molecules is provided; the method comprises the following steps: a first lysis step, during which first lysis reactions are enzymatically induced and occur on at least part of the organic molecules of vegetal matter; a first separation step, which follows the first lysis step and during which at least part of the organic molecules of larger dimensions are separated from the vegetal matter,- a second lysis step following the first separation step during which second lysis reactions, different from the first lysis reactions, are enzymatically induced and occur on at least part of the organic molecules of the vegetal matter.

It is important to point out that the first and the second lysis reactions are induced using different enzymatic preparations; performing the first and the second lysis steps separately the mutual inhibition of the different enzymatic preparations can be avoided. Moreover, the separation of the organic molecules of larger dimensions before the second lysis step reduces the risk that smaller organic molecules inhibits the enzymes during the first lysis step. These two aspects in combinations permit to obtain a surprisingly efficient molecular disgregation of the organic matter. In particular, the first and the second lysis reactions are hydrolysis reactions occurring in presence of water.

According to some embodiments, the first lysis reactions are endo-lysis reactions. Advantageously the endo-lysis reactions are induced with a first enzymatic preparation having at least one enzymatic activity chosen in the group consisting of: amylase, cellulase, protease, pectinase, xylanase . According to some advantageous embodiments, the first enzymatic preparation has at least three (or at least two or at least four) enzymatic activities chosen in the group consisting of: amylase, cellulase, protease, pectinase, xylanase. Advantageously, the first enzymatic preparation has the following enzymatic activities: amylase, cellulase, protease, pectinase, xylanase .

According to advantageous embodiments, the first enzymatic preparation has an amilase activity between 340 and 2300 KU/L. According to advantageous implementation the first enzymatic preparation has a xylanase activity between 40 and 280 KU/L. According to advantageous implementation the first enzymatic preparation has a pectinase activity between 10 and 700 KU/L.

According to advantageous implementation the first enzymatic preparation has a cellulase activity between 1,2 and 8,4 U/L.

According to advantageous implementation the first enzymatic preparation has a protease 's activity between 1 and 3000 KU/L. Advantageously, the first lysis step is performed stirring the vegetal matter in presence of the first enzymatic preparation for from 2 to 20 hours, preferably at a temperature from 10 to 90 ⁰C.

It should be noted that, during the first separation step, the first enzymatic preparation is separated from the vegetal matter that undergoes to the second lysis step.

According to some embodiments, the second lysis reactions are exo- lysis reactions. Advantageously, the exo-lysis reaction are induced by an enzymatic preparation having at least one enzymatic activity chosen in the group consisting of: glucosidase, arylesterase, maltase, cellobiases. According to some advantageous embodiments, the second enzymatic preparation has at least two (or at least three) of the enzymatic activities chosen in the group consisting of: glucosidase, arylesterase, maltase, cellobiase. Advantageously, the second enzymatic preparation has the following enzymatic activities: glucosidase, arylesterase, maltase, cellobiase.

According to advantageous embodiments, the second enzymatic preparation has a glucosidase activity between 90 and 630 KU/L.

According to advantageous embodiments, the second enzymatic preparation has a arylesterase activity between 460 and 3220 KU/L.

Advantageously, the second step of lysis is performed stirring the vegetal matter in presence of the second enzymatic preparation for from 2 to 20 hours, preferably at a temperature from 10 to 90 ⁰C.

Advantageously, the first separation step provides for the mechanical filtration of the vegetal matter. According to some embodiments, the vegetal matter is filtrated using a first membrane having a porosity lower than 6 μm.

According to some embodiments, the first membrane has a porosity from 2 to 5 μm. According to some embodiments, the first membrane has a porosity lower than 110 KDa (advantageously, from 110 KDa to 80 KDa, in particular of approximately 100 KDa) . Advantageously, the first membrane has a porosity lower than 11 KDa (advantageously, from 11 KDa to 8 KDa, in particular of approximately 10 KDa) .

According to' some embodiments, during the first separation step, an overpressure of at least 2 bar, preferably from 3 to 4 bar, in particular of 3.5 bar, is applied on the vegetal matter upstream from the first membrane. Advantageously, a negative pressure (suction) (in particular a pressure lower than 1 bar) is applied to the vegetal matter permeated through the first membrane.

According to some embodiments, the first separation step comprises a micro- filtration step, during which the vegetal matter is filtrated through the first membrane having a porosity lower than 6 μm (advantageously, from 2 to 5 μm) ; and a first ultrafiltration step, that follows the micro-filtration step, during which the vegetal matter is filtered through a second membrane having a porosity lower than 110 KDa (advantageously, from 110 KDa to 80 KDa, in particular of approximately 100 KDa) . According to some embodiments, the first separation step comprises, in addition or as an alterative to the first ultra- filtration step, a second ultra- filtration step, which is, preferably, subsequent to the first ultra-filtration step and/or to the micro- filtration step and during which the vegetal matter is filtrated through a membrane having a porosity lower than 11 KDa (advantageously, from 11 KDa to 8 KDa, in particular of approximately 10 KDa) .

According to some embodiments, the above- described method comprises a second separation step, which follows the second lysis step and during which at least part of further organic molecules of larger dimensions are separated from the vegetal matter. Advantageously, during the second separation step the vegetal matter is filtered through a third membrane having a porosity lower than the first ad/or the second membrane .

According to some embodiments, the third membrane has a porosity lower than 11 KDa (advantageously from 11 KDa to 8 KDa, in particular of approximately 10 KDa) .

According to some embodiments, the third membrane has a porosity lower than 1.1 KDa (advantageously from 1.1 KDa to 0.8 KDa, in particular of approximately 1 KDa) . According to some embodiments, during the second separation step, an overpressure of at least 2 bar, preferably from 3 to 4 bar, in particular of 3.5 bar, is applied on the vegetal matter upstream from the third membrane. Advantageously, a negative pressure (suction) (in particular a pressure lower than 1 bar) is applied to the vegetal matter permeated through the third membrane .

According to some embodiments, further lysis steps and/or separation steps are provided. According to some embodiments, the above- mentioned method comprises a concentration step, which follows the second separation step and during which a liquid component, in particular water, is separated from a treated vegetal matter (that is to say, substantially a molecular disassembly of the vegetal matter subjected to the first lysis step) , in particular by the means of reverse osmosis. The so obtained molecular disassembly can be, for example, used to enrich foods.

According to some embodiments, the above- disclosed method comprises a third separation step, which follows the second separation step and during which the components of the vegetal matter are separated from each other. In this way it is possible, for instance, to obtain sugars, amino- acids, fatty acids, glycerol and/or phytocomponents .

According to some embodiments, the above disclosed method comprises a step of fuel generation, which follows the second separation step and during which the vegetal matter is at least partially transformed in fuel with a microbial process.

According to some advantageous embodiments, the above-mentioned method comprises a step of thermal treatment, during which the vegetal matter is mixed with 3 to 12 folds by weight of water (in particular deionised water) and stirred for 30 to 120 minutes at a temperature between 60⁰C and 90⁰C, the step of thermal treatment is precedent to the first lysis step. Advantageously, the thermal treatment step is performed in presence of preservatives; in particular m-parahydroxybenzoate 0.01-1 % (weight on volume) and/or citric acid 1-5% (weight on volume) and/or lactic acid 1-6% (weight on volume) and/or EDTA 0.1-3 % (weight on volume) .

According to advantageous embodiments, before the thermal treatment step, a mechanical treatment step is provided, during which the vegetal matter is processed in order to get particles having small dimension.

Referring to figure 1, 1 indicates the overall plant to implement some embodiments of the above- disclosed method.

The plant 1 comprises a device 2 for feeding the vegetal matter to an homogenizer 3, in the area of which the vegetal matter undergoes a thermal and mechanical treatment; and a reactor 4, that is placed downstream from and connected to the homogenizer 3.

Plant 1 comprises, moreover, a reactor 5, which is placed downstream from and connected to reactor 4 through a separation unit 6; and a recirculation unit 7 for conveying the organic molecules of larger dimension separated from the vegetal matter in the area of unit 6 to reactor 4.

A separation unit 8 is located downstream from reactor 5, which separation unit 8 is connected to a device 9 for storing and/or conveying the treated vegetal matter, and to a recirculation unit 10 for conveying the further organic molecules of larger dimension separated from the vegetal matter in the area of unit 8 to reactor 5 are placed.

In figure 2 a plant 1' is shown, which plant 1' differs from plant 1 of figure 1 in that it comprises an osmosis device 11, which is placed downstream from device 9 and it is designed to separate the treated vegetal matter (that is to say, substantially a molecular disassembled of the vegetal matter fed by device 2) from the solvent (specifically, water) by means of reverse osmosis.

Moreover, plant 1' includes a recirculation unit 12 which is designed to convey the solvent separated by the reverse osmosis unit 11 to the homogenizer 3. In figure 3, a plant 1'' is depicted, which plant 1' ' differs from plant 1 of figure 1 in that it comprises a separation device 13 (known per se) (for example, comprising or consisting of a chromatographic column) , it is placed downstream from device 9 and it is designed, in particular, to separate sugars, aminoacids, fatty acids, glycerol and/or phyto-compounds from the vegetal matter.

In figure 4, a plant 1''' is depicted, which plant 1' ' ' differs from plant 1 of figure 1 in that it comprises a device 14, which is placed downstream of device 9 and it is designed to produce biofuels with per se known techniques (specifically, fermentation) . The plant 1' ' ' comprises, additionally, a unit 15 for the production of electricity through a biofuel cell (a biofuel cell of microbial or enzymatic kind) which is fed by device 9. The use of the vegetal matter of device 9 to feed a bio-fuel cell is particularly advantageous because this vegetal matter is substantially sterile.

According to the not shown embodiments, plant 1 comprises a device similar to device 14 placed downstream from a device analogous to device 13.

According to some embodiments, in the method above-described, the first lysis and the second lysis steps occur in reactor 4 and in reactor 5, respectively, which are connected to each other by the separation unit 6; the method comprises a first recirculation step, during which the larger dimension organic molecules are separated from the vegetal matter during the first separation step in the separation unit 6 and conveyed again to the first reactor 4.

Advantageously, the method comprises a second separation step, which occurs in the separation unit 8 placed downstream from the second reactor 5 and during which at least part of further larger dimension organic molecules are separated from the vegetal matter; and a second recirculation step, during which the further larger dimension organic molecules, separated from the vegetal matter during the second separation step, are conveyed again to the second reactor 5.

According with another aspect of the present invention, organic molecules, obtained with a method as above defined, are provided.

It is important to stress that the method according with the present invention allows to get achieve at least the following advantages in comparison with the state of the art. - The use of agro-industrial by-products as a low cost starting material for the production of functional beverages and/or "functionalizing" additives. During wine making industry, olive oil production, milling industry and canning factory processes are also obtained by-products such as skins, brans and vegetation waters that often do not have market and need to be detoxified and treated. It is possible, for example, to produce low cost "antioxidant" juices, additives for food and cosmetic industries, active principles for the detergence industry.

- The use of agro-industrial by-products as a low cost starting material for the production of high added value compounds . - The use agro-industrial by-products as a low cost starting material for the production of energy and/or biofuels . The method according with the present invention permits a potentially complete elimination of byproducts straightening out on one side the industrial problem of the treatment costs and on the other side generating an economical value.

Other characteristics of the present invention will emerge from the following description of a purely illustrative and not limiting example.

Example 1 Step 1 thermal pre-treatment

Wheat bran, loaded in a stirred tank, is added with an amount of deionized water equal to 8 times the amount of the treated bran by weight, stirred with a propeller stirrer or other appropriate stirring system, then added with appropriate preservatives (m-parahydroxybenzoate 0.6% w/v and/or citric acid 3 % w/v and/or lactic acid 3% w/v and/or EDTA 1.5% w/v). The suspension is thermally treated at a temperature of 75°C for 75 minutes. Step 2 hydrolysis pre-treatment with endo-enzymes The suspension is added with selected enzymatic preparations having the following activities: •amylase 1300KU/L approximately; •xylanase 140 KU/L approximately; *pectinase 400 KU/L approximately;

•cellulase 5.2 U/L approximately; •protease 1500 KU/L approximately; The suspension is stirred for 11 hours at 50⁰C. Step 3 micro-filtration step at 2-5 μm

The liquid is cooled down at room temperature and undergoes a micro- filtration process through a tubular ceramic membrane having a porosity of 3.5 μm, channel diameter 7 mm, length 250 mm, surface area 50m².

Flow rate: 1000 L/hour

Incoming pressure : 2.5 bar

Outcoming pressure: 2.3 bar Permeate pressure: < 1 bar

Flow rate on the membrane : 7 m/sec

The starting volume is 3 L that yields 0.47 L of concentrate and 2.5 L of microfiltered permeate.

The concentrate is sent again to step 1 and mixed with 2.21 L of new suspension.

Step 3 ultra-filtration at 100 KDa

2.5 L of permeate undergoes an ultra-filtration process through a tubular ceramic membrane having a porosity of 100 KDa, channel diameter 7 mm, length 250 mm, surface area 50m².

Flow rate: 1000 L/hour

Incoming pressure : 3.5 bar

Outcoming pressure: 3.3 bar

Permeate pressure: < 1 bar Flow rate on the membrane: 2 m/sec

This step yields 0.32 L of concentrate, that is sent again to step 1 to replace the 3 L that have to be micro-filtrated again, and 2.17 L of permeate. Step 4 hydrolysis treatment with endo-enzymes and exo-enzymes the 2.17 L of permeate undergoes to an enzymatic hydrolysis treatment with selected enzymatic preparations having the following activities: •amilase 1300KU/L approximately; •xylanase 160 KU/L approximately; •pectinase 400 KU/L approximately; •cellulase 4.8 U/L approximately; "protease 1500 KU/L approximately;

•aryl-esterase 1900 KU/L approximately; •clucosidase 380 U/L approximately; The liquid is stirred for 11 hours at 50⁰C. Step 5 ultra-filtration at 10 KDa 2.17 L of hydrolyzed product undergoes to an ultra-filtration process through a tubular ceramic membrane having a porosity of 10 KDa, channel diameter 7 mm, length 250 mm, surface area 50m².

Flow rate: 1000 L/hour Incoming pressure: 3.5 bar Outcoming pressure : 3.3 bar Permeate pressure : < 1 bar Flow rate on the membrane: 2 m/sec This step yields 0.28 L of concentrate, that is sent again to step 4 to undergo again the hydrolisis step, and 1.89L of permeate.

Step 6 hydrolysis treatment with exo-enzymes The 1.89L of permeate undergoes an enzymatic hydrolysis treatment with selected enzymatic preparations having the following activities:

•aryl-esterase 1900 KU/L approximately; «clucosidase 335 U/L approximately;

The liquid is stirred for 11 hours at 50⁰C. Step 7 ultra-filtration at 1 KDa

1.89 L of hydrolyzed product undergoes an ultrafiltration process through a tubular ceramic membrane having a porosity of 1 KDa, channel diameter 7 mm, length 250 mm, surface area 50m². Flow rate: 1000 L/hour Incoming pressure: 3.5 bar Outcoming pressure : 3.3 bar Permeate pressure: < 1 bar

Flow rate on the membrane: 2 m/sec This step yields 0.24 L of concentrate, that is sent again to step 6 to undergo again to the hydrolisis and ultra-filtration step, and 1.65 L of permeated matter.

This permeated matter is therefore enriched with chemical compounds with a weight lower than 1000 g/mole . These molecular weights are in the size range of the oligomers that constitute the primary fibrous structures of vegetal matter of the starting byproduct .

For this reason this product can be considered a real molecular disgregate. Typical aspect:

• Colour: yellow, transparent

• Odour: characteristic, fruity

• pH approximately 3.5 • Simple sugars: 40-60 g/L

• Soluble phenols: 200-400 mg/L

• Antioxidant activity as ORAC/L: 700- 1200 TE

Steps 2, 3 and 5 can yield useful concentrates characterized by chemical mixtures with specific molecular weights that can be taken outside the process .

The molecular disaggregate can undergo other hydrolytic and ultra-filtration steps with a molecular cut-off lower than 1 KDa up to the reverse osmosis process in order to recover water and salts.

Claims

1- Method for the treatment of vegetal matter comprising organic molecules, the method comprises the following steps: a first lysis step, during which first lysis reactions are enzymatically induced on at least part of the organic molecules of the vegetal matter; a first separation step, which follows the first lysis step and during which at least part of the larger dimension organic molecules are separated from the vegetal matter; a second lysis step, which follows the first separation step and during which second lysis reactions, different from the first lysis reactions, are enzymatically induced on at least part of the organic molecules of the vegetal matter.

2- Method according to claim 1, wherein the first lysis and the second lysis step occur in a first and in a second reactor respectively, which are connected by a first separation unit; the method comprises a first recirculation step, during which the larger dimensions organic molecules separated from the vegetal matter during the first separation step in the first separation unit are conveyed again to the first reactor.

3- Method according to claim 2, and comprising a second separation step, occurring in a second separation unit placed downstream from the second reactor and during which at least part of further larger dimensions organic molecules are separated from the vegetal matter; and a second recirculation step, during which the further larger dimensions organic molecules separated from the vegetal matter during the second separation step are conveyed again to the second reactor.

4- Method according to any one of the foregoing claims, wherein the first lysis reactions are endo- lysis reactions.

5- Method according to claim 4, wherein the endo- lysis reactions are induced by a first enzymatic preparation having at least one activity chosen in the group consisting of: amylase, cellulase, protease, pectinase xylanase . 6- Method according to claim 5, wherein the first enzymatic preparation has at least three activities chosen in the group consisting of: amylase, cellulase, protease, pectinase, xylanase.

7- Method according to any one of the foregoing claims, wherein the second lysis reactions are exo- lysis reactions.

8- Method according to claim 7, wherein the exo- lysis reactions are induced by a second enzymatic preparation having at least one of the activity chosen in the group consisting of: glucosidase, arylesterase , maltase, cellobiases .

9- Method according to claim 7, wherein the second enzymatic preparation has at least two activities of the following group of enzymes: glucosidase, arylesterase, maltase, cellobiases.

10- Method according to any one of the foregoing claims, wherein the first and the second lysis occur in presence of water.

11- Method according to any of the above claim, wherein the first separation step provides the mechanical filtration of the vegetal matter.

12- Method according to claim 11, wherein during the first separation step, the vegetal matter is filtered through a first membrane having a porosity lower than 6 μm.

13- Method according to claim 12, wherein the first membrane have a porosity from 110 KDa to 80 KDa.

14- Method according to claim 12, wherein the first membrane have a porosity from 11 KDa to 8 KDa.

15- Method according to any of the claims from 12 to 14, wherein during the first separation step, a pressure of at least 2 bar is applied to the vegetal matter upstream from the first membrane; a negative pressure is applied to the vegetal matter through the membrane .

16- Method according to claim 12 or 15, wherein the first separation step comprises a micro- filtration step, during which the vegetal matter is filtered through a first membrane having a porosity from 2 to 5 μm; and a first ultra-filtration step which follows the micro-filtration step and during which the vegetal matter is filtered through a second membrane having a porosity from 110 to 80 KDa.

17- Method according to any one of the foregoing claims, and comprising a second separation step, which follows the second lysis step and during which at least part of further larger dimension organic molecules are separated from the vegetal matter.

18- Method according to claim 17, wherein during the first separation step the vegetal matter is filtered through a first membrane and/or a second membrane; during the second separation step the vegetal matter is filtered through a third membrane having porosity lower than the first and/or the second membrane .

19- Method according to claim 18, wherein the third membrane has a porosity lower than 11 KDa.

20- Method according to claim 18, wherein the third membrane has a porosity lower than 1.1 KDa. 21- Method according to any one of claims 17 to 19, wherein during the second separation step, a pressure of at least 2 bar is applied on the vegetal matter upstream from the third membrane; a negative pressure is applied to the vegetal matter through the third membrane.

22- Method according to any of the claims from 17 to 21, and comprising a concentration step which follows the second separation step and during which a liquid component, particularly water, is separated from a treated vegetal matter, particularly by means of reverse osmosis.

23- Method according to any of the claims from 17 to 22, and comprising a third separation step, which follows the second separation step and during which components of the vegetal matter are mutually separated.

24- Method according to any one of claims 17 to 22, and comprising a fuel generation step, which follows the second separation step and during which the vegetal matter is at least partially transformed in fuel through a microbial process.

25- Method according to any of the claims from 17 to 22, and comprising an energy production step, which follows the second separation step and during which the vegetal matter is used as fuel .

26- Organic molecules obtained through a method according to any one of the foregoing claims.