Method for extracting xyloglucan from primary wall of plant cell

Abstract

The invention relates to a method for extracting xyloglucan contained in a primary wall of a plant cell, and more particularly, the plant is a dicotyledonous plant. In particular, the method of the present invention comprises a relatively limited number of steps and uses compounds that are low toxic to humans and the environment.

Description

Technical Field

[0001] The present invention relates to the field of extracting co-hemicellulose from the primary walls of plant cells. Notably, the present invention relates to a method for extracting xyloglucan contained in the primary walls of plant cells. In this regard, the present invention provides a method for extracting xyloglucan in polymeric form that complies with environmental constraints and provides a yield sufficient for industrial scale application. Background Art

[0002] Tamarind seeds are often the preferred source of xyloglucan due to their abundance, rich xyloglucan content, and the ease of extracting xyloglucan from tamarind seeds.

[0003] However, it is now desirable to diversify the supply sources. Among the interesting and / or alternative supply sources, agricultural by-products are particularly noteworthy. It is worth noting that these by-products include a large amount of plant cell primary walls containing xyloglucan.

[0004] For example, among agricultural by-products considered for xyloglucan extraction, apple pomace or citrus fruit by-products from juice extraction are of particular interest.

[0005] In this regard, document [1] cited at the end of the specification discloses a method for extracting xyloglucan from the primary walls of apple pomace cells.

[0006] However, this extraction method has relatively more steps and lower yield and purity, making it less suitable for industrial implementation of xyloglucan extraction.

[0007] Furthermore, the extraction method considered in the literature [1] also includes a delignification step using sodium chlorite (NaClO2), the toxicity of which is well known, especially for its environmental toxicity.

[0008] Finally, the amount of sodium hydroxide used in the implementation of this method is relatively large.

[0009] The object of the present invention is therefore to propose a method for extracting xyloglucan from the primary walls of plant cells which comprises a limited number of steps compared to the methods known from the prior art and which allows at the same time an efficient extraction (high yield and high purity) of xyloglucan in polymeric form (high molecular weight).

[0010] Another object of the present invention is to provide a method for extracting xyloglucan from the primary wall of plant cells, which has a high yield and is suitable for industrial implementation.

[0011] Another object of the present invention is to provide a method for extracting xyloglucan from the primary wall of plant cells, wherein the amount of alkali used in the method is lower than that in the methods known in the prior art. Summary of the Invention

[0012] The object is achieved at least in part by a method for extracting xyloglucan contained in the primary wall of plant cells, the method comprising the steps of:

[0013] a) a first step comprising mixing natural or extracted plant cell primary walls with a first aqueous solution, and adding a strong base having a concentration between 0.1 mol / L and 0.75 mol / L, advantageously between 0.25 mol / L and 0.5 mol / L,

[0014] performing a first step to form a first liquid residue and a first solid product, the first solid product primarily comprising holocellulose;

[0015] b) The second step: mixing the first product with a second solution containing a strong base with a concentration between 1 mol / L and 3 mol / L, advantageously between 2 mol / L and 3 mol / L. The second step separates the cellulose and xyloglucan contained in the first product.

[0016] According to one embodiment, the first step a) is divided into two consecutive sub-steps, which include:

[0017] a1) a first sub-step comprising mixing the plant cells with a first aqueous solution, the pH value of the mixture of the first aqueous solution and the plant cells being between 3 and 7, advantageously between 4 and 7, even more advantageously between 4 and 5;

[0018] a2) A second sub-step comprising adding a strong base having a concentration between 0.1 mol / L and 0.75 mol / L, advantageously between 0.25 mol / L and 0.5 mol / L, to the mixture obtained in the first sub-step a1).

[0019] According to one embodiment, the first aqueous solution contains an acidic substance, so that the pH value of the mixture of the first aqueous solution and the plant cells is between 3-7, advantageously between 4-7, and even more advantageously between 4-5.

[0020] According to one embodiment, the acidic substance includes at least one substance selected from citric acid, acetic acid, and nitric acid.

[0021] According to one embodiment, the presence of acidic compounds in the plant cells is such that the pH value of the mixture of the first aqueous solution and the plant cells is between 3-7, advantageously between 4-7, even more advantageously between 4-5.

[0022] According to one embodiment, the first sub-step a1) is carried out at a temperature higher than 80° C. and for a duration greater than 30 minutes.

[0023] According to one embodiment, the second sub-step a2) is carried out at a temperature higher than 60° C. and for a duration greater than 1 hour.

[0024] According to one embodiment, the second step b) is carried out at a temperature higher than 50° C. and for a duration greater than 1 hour.

[0025] According to one embodiment, the strong base includes at least one compound selected from sodium hydroxide (NaOH) and potassium hydroxide (KOH).

[0026] According to one embodiment, the method does not use sodium chlorite, calcium chloride or any other oxidizing agent capable of removing lignin.

[0027] According to one embodiment, the plant cell is a dicotyledonous plant cell.

[0028] According to one embodiment, the dicotyledonous plant comprises one selected from apple or citrus fruit.

[0029] According to one embodiment, the method is limited to performing only the first step a) and the second step b). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Other features and advantages of the present invention will be explained in detail with reference to the following drawings, in which:

[0031] [ Figure 1 ][ Figure 1 ] is a schematic diagram of a method for extracting xyloglucan contained in the primary wall of plant cells;

[0032] [ Figure 2 ][ Figure 2 ] The relationship between the yield of xyloglucan extracted from the first product (vertical axis) and the concentration of the strong base (particularly sodium hydroxide, mol / L) (horizontal axis) is shown in the form of a graph. It is worth noting that the graph shows the yield at different temperatures, more particularly the yield at 20°C, 35°C, and 70°C;

[0033] [ Figure 3 ][ Figure 3 ] shows in a graphical form the variation of the molar mass of the xyloglucan obtained in step b) with the concentration of the strong base at different temperatures (20°C, 35°C, 70°C) of step b). It is worth noting that the vertical axis represents the molar mass (10 3 g / mol), the horizontal axis represents the concentration of the strong base concentration (particularly sodium hydroxide, mol / L), more particularly, the "circle" pattern represents the molar mass obtained at 20°C, the "triangle" represents the molar mass obtained at 35°C, and the "diamond" pattern represents the molar mass obtained at 70°C. DETAILED DESCRIPTION

[0034] The present invention relates to a method for extracting xyloglucan Xg contained in the primary wall of plant cells, more particularly, the plant is a dicotyledonous plant.

[0035] The methods of the present invention particularly involve a relatively limited number of steps and utilize compounds of limited toxicity to humans and the environment.

[0036] It is noteworthy that the method of the present invention comprises performing the following steps:

[0037] a) The first step consists of the following two consecutive sub-steps:

[0038] a1) a first sub-step comprising mixing the plant cells with a first aqueous solution, the pH value of the mixture of the first aqueous solution and the plant cells being between 3 and 7, advantageously between 4 and 7, even more advantageously between 4 and 5;

[0039] a2) a second sub-step, which comprises adding a strong base having a concentration between 0.1 mol / L and 0.75 mol / L, advantageously between 0.25 mol / L and 0.5 mol / L, to the mixture obtained in the first sub-step a1);

[0040] Performing the two sub-steps a1) and a2) to form a first liquid residue PRL and a first solid product PPS, wherein the first solid product PPS mainly comprises holocellulose;

[0041] b) The second step: mixing the first product with a second solution, wherein the second solution contains a strong base with a concentration between 1 mol / L and 3 mol / L, advantageously between 2 mol / L and 3 mol / L. The second step separates the cellulose Cell and the xyloglucan Xg contained in the first product.

[0042] The implementation of sub-step a1) is optional in some cases, so the first step can be limited to mixing the plant cells with the first aqueous solution and adding thereto a strong base at a concentration between 0.1 mol / L and 0.75 mol / L, advantageously between 0.25 mol / L and 0.5 mol / L. This is particularly the case when the plant cells are first treated, in particular for pectin extraction.

[0043] In the remainder of the description, the first step a) will be considered to be carried out as two sub-steps. However, a person skilled in the art, based on the present description, will understand that the first step comprises mixing natural or extracted primary cell walls with a first aqueous solution and adding a strong base thereto at a concentration between 0.1 mol / L and 0.75 mol / L, advantageously between 0.25 mol / L and 0.5 mol / L. The first step is carried out to form a first liquid residue and a first solid product, the first solid product mainly comprising holocellulose;

[0044] "Natural" can be understood as the primary wall of untreated plant cells. In other words, "natural" refers to the primary wall of plant cells that has not been chemically treated, in particular, has not been treated with pectin extraction.

[0045] "Extract" refers to the primary walls obtained from unprocessed plant cells by a first step treatment, such as a pectin extraction process.

[0046] therefore,[ Figure 1 ] is a schematic diagram of a method for extracting xyloglucan Xg contained in the primary wall of plant cells.

[0047] It is worth noting that the method of the present invention comprises performing a first step a). It is worth noting that the first step a) comprises performing a first sub-step a1) and a second sub-step a2).

[0048] In this regard, the first sub-step a1) comprises mixing the plant cells with a first aqueous solution. It is noteworthy that the pH value of the mixture of the first aqueous solution and the plant cells is between 3 and 7, advantageously between 4 and 7, and even more advantageously between 4 and 5. According to one embodiment, the first aqueous solution contains an acidic substance, such that the pH value of the mixture of the first aqueous solution and the plant cells is between 3 and 7, advantageously between 4 and 7, and even more advantageously between 4 and 5.

[0049] For example, the first aqueous solution may include citric acid and / or acetic acid, and / or nitric acid. However, the present invention is not limited to consideration of these three acids, and those skilled in the art may consider using any other weak or strong acid that is non-toxic to humans and the environment.

[0050] Alternatively, the presence of the acidic compound in the plant cells is such that the pH value of the mixture of the first aqueous solution and the plant cells is between 3-7, advantageously between 4-7, even more advantageously between 4-5.

[0051] The first sub-step a1) can advantageously be carried out at a temperature higher than 70° C., advantageously higher than 80° C., even more advantageously higher than 85° C. Furthermore, the duration of the first sub-step a1) can be higher than 15 minutes, advantageously higher than 30 minutes, even more advantageously higher than 45 minutes.

[0052] For example, the first sub-step a1) can be carried out at a temperature of 90° C. and for a duration of 1 hour.

[0053] Carrying out sub-step a1) allows in particular the separation of holocellulose on the one hand and of a supernatant on the other hand. This supernatant comprises free sugars, oligosaccharides and pectin.

[0054] "Mild" acidic conditions (weak acidity, pH value higher than 3) can preserve the structure of holocellulose, especially the xyloglucan Xg contained therein. It should be understood that holocellulose includes cellulose Cell and xyloglucan Xg (xyloglucan Xg is a co-hemicellulose).

[0055] Carrying out the first sub-step a1) at a temperature above 70° C. also allows the starch that may be present in the primary walls of the plant cells to be dissolved in the supernatant.

[0056] However, some pectins that are likely to be present in the primary walls of plant cells are not necessarily soluble in acidic environments. Therefore, according to the present invention, it is proposed to dissolve these pectins in the supernatant by subjecting the mixture to alkaline conditions (sub-step a2).

[0057] Thus, the first sub-step a1) is followed by a second sub-step a2) which comprises adding a strong base to the mixture formed in the first sub-step a1).

[0058] It is worth noting that the second sub-step a2) is carried out so that the concentration of the strong base is between 0.1 mol / L and 0.75 mol / L, advantageously between 0.25 mol / L and 0.5 mol / L.

[0059] The strong base in question may include at least one substance selected from sodium hydroxide (NaOH) and potassium hydroxide (KOH).

[0060] Furthermore, the second sub-step a2) may be carried out at a temperature higher than 60°C, advantageously higher than 70°C, even more advantageously higher than 75°C.

[0061] Furthermore, the duration of the second sub-step a2) may be greater than 30 minutes, advantageously greater than 45 minutes, even more advantageously greater than 1 hour.

[0062] For example, the second sub-step a2) can be performed at a temperature of 80° C. and for 2 hours.

[0063] In particular, the second sub-step a2) results in the extraction of pectins, proteins.

[0064] Without being bound by the following explanation, the inventors believe that the second sub-step a2) allows the demethylation of galacturonic acid in the undissolved pectin during the first sub-step a1), for example by saponification. Due to this demethylation, the solubility of the pectin in the aqueous medium increases.

[0065] The mixture may contain calcium and magnesium ions which are likely to interact with the carboxylic acid functional groups in the pectin, thereby causing the latter to gel.

[0066] In order to prevent gelling of the pectin, it is also possible to consider adding a chelating agent during the second sub-step a2), which is intended to preferentially form complexes with calcium and magnesium ions, thereby making these ions unavailable for gelling of the pectin. Such chelating agents may include cyclohexanediaminetetraacetic acid (CDTA) or ethylenediaminetetraacetic acid (EDTA).

[0067] Therefore, the two sub-steps a1) and a2) are performed to form a first liquid residue PRL (supernatant) and a first solid product PPS. The first solid product PPS mainly includes holocellulose (i.e., a complex of cellulose Cell / xyloglucan Xg), while the first liquid residue PRL includes free sugars, oligosaccharides, and pectin originally present in the primary wall of plant cells.

[0068] Xyloglucan Xg has a strong affinity for cellulose Cell. Therefore, in order to separate these two substances, the present invention proposes to proceed to the second step b).

[0069] It is noteworthy that the second step b) comprises mixing the first solid product ("holocellulose") with a second solution comprising a strong base having a concentration between 1 mol / L and 3 mol / L, advantageously between 2 mol / L and 3 mol / L, for example, 2.5 mol / L. This second step allows the separation of cellulose Cell and xyloglucan Xg contained in the first solid product PPS.

[0070] The second step b) may be carried out at a temperature higher than 20°C, advantageously higher than 35°C, even more advantageously higher than 50°C.

[0071] The duration of the second step b) may be greater than 30 minutes, advantageously greater than 45 minutes, even more advantageously greater than 1 hour.

[0072] The purity of the cellulose Cell thus obtained is higher than 75%, or even higher than 85%, while the purity of the xyloglucan Xg is higher than 80%, or even higher than 90%.

[0073] Furthermore, the extraction yield of xyloglucan Xg can reach a value of approximately 5%, or even higher, compared to the dry mass of plant cells initially considered during the implementation of the method of the present invention.

[0074] Furthermore, the xyloglucan Xg is subjected to minimal or no chemical influence during the first and second steps.

[0075] Plant cells contemplated for use in practicing the methods of the present invention may include plant cells of dicotyledonous plants, more particularly plant cells of apple or citrus fruit.

[0076] In addition, the proposed method can be limited to performing steps a) and b), which are easy to implement and require only relatively mild chemical conditions, and can keep xyloglucan Xg in polymer form while obtaining high yield and high purity.

[0077] Finally, the proposed method does not use any chemicals (such as sodium chlorite NaClO2) that may be toxic to the environment.

[0078] Particularly advantageously, it is conceivable to carry out step b) at moderate temperatures.

[0079] Notably, the second step b) can be carried out at a temperature of 10-50°C, advantageously 10-35°C, even more advantageously 10-25°C, even more advantageously 15-25°C.

[0080] in this regard,[ Figure 2 The graph shows the relationship between the mass extraction yield of xyloglucan extracted from the first product (hereinafter referred to as "yield," "%") (vertical axis) and the concentration of a strong base, particularly sodium hydroxide (mol / L) (horizontal axis). In particular, the graph shows the yield at different temperatures, more specifically at 20°C, 35°C, and 70°C. It is clear from the graph that the concentration of the strong base required to achieve a given yield decreases as the temperature at which step b) is carried out decreases.

[0081] The temperature at which step b) is carried out is particularly advantageous in several respects. Notably, it helps to limit energy consumption and the consumption of strong base.

[0082] Furthermore, the last aspect is to relax the extraction conditions by limiting the concentration of strong base. It is worth noting that considering temperatures below 35°C and lower strong base concentrations, the extraction conditions are less harsh and higher molar mass xyloglucans can be obtained.

[0083] in this regard,[ Figure 3 ] shows in a graphical manner the variation of the molar mass of the xyloglucan obtained in step b) with the concentration of the strong base at different temperatures (20°C, 35°C, 70°C) of step b). It is worth noting that the vertical axis represents the molar mass (10 3g / mol), and the horizontal axis represents the concentration of sodium hydroxide (mol / L). More specifically, the "circle" pattern represents the molar mass obtained at 20°C, the "triangle" pattern represents the molar mass obtained at 35°C, and the "diamond" pattern represents the molar mass obtained at 70°C.

[0084] Therefore, advantageously, the concentration of the strong base in the second solution is between 1.5 mol / L and 2.5 mol / L, advantageously between 2 mol / L and 2.5 mol / L.

[0085] Also advantageously, step b) can be performed under the following conditions:

[0086] If the temperature for carrying out the second step is higher than 35°C, the concentration of the strong base is higher than 2 mol / L, advantageously between 2 mol / L and 2.5 mol / L,

[0087] If the temperature at which the second step is performed is lower than 35° C., the concentration of the strong base is lower than 2.2 mol / L.

[0088] Exemplary implementations of the extraction methods of the present invention are described below.

[0089] Exemplary implementation of the extraction method:

[0090] In this example, 30 g of apple pomace powder was dried in an oven at 60° C. for 8 hours.

[0091] In this embodiment, the first sub-step includes mixing apple pomace with 900 mL of deionized water in a 2 L three-necked flask and heating the mixture in an oil bath at a temperature of about 90° C. for 1 hour.

[0092] In order to perform the second sub-step, the temperature of the mixture was lowered to 70° C., and 18 g of sodium hydroxide (0.5 mol / L sodium hydroxide) was added to the mixture with stirring.

[0093] The second sub-step in this example involves heating the mixture at 80° C. for 2 hours with mechanical stirring.

[0094] After the second sub-step, the mixture was centrifuged at approximately 8000 g for 15 minutes. The solid matter (holocellulose) was recovered, redispersed in deionized water, and then the centrifugation was repeated three times to obtain a better washing effect.

[0095] The mass yield of the recovered holocellulose at the end of the second sub-step was 20 m%. After dialysis, the glycosidic composition of the first liquid residue (PRL) indicated the presence of starch, pectin, and other co-hemicelluloses. Furthermore, CP-MAS solid-state NMR analysis of the dried first liquid residue indicated the presence of significant amounts of protein and lipid polymers. Most aromatic compounds (lignin and tannins) appeared to be present in the first liquid residue.

[0096] The holocellulose obtained at the end of the second sub-step was then processed in a second step. Thus, the holocellulose was redispersed in 200 mL of sodium hydroxide solution containing a total of 20 g (approximately 1 / 30 w / w) at 70°C. The dispersion was heated at 70°C for 2 hours with mechanical stirring.

[0097] The dispersion was then centrifuged at approximately 10,000 g for 30 minutes to separate the supernatant (Xg) and the residue (Cell).

[0098] The residue (Cell) was redispersed under the same conditions and centrifuged again.

[0099] The supernatant (X g) was collected and dialyzed against deionized water until the conductivity of the dialyzed water stabilized at that of deionized water.

[0100] The residue (Cell) was washed by redispersion in deionized water and continuous centrifugation under the same conditions as before.

[0101] The glycoside composition of the supernatant (X g) obtained by hydrolysis with trifluoroacetic acid showed that the main component was xyloglucan (glucose, xylose, galactose, fucose, arabinose). CP-MAS NMR analysis showed that the protein content in the xyloglucan fraction and the cellulose fraction was negligible. The glycoside composition of the cellulose obtained by sulfuric acid hydrolysis of the solid fraction was mainly glucose, with other small amounts of residual sugars (mannose, xylose), indicating the presence of residual co-hemicellulose.

[0102] The yield of the final supernatant fraction (Xg) was 5 m% of the initial dry matter mass.

[0103] Upon analysis, the purity of the final supernatant fraction of xyloglucan was estimated to be 95%.

[0104] The implementation of the above extraction method demonstrates that xyloglucan and cellulose can be obtained in a simple manner at relatively high purity.

[0105] The inventors were able to characterize the cellulose obtained by the extraction method of the invention.

[0106] Remarkably, the inventors were able to note that despite the high concentration of sodium hydroxide considered for step b), it was still possible to extract cellulose I. Crystallinity studies have been carried out using X-ray diffraction or CP-MAS solid state NMR methods.

[0107] The cellulose purity was determined by solid-state NMR (CP-MAS) and acid hydrolysis and sugar analysis (glycoside composition). Solid-state NMR revealed that the residual cellulose fraction contained no protein, fatty acids, or pectin. The glycoside composition indicated that some hemicellulose remained and was adsorbed on the cellulose. These co-hemicelluloses are considered to contribute to cellulose utilization, as they are crucial for the rheological thickening behavior of the cellulose microfibrils.

[0108] Therefore, the purity of the cellulose component was estimated to be about 85%, with the remaining 15% of impurities being mainly co-hemicelluloses still adsorbed on the cellulose.

[0109] Given the observed purity, the cellulose thus extracted can be effectively utilized. Notably, this so-called thin-walled cellulose (i.e., primary-wall cellulose) is obtained in the form of cellulose microfibrils. These microfibrils are highly purified and can be used in a variety of applications, particularly in the materials and paper industries.

[0110] From the conceivable applications, it is possible to use additives based on cellulose microfibrils in coatings or molding compositions for composite materials or mastics.

[0111] It is also conceivable to use these cellulose microfibrils to improve the strength and holding power of paper (see document [3] cited at the end of the description).

[0112] Cellulose microfibrils can also be used in the cosmetics industry (reference [4] cited at the end of the specification).

[0113] The inventors have also been able to find that the xyloglucan extracted by the principles of the present invention can have a purity of 90%-95% (the latter being determined in particular by glycoside composition, solid state NMR (CP-MAS), Bradford assay and 1 obtained by H liquid NMR).

[0114] The glycoside composition of the co-hemicellulose fraction (Xg) obtained at the end of the extraction was as follows:

[0115] - Glucose: 39.2 mol%;

[0116] -Xylose: 31.6 mol%;

[0117] -Galactose: 12.2 mol%;

[0118] - Fucose: 6.7 mol%;

[0119] -arabinose: 6 mol%;

[0120] -Mannose: 4.4 mol%;

[0121] The xyloglucan obtained at the end of the extraction contained lower proportions of fucose and arabinose, in line with the Xg expected for the primary wall.

[0122] Depending on certain extraction conditions, at the end of the extraction, the molar mass (Mn) of xyloglucan measured by size exclusion chromatography is between 60 kDa and 100 kDa.

[0123] The residual protein level in the xyloglucan was determined by Bradford assay and estimated to be approximately 3 g / kg material, ie a protein content of approximately 0.3 m%.

[0124] The xyloglucan extracted from the primary wall by the extraction method of the present invention can be used to synthesize xyloglucan oligomers. It is worth noting that, in the context of oligomer synthesis, the xyloglucan can be subjected to enzymatic hydrolysis, given its purity. The purity of the xyloglucan extracted according to the content of the present invention is beneficial to the enzymatic activity of the enzymes used, in particular glucanase-type enzymes. The xyloglucan oligomers can be used as compounds that utilize their enzymatic activity, similar to the uses described in the document [5] cited at the end of the specification.

[0125] Xyloglucan extracted from primary wall can also be used as an additive for packaging film forming, especially for improving its mechanical properties (see reference [6] cited at the end of the specification).

[0126] Of course, the invention is not limited to the embodiments described and various different embodiments can be envisaged without departing from the scope of the invention as defined in the claims.

[0127] References

[0128] [1] Ma, Y., Luo, J. & Xu, Y. "Co-preparation of pectin and cellulose from apple pomace by a sequential process", J Food Sci Technol 56, 4091-4100 (2019);

[0129] [2]FR2867193A1;

[0130] [3]US9399838B2;

[0131] [4] EP0820267A1; <h2 style=";text-align:left;direction:ltr">

[0132] <h2 style=";text-align:left;direction:ltr"> [5]EP01972226A;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0133] <h2 style=";text-align:left;direction:ltr"> [6]US9534096B2.

Claims

1. A method for extracting xyloglucan contained in the primary wall of dicotyledonous plant cells, the method comprising the following steps: a) a first step comprising mixing natural or extracted plant cell primary walls with a first aqueous solution, and adding a strong base having a concentration between 0.1 mol / L and 0.75 mol / L, advantageously between 0.25 mol / L and 0.5 mol / L, performing a first step to form a first liquid residue and a first solid product, the first solid product primarily comprising holocellulose; b) a second step: mixing the first product with a second solution comprising a strong base having a concentration between 1 mol / L and 3 mol / L, advantageously between 2 mol / L and 3 mol / L, wherein the second step separates the cellulose and xyloglucan contained in the first product.

2. The method according to claim 1, wherein The first step a) is divided into two consecutive sub-steps, which include: a1) a first sub-step comprising mixing the plant cells with the first aqueous solution, the pH value of the mixture of the first aqueous solution and the plant cells being between 3 and 7, advantageously between 4 and 7, even more advantageously between 4 and 5; a2) A second sub-step, which consists in adding said strong base to the mixture obtained at the end of the first sub-step a1) at a concentration between 0.1 mol / L and 0.75 mol / L, advantageously between 0.25 mol / L and 0.5 mol / L.

3. The method according to claim 2, wherein: The first aqueous solution contains an acidic substance, so that the pH value of the mixture of the first aqueous solution and the plant cells is between 3-7, advantageously between 4-7, and even more advantageously between 4-5.

4. The method according to claim 3, wherein the acidic substance comprises at least one substance selected from citric acid, acetic acid and nitric acid.

5. The method according to claim 2, wherein: The presence of acidic compounds in the plant cells causes the pH value of the mixture of the first aqueous solution and the plant cells to be between 3 and 7, advantageously between 4 and 7, even more advantageously between 4 and 5.

6. The method according to any one of claims 2 to 5, wherein The first sub-step a1) is carried out at a temperature higher than 80° C. and lasts for more than 30 minutes.

7. The method according to any one of claims 2 to 6, wherein Said second sub-step a2) is carried out at a temperature exceeding 60° C. and for a duration exceeding 1 hour.

8. The method according to any one of claims 2 to 7, wherein Said second step b) is carried out at a temperature higher than 50° C. and lasts for more than 1 hour.

9. The method according to any one of claims 2 to 8, wherein The strong base includes at least one compound selected from NaOH and KOH.

10. The method according to any one of claims 2 to 9, wherein The method does not use sodium chlorite or calcium chloride.

11. The method according to any one of claims 2 to 10, wherein The dicotyledonous plant comprises one selected from apple or citrus fruit.

12. The method according to any one of claims 1 to 11, wherein The method is limited to performing the first step a) and the second step b).

13. The method according to any one of claims 1 to 12, wherein Said second step b) is carried out at a temperature of 10-50°C, advantageously 10-35°C, even more advantageously 10-25°C, even more advantageously 15-25°C.

14. The method according to claim 13, wherein The concentration of the strong base in the second solution is between 1.5 mol / L and 2.5 mol / L, advantageously between 2 mol / L and 2.5 mol / L.

15. The method according to any one of claims 1 to 12, wherein If the temperature of the second step is higher than 35°C, the strong base concentration is higher than 2 mol / L, advantageously between 2 mol / L and 2.5 mol / L. If the temperature of the second step is lower than 35°C, the strong base concentration is lower than 2.2 mol / L.

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