FR3160297A1 - Biostimulation and/or biocontrol composition for plants, use of said composition in agriculture and process for obtaining said composition - Google Patents

Abstract

The present invention relates to a biostimulation and/or biocontrol composition for plants, this composition comprising compounds of interest from at least one algae and a DES-type solvent used to extract the compounds of interest, the algae being selected from the group consisting of kelp, ulva, fucales, gigartinales, and mixtures thereof. The invention also relates to the use of this composition in agriculture for the biostimulation and/or biocontrol of plants. The invention also relates to a method for obtaining this composition.

Description

Biostimulation and/or biocontrol composition for plants, use of said composition in agriculture and process for obtaining said composition

The present invention belongs to the technical field of compositions for agriculture, and in particular biostimulation and biocontrol compositions for plants comprising compounds of interest derived from algae, and natural solvents of the DES type.

Prior art

Societal interest in environmental and health issues continues to grow, particularly in the field of agriculture. Awareness of the harmful effects of using synthetic products on the environment has led to the development of so-called "sustainable" or "green" agriculture, which increasingly uses natural biological resources.

Algae extracts are thus known which contain active compounds of interest in agriculture, for example to protect or stimulate the growth of plants.

Patent application FR3120768 A1 teaches in particular the use of a red algae extract as a natural biological resource to treat soil in order to promote plant growth, this red algae extract making it possible to reduce the access of nematodes, worm-like animals, to plant roots. This red algae extract is obtained by aqueous extraction.

It is also known to use non-toxic solvents, such as water, to extract compounds of interest from algae. However, in general, the extraction yields of such compounds are disadvantageously low, whether the extraction solvent is a natural solvent or a synthetic chemical solvent, and these compounds of interest exhibit relative stability over time.

Furthermore, although compounds of interest extracted from algae using non-toxic solvents, such as water, as illustrated above, are known to those skilled in the art, these algal compounds are, in the majority of cases, extracted with synthetic solvents. Indeed, the solvents most frequently used in the field of extraction of active compounds of interest, but also more generally in the field of chemistry and biochemistry, are organic solvents, such as hexane, benzene or even acetone.

However, these synthetic solvents do not comply with the principles of green chemistry, the aim of which is to implement safe, clean and energy-efficient chemical processes.

These synthetic solvents have many disadvantages: among others, they are generally/essentially derived from petrochemicals, volatile in nature and have low selectivity and a restricted range of polarity.

Furthermore, although some organic solvents are considered non-toxic, such as ethanol, glycerol, propylene glycol or butylene glycol, and more broadly mono- and polyhydroxylated alcohols, these are not always effective in optimally extracting certain compounds of interest, particularly the less polar molecules. Similarly, hydrophobic solvents, such as caprylic/capric triglyceride or sunflower oil, do not provide optimal extraction efficiency for compounds of interest from plants.

Furthermore, in agriculture, while it is possible to use organic solvents, few solvents are used, resulting in a lack of solutions. Furthermore, extraction using organic solvents is rarely used in this field due to human and environmental safety concerns. Aqueous, acidic, and basic extracts also represent the majority of extracts obtained, which is relatively limiting.

To solve the above-mentioned problems, it is known to those skilled in the art to use deep eutectic solvents (DES) to extract active compounds of interest from natural resources, and in particular from algae.

For example, the publication “Green extraction of value-added compounds from microalgae: a short review on natural deep eutectic solvents (NaDES) and related pre-treatments”, by Mehariya Sanjeet et al, teaches the use of DES-type solvents to extract compounds of interest from microalgae, these compounds of interest being able to be used in the pharmaceutical and cosmetology sectors.

DES solvents are sustainable solvents derived from renewable resources, which are easy to prepare, requiring no purification step. They are usually composed of two or three compounds capable of associating together via hydrogen bonds. They are typically described as a mixture of at least one hydrogen bond acceptor and one hydrogen bond donor that, at a certain stoichiometry, forms a eutectic, which has a lower melting point than either of the constituents. When the constituents of these solvents are naturally occurring compounds, such as primary plant metabolites, they are called natural deep eutectic solvents (usually referred to by the acronym NaDES).

These alternative solvents, in addition to improving selectivity and expanding the polarity range, are typically used to reduce the toxicity, volatility and hazard of solvents, and thus better comply with the principles of green chemistry.

In the field of cosmetology, extracts and compositions comprising algal compounds of interest have been prepared with DES-type solvents as extraction solvents in order to meet a need for compositions based on the principles of green chemistry. The use of natural DES solvents has made it possible to reduce the conventional use of synthetic chemical solvents, to reduce the negative impact linked to the use of synthetic compounds.

However, in these prior art cosmetic compositions, DESs were not used for the purpose of providing the compositions with a specific cosmetic effect. The particular presence of DESs in the cosmetic compositions was not an end in itself: their use had the sole objective of being used as more natural extraction solvents that were less harmful to the environment and human health.

However, the Applicant has surprisingly discovered that compositions comprising compounds of interest extracted from specific algae and from DES-type solvents are of interest in the field of agriculture, in particular for stimulating the growth of plants and/or for protecting them. In this field, there are currently no real solutions for optimally extracting certain compounds of interest, let alone from natural resources. Furthermore, in addition to the effects associated with the active compounds of interest in the compositions of the invention, the Applicant has discovered that the DES used for the extraction of these compounds of interest and also present in these compositions, themselves have beneficial effects on the biostimulation and biocontrol of plants.

To this end, those skilled in the art have developed new compositions for use in agriculture which make it possible to reduce the harmful impacts on the environment and human health associated with the agricultural compositions of the prior art, and which exhibit improved effectiveness in the biocontrol and/or biostimulation of plants.

More particularly, the subject of the present invention is a biostimulation and/or biocontrol composition for plants, this composition comprising compounds of interest from at least one algae and a DES type solvent used for the extraction of these compounds of interest, characterized in that the algae is chosen from the group consisting of laminaria, ulva, fucales, gigartinales and their mixtures.

For the purposes of the present invention, compounds of interest from at least one algae are understood to mean any type of compounds, molecules and metabolites of interest from at least one algae.

The compositions according to the invention make it possible to reduce the negative impact observed both on the environment and on human health when synthetic chemical compounds are typically used in agriculture. The compositions according to the invention comprise active compounds of interest of algal origin, which are derived from natural biomass, as well as DES (and more particularly NaDES) which are natural compounds which have also been used for the extraction of these compounds of interest. In addition, the DES used as solvents in the invention have a much lower toxicity than organic solvents. They are neither volatile nor flammable, while being biodegradable and biocompatible.

The compositions according to the invention act on the biostimulation of plants. They are capable of stimulating plants, to optimize their natural growth and survival processes. In particular, they allow their nutrition to be optimized, and their resistance to abiotic stress to be improved. These abiotic stresses are caused by the environmental conditions in which the plants are placed, in particular when these conditions are extreme, such as in the event of drought, frost, or significant temperature variations. The compositions according to the invention can therefore advantageously be used in addition to the fertilizers and fertilizers usually used in agriculture on plants. They are used to help and support the plant throughout its development, they can therefore be applied at any time in the life of a plant (for example, at the beginning of the cycle, during growth, or at the end of the cycle).

The compositions according to the invention also act on the biocontrol of plants, that is to say, they act on the plants themselves to enable them to better defend themselves against pathogenic agents. They can also directly neutralize pathogens present on/in plants. In other words, the compositions according to the invention enable plants to combat biotic stresses, which are caused by living organisms, such as pathogens. They therefore also make it possible to reduce, or even replace, synthetic pesticide, herbicide and fungicide agents, as well as phytosanitary products, sprayed on plants.

The compositions according to the invention can be used to stimulate the breaking of dormancy in plants. They can be applied to plant seeds, or directly to a plant, for example to the buds of a plant. This makes it possible to "awaken" the germination and growth of seeds and/or plants "dormant" during the winter period.

The compositions according to the invention can also be used for seed coating. In this case, they are applied to plant seeds. This protects the seeds to be sown and improves the germination and growth of the plants that are produced from these seeds.

The compositions according to the invention can be applied to soil, to plant seeds or directly to a plant in a single application, or in several applications (for example, in three, four or five applications on the same area of soil, on the same seeds or on the same plant). Each application can be more or less spaced out in time, depending on the composition and/or depending on the plant to be treated.

Thus, within the framework of the present invention, the biostimulation and biocontrol effects on plants are ensured both by the compounds of interest derived from algae and by DES-type solvents:

- regarding DES type solvents, these have a strong solvating and extracting power for compounds of interest from algae; they are also present in nature for the survival of species in extreme conditions, and allow the storage and transport of molecules that are not soluble in water, or when there is little water. They are therefore very good extraction solvents with improved extraction yields compared to the use of conventional extraction solvents, such as water or synthetic solvents;

- with regard to the algal compounds of interest, the algae are specifically chosen from the group consisting of kelp, ulva, fucales, gigartinales and their mixtures, in order to extract target compounds of interest which are identified as being active in the biostimulation and biocontrol of plants; in addition, the compounds of interest from algae extracted by DESs have improved stability compared to the compounds of interest from algae extracted by conventional extraction solvents. DESs have protective properties with respect to the compounds of interest extracted from algae.

- in the compositions of the invention, the effects of the algal compounds of interest and the DES are juxtaposed to ensure a synergistic effect of optimal biostimulation and/or biocontrol.

The compositions according to the invention therefore have improved efficacy compared to the compositions of the prior art used in agriculture for stimulating the growth and protecting plants, in particular due to the presence of active compounds of interest having improved stability over time. In addition, the compositions according to the invention may comprise a larger quantity of these compounds of interest, since it is easier to extract interesting quantities of algal compounds of interest during an extraction protocol.

The compositions according to the invention can be applied directly to soil, seeds or plants. They can also be incorporated into agricultural products, which can be solid or liquid, for example biostimulant products, in varying concentrations.

Advantageously, the algae used in the compositions of the invention is a macroalga.

But the compositions of the invention can also comprise compounds of interest extracted from microalgae.

Advantageously, the macroalgae from which the compounds of interest are extracted is chosen from Ascophyllum nodosum, Mastocarpus stellatus, Chondrus crispus, Ulva lactuca, Fuccus serratus, Fucus vesiculosus, Laminaria digitata and their mixtures.

The inventors tested these specific macroalgae and succeeded in extracting compounds of interest, useful in the biostimulation and biocontrol of plants in agriculture.

The species Ascophyllum nodosum, Fuccus serratus, Fucus vesiculosus and Laminaria digitata are brown macroalgae.

The species Mastocarpus stellatus and Chondrus crispus are red macroalgae.

And the species Ulva lactuca is a green macroalgae.

Preferably, the DES type solvent may be a NaDES.

Compositions that include NaDES have a beneficial impact on the environment and human health, as these compounds are natural.

Preferably, the NaDES of the compositions according to the invention may be chosen from DES Proline/Fructose/Glycerol (1:1:1), DES Urea/Proline (1:1), DES Betaine/D-glucose (1:1), DES Betaine/D-Sorbitol (1:1), DES Lactic Acid/Glycerol (1:1), DES Glycine/Arginine/Sorbitol (1:1:3) and DES Lactic Acid/Dodecanol (1:1).

In the following description, the proportions of the different components of DES are noted both in the form (1:1) and in the form 1/1.

These NaDES also contain water, in varying amounts. The water content in these solvents impacts the extraction of the compounds of interest.

Advantageously, the algal compounds of interest present in the compositions according to the invention may be phytohormones, polyphenols, sugars and sugar derivatives comprising monosaccharides (for example glucose), oligosaccharides and polysaccharides, and in particular hydrolyzed polysaccharides, organic acids, amino acids, mycosporin like amino acid (MMA), digeneasides and/or hydrophobic compounds comprising carotenoids and chlorophylls.

As polyphenols which can be used in the compositions according to the invention, mention may in particular be made of phlorotannins and/or mannitol.

As hydrolyzed polysaccharides which can be used in the compositions according to the invention, mention may in particular be made of laminarans, sulfated or non-sulfated fucans, sulfated or non-sulfated galactans, sulfated or non-sulfated arabinogalactans, sulfated or non-sulfated mannans, furcellaranes, carrageenans and their mixtures.

As phytohormones which can be used in the compositions according to the invention, mention may in particular be made of cytokinins, auxins, abscisic acid, giberellins, ethylene, polyamines, brassinosteroids, oxilipins, salicylates or even signal peptides.

The present invention also relates to the use of the composition defined above in agriculture for the biostimulation and/or biocontrol of plants.

Advantageously, this use may comprise the application of the composition of the invention to the soil, or to a seed of a plant, or directly to a plant.

The composition according to the invention can thus be used in several ways in agriculture. In particular, it can be used at different stages of a plant's growth: when the plant is at the seed stage or when it has already started to grow.

The present invention also relates to a process for obtaining a composition according to the invention, this process comprising the following steps:
- harvesting at least one algae;
- possibly, drying of the harvested seaweed;
- possibly, grinding of the harvested seaweed, whether dry or fresh;
- bringing the harvested algae into contact with a DES-type solvent.

The method according to the invention is in itself more effective than the methods known from the prior art since it makes it possible to obtain better extraction yields of the compounds of interest from algae.

Advantageously, the method according to the invention further comprises the following steps:
- filtration of the algae residues after contacting the algae with the DES type solvent, to obtain an extract;
- vacuum filtration of the extract, or on a cartridge, or on a membrane, or by means of a filter press, to obtain a final extract;
- recovery of the final extract in liquid form, the extract corresponding to the biostimulation and/or biocontrol composition for plants.

The vacuum filtration step of the process according to the invention may correspond to filtration on a Büchner funnel with filter paper.

Detailed description of the invention

Other advantages and features of the present invention will result from the description which follows, given by way of non-limiting example and made with reference to the examples.

EXAMPLES

EXAMPLE 1: Process for preparing NaDES used in the agricultural compositions of the invention

The NaDES prepared and used in the invention are listed below:

Lactic acid/Glycerol 1/1 at 30% water;

Glycine/Arginine/Sorbitol 1/1/3 at 30% water;

Proline/Fructose/Glycerol 1/1/1 at 30% water;

Urea/Proline 1/1 at 30% water;

Betaine/D-glucose 1/1 at 30% water;

Betaine/D-Sorbitol 1/1 at 30% water;

Lactic acid/Dodecanol 1/1 at 6.6% water: given this water content, this DES is a hydrophobic compound.

Their preparation process is as follows:
- mixing in stoichiometric quantities of each of the components of a NaDES, by heating at 60°C, for 4 hours, with strong mechanical stirring;
- recovery of a homogeneous liquid mixture, constituting NaDES;
- measurement of the water content of NaDES by coulometric titration on a Karl-Fisher titrator;
- for hydrophilic NaDES solvents, homogenization to 30% water (mass) by adding water measured accordingly.

EXAMPLE 2: Process for preparing the agricultural compositions of the invention

The macroalgae used in the invention are listed below:

Laminaria digitata;

Ascophyllum nodosum;

Mastocarpus stellatus;

Chondrus crispus (also called “red lichen”);

Ulva lactuca;

Fuccus serratus.

The compositions according to the invention which comprise compounds of interest from at least one macroalgae and one of the following NaDES: Proline/Fructose/Glycerol (1:1:1), Urea/Proline (1:1), Betaine/D-glucose (1:1), Betaine/D-Sorbitol (1:1), Lactic acid/Glycerol (1:1), Glycine/Arginine/Sorbitol (1:1:3), are prepared by implementing a hydrophilic extraction process.

The compositions according to the invention which comprise compounds of interest from at least one macroalgae and NaDES Lactic Acid/Dodecanol (1:1) are prepared by implementing a hydrophobic extraction process.

Process for preparing the compositions according to the invention using hydrophilic extraction:
-optionally, drying at least one macroalgae and/or grinding this macroalgae to obtain flakes 0.01 to 5 cm wide, preferably 0.05 to 2.5 cm, more preferably 0.1 to 1.5 cm;
- bringing these macroalgae flakes into contact with the chosen NaDES, with stirring, for a period of 10 minutes, at a temperature of 30°C, where the quantity of macroalgae (dried or not) is between 2 and 6% by weight (i.e. between 0.5g/20ml and 1g/20ml) relative to the total weight of the macroalgae/NaDES mixture;
-centrifugation of the macroalgae residues formed in the previous step for 20 minutes, at 20°C, at 6000 rpm (acronym for “rotation per minute” in English, corresponding to the unit “revolution per minute” in French);
-filtration of the residual supernatant formed in the previous step on an MCE filter (acronym for “cellulose ester membrane”) with a porosity of 5 µm;
-filtration of the final extract obtained on an MCE filter with a porosity of 0.22 µm;
-recovery of the final extract in liquid form, this extract constituting the composition according to the invention.

Process for preparing the compositions according to the invention using hydrophobic extraction:

The process for preparing compositions according to the invention which implements a hydrophobic extraction comprises the same steps as the process described previously implementing a hydrophilic extraction.

These hydrophilic extraction and hydrophobic extraction processes are identical when microalgae are used to prepare the agricultural compositions of the invention.

For the rest of the examples, the preparation of macroalgae in order to extract the active compounds of interest (when this was necessary) is specified in the examples.

EXAMPLE 3: preparation of a first example of composition according to the invention from macroalgae Laminaria digitata and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 1.

Preparation of macroalgae Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention Dried and crushed 0.283 Lactic Acid/Glycerol 1/1 5,610 Incubation of 3 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 4: preparation of a first example of composition according to the invention from macroalgae Laminaria digitata and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 2.

Preparation of macroalgae Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention Dried 0.269 Lactic Acid/Glycerol 1/1 5,667 Incubation of 3 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 5: preparation of a first example of composition according to the invention from macroalgae Laminaria digitata and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 3.

Preparation of macroalgae Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention Dried and crushed 1,018 Lactic Acid/Glycerol
1/1 22,228 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 6: preparation of a first example of composition according to the invention from macroalgae Laminaria digitata and DES Glycine/Arginine/Sorbitol 1/1/3

The protocol of the composition preparation process is detailed in Table 4.

Preparation of macroalgae Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention Dried and crushed 0.273 Glycine/Arginine/Sorbitol 1/1/3 5,514 Incubation of 1 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 7: preparation of a first example of composition according to the invention from macroalgae Laminaria digitata and DES Proline/Fructose/Glycerol 1/1/1

The protocol of the composition preparation process is detailed in Table 5.

Preparation of macroalgae Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention Dried and crushed 0.577 Proline/Fructose/Glycerol 1/1/1 11,374 Incubation of 3 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 8: preparation of a first example of composition according to the invention from macroalgae Laminaria digitata and DES Urea/Proline 1/1

The protocol of the composition preparation process is detailed in Table 6.

Preparation of macroalgae Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention Dried and crushed 0.99 Urea/Proline 1/1 23,268 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 9: preparation of a first example of composition according to the invention from macroalgae Laminaria digitata and DES lactic/Dodecanol 1/1

The protocol of the composition preparation process is detailed in Table 7.

Preparation of macroalgae Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention Dried and crushed 0.998 Lactic acid/Dodecanol 1/1 17,313 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 10: preparation of a second example of composition according to the invention from macroalgae Ascophyllum nodosum and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 8.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 0.286 Lactic Acid/Glycerol 1/1 5,667 Incubation of 3 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 11: preparation of a second example of composition according to the invention from macroalgae Ascophyllum nodosum and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 9.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,003 Lactic Acid/Glycerol 1/1 21,934 Incubation of 7 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 12: preparation of a second example of composition according to the invention from macroalgae Ascophyllum nodosum and DES Glycine/Arginine/Sorbitol 1/1/3

The protocol of the composition preparation process is detailed in Table 10.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1.04 Glycine/Arginine/Sorbitol 1/1/3 23,350 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 13: preparation of a second example of composition according to the invention from macroalgae Ascophyllum nodosum and DES Proline/Fructose/Glycerol 1/1/1

The protocol of the composition preparation process is detailed in Table 11.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 0.515 Proline/Fructose/Glycerol 1/1/1 11,270 Incubation of 3 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 14: preparation of a second example of composition according to the invention from macroalgae Ascophyllum nodosum and DES Proline/Fructose/Glycerol 1/1/1

The protocol of the composition preparation process is detailed in Table 12.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,017 Lactic acid/Dodecanol 1/1 17,065 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 15: preparation of a second example of composition according to the invention from macroalgae Ascophyllum nodosum and DES Urea/Proline 1/1

The protocol of the composition preparation process is detailed in Table 13.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,074 Urea/Proline 1/1 23.27 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 16: preparation of a third example of composition according to the invention from macroalgae Fuccus serratus and DES Urea/Proline 1/1

The protocol of the composition preparation process is detailed in Table 14.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 0.933 Urea/Proline 1/1 23,187 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 17: preparation of a third example of composition according to the invention from macroalgae Fuccus serratus and DES Proline/Fructose/Glycerol 1/1/1

The protocol of the composition preparation process is detailed in Table 15.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,059 Proline/Fructose/Glycerol 1/1/1 24,075 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 18: preparation of a third example of composition according to the invention from macroalgae Fuccus serratus and DES Glycine/Arginine/Sorbitol 1/1/3

The protocol of the composition preparation process is detailed in Table 16.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,072 Glycine/Arginine/Sorbitol 1/1/3 22,160 Incubation of 7 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 19: preparation of a third example of composition according to the invention from macroalgae Fuccus serratus and DES Lactic Acid/Dodecanol 1/1

The protocol of the composition preparation process is detailed in Table 17.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,047 Lactic acid/Dodecanol 1/1 17,686 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 20: preparation of a third example of composition according to the invention from macroalgae Fuccus serratus and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 18.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,001 Lactic Acid/Glycerol 1/1 22,251 Incubation of 5 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 21: preparation of a fourth example of composition according to the invention from macroalgae Ulva lactuca and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 19.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,046 Lactic Acid/Glycerol 1/1 23,904 Incubation of 0.5 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 22: preparation of a fourth example of composition according to the invention from macroalgae Ulva lactuca and DES Glycine/Arginine/Sorbitol 1/1/3

The protocol of the composition preparation process is detailed in Table 20.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,082 Glycine/Arginine/Sorbitol
1/1/3 22,855 Incubation of 3 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 23: preparation of a fourth example of composition according to the invention from macroalgae Ulva lactuca and DES Lactic Acid/Dodecanol 1/1

The protocol of the composition preparation process is detailed in Table 21.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 0.983 Lactic acid/Dodecanol 1/1 17,563 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 24: preparation of a fourth example of composition according to the invention from macroalgae Ulva lactuca and DES Proline/Fructose/Glycerol 1/1/1

The protocol of the composition preparation process is detailed in Table 22.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 0.813 Proline/Fructose/Glycerol 1/1/1 17,022 Incubation of 3 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 25: preparation of a fourth example of composition according to the invention from macroalgae Ulva lactuca and DES Urea/Proline 1/1

The protocol of the composition preparation process is detailed in Table 23.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 0.936 Urea/Proline 1/1 23,119 Incubation of 7 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 26: preparation of a fifth example of composition according to the invention from macroalgae Mastocarpus stellatus and Chondrus crispus and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 24.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1.2 Lactic Acid/Glycerol 1/1 23,767 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 27: preparation of a fifth example of composition according to the invention from macroalgae Mastocarpus stellatus and Chondrus crispus and DES Lactic Acid/Glycerol 1/1

The protocol of the composition preparation process is detailed in Table 25.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1 Lactic Acid/Glycerol 1/1 22,271 Incubation of 3 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 28: preparation of a fifth example of composition according to the invention from macroalgae Mastocarpus stellatus and Chondrus crispus and DES Glycine/Arginine/Sorbitol 1/1/3

The protocol of the composition preparation process is detailed in Table 26.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,086 Glycine/Arginine/Sorbitol 1/1/3 23,369 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 29: preparation of a fifth example of composition according to the invention from macroalgae Mastocarpus stellatus and Chondrus crispus and DES Proline/Fructose/Glycerol 1/1/1

The protocol of the composition preparation process is detailed in Table 27.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 0.756 Proline/Fructose/Glycerol 1/1/1 17,586 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 30: preparation of a fifth example of composition according to the invention from macroalgae Mastocarpus stellatus and Chondrus crispus and DES Lactic Acid/Dodecanol 1/1

The protocol of the composition preparation process is detailed in Table 28.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 1,042 Lactic Acid/Dodecanol
1/1 17,765 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 31: preparation of a fifth example of composition according to the invention from macroalgae Mastocarpus stellatus and Chondrus crispus and DES Urea/Proline 1/1

The protocol of the composition preparation process is detailed in Table 29.

Macroalgae content (in g) Nature of DES DES content (in g) Extraction protocol for obtaining a composition according to the invention 0.999 Urea/Proline 1/1 23,265 Incubation of 10 mL of the macroalgae/DES mixture at 30°C for 1 hour

EXAMPLE 32: analysis of the antioxidant properties of the compositions according to the invention

Example 32 a: 2,2 diphenyl-1-picrylhydrazyl assay (hereinafter referred to as DPPH) to determine the antioxidant power of the compositions according to the invention

Samples of compositions according to the invention:

Samples of compositions prepared according to the preceding examples 6, 15 and 28 were taken and filtered on a 0.22 µm MCE syringe filter.

Reagent solution:

The reagent was prepared by mixing 9.86 mg of DPPH with methanol in a 50 mL volumetric flask. It was then stirred in an ultrasonic bath protected from light for 15 min.

Control solutions:

Two positive control solutions corresponding to the IC 50 (acronym for median inhibitory concentration) of quercetin and gallic acid were prepared.

The first control solution (solution C1) was prepared by mixing 3.62 mg of quercetin with methanol in a 20 mL volumetric flask (5.35.10-4 mol/L).

And the second control solution (solution C2) was prepared by first preparing a stock solution of gallic acid, by mixing 3.4 mg of gallic acid with methanol in a 20 mL volumetric flask; then 10 mL of this solution was then taken to be mixed with methanol in a 20 mL volumetric flask (5.10-4 mol/L).

Dosage protocol:

The assay was performed in a 96-well plate with 5 to 8 identical wells filled according to the scheme detailed in Table 30 below.

Reference well White witness wells DPPH control well White wells methanol Sample well Volume of reference solution (C1 or C2) (in μL) 10

- - - - Sample volume according to the invention (in μL) - 10 - - 10 Volume of methanol (in μL) 100 200 110 210 100 Volume of reagent solution (in μL) 100 - 100 - 100

The 96-well plate was then incubated at 25°C, with shaking (80 rpm), away from light, for 10 min. It was then placed in a plate reader, with shaking at 200 rpm, for 5 min, and the absorbance was read at 540 nm. The percentage of disappearance of DPPH was calculated according to the following formula:

% disappearance = 100 – [(A corrected sample * 100)/A corrected DPPH control]

where A corrected sample corresponds to the average absorbance of the sample minus the average absorbance of the control blank, and A corrected DPPH control corresponds to the average absorbance of the DPPH control minus the average absorbance of the methanol blank. The percentages of disappearance of the references serve as calibration; they must be close to 50%.

Results :

The results of this DPPH assay for the compositions of Examples 6, 15 and 28 are detailed in Table 31 below. The values in the table for each NaDES/macroalgae pair are expressed as % disappearance of DPPH.

macroalgae
NaDES Laminaria digitata Chondrus crispus Ascophyllum nodosum Glycine/Arginine/Sorbitol 1/1/3 57.2 57.2 - Urea/Proline 1/1 - - 47.4

These results illustrate that the compositions comprising the combination Laminaria digitata /Glycine/Arginine/Sorbitol (1:1:3), the combination Chondrus crispus /Glycine/Arginine/Sorbitol (1:1:3), and the combination Ascophyllum nodosum /Urea/Proline (1:1) were able to reduce the chemical radical DPPH° by transforming it into DPPH-H by transfer of a hydrogen. The radical DPPH° is colored (purple), while DPPH-H is colorless. It is therefore possible to observe the reduction of DPPH° into DPPH-H by spectrophotometry. This reduction of DPPH° into DPPH-H by the tested compositions clearly demonstrates their antioxidant properties.

Example 32 b: FRAP assay (acronym for ferric ion reducing antioxidant power) to determine the total antioxidant activity of compositions comprising hydrophilic NaDES

This test measures the reduction capacity of a ferric complex to the ferrous form (Fe ²⁺ ).

Samples of compositions according to the invention:

Samples of compositions prepared according to Examples 3 to 31 were filtered through a 0.22 µm MCE syringe filter.

FRAP working solution:

This solution was prepared just before the dosage, by mixing three solutions A, B and C, according to the following proportions, by volume: 10/1/1 (V/V/V).

Solution A is a 300 mmol/L sodium acetate (NaOAc) buffer solution; it was prepared by mixing 738 mg of sodium acetate with 4.86 g (4.63 mL) of glacial acetic acid in a 300 mL flask. The flask was filled to the mark with distilled water.

Solution B corresponds to a 10 mM solution of 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM sodium chloride (HCl); it was prepared by mixing 9.4 mg of TPTZ and 2.9 mL of water, then adding 100 µL of a 3.7% HCl solution.

Solution C corresponds to a solution of iron (III) chloride hexahydrate (FeCl ₃ .6H ₂ O (20 mmol/L); it was prepared by mixing 16.2 mg and 3 mL of water.

Standard range:

A 4000 μmol/L iron sulfate (FeSO ₄ ) stock solution was prepared by weighing 11.12 mg of iron (II) sulfate heptahydrate (FeSO ₄ .7H ₂ O) into a 10 mL flask, which was topped up with solution A described previously. Then a standard range (from 50 μmol/L to 4000 μmol/L) of iron sulfate was prepared by diluting this FeSO4 stock solution.

Control solution:

A positive control solution of ascorbic acid was prepared by mixing 5 mg of ascorbic acid with the reference solvent (DES, water, etc.) in a 5 mL volumetric flask (2.27 mol/L).

Dosage protocol:

The assay was carried out in a 96-well plate with 3 identical wells filled according to the diagram detailed in Table 32 below.

White Wells White Wells
FeSO ₄ range White control wells White sample wells Volume of buffer solution A (in μL) 160 150 150 150 Sample volume (in μL) - - - 10 Volume of control solution (in μL) - - 10 - Volume of standard FeSO ₄ solution (in μL) - 10 - -

The 96-well plate was then placed in a plate reader, and an initial reading of the plate at 593 nm was taken to measure the blank (MB value).

In each well, 100 μL of the FRAP working solution was added, then the plate was shaken in an incubator, at 25°C, protected from light, for 15 min. A second reading of the plate at 593 nm was made to measure the FRAP antioxidant activity (MF value).

The result comes from the subtraction of the blank measurement from the FRAP dosage measurement (MF – MB) and is expressed in μmol/100g of starting material (macroalgae) equivalent Fe (II) using the straight line obtained with the FeSO ₄ standards.

Results :

The results of this FRAP assay for the compositions of Examples 7, 13 to 16, 21, 24, 26, 27 and 29 are detailed in Table 33 below. The values in the table for each NaDES/macroalga pair correspond to the concentration in Fe (II) equivalent, and are expressed in µmol/100g of matrix (macroalga).

macroalgae
NaDES Ascophyllum nodosum Chondrus crispus Ulva lactuca Fucus serratus Laminaria digitata Proline/Fructose/Glycerol 1/1/1 7102.52 5341.01 4322.26 - 6593.55 Lactic Acid/Glycerol 1/1 - 1849.60 1763.17 - - Urea/Proline 1/1 2104.90 - - 2236.03 -

These results illustrate that the compositions comprising the respectively tested macroalgae/NaDES combinations were able to reduce ferric ions (Fe ³⁺ ) to ferrous ions (Fe ²⁺ ). Ferric ions are colorless, while ferrous ions are intensely blue. It is therefore possible to observe this reduction by spectrophotometry. This reduction of ferric ions to ferrous ions by the tested compositions clearly demonstrates their antioxidant properties.

These antioxidant properties (also called anti-radical) are very relevant for the biostimulation and biocontrol of plants in agriculture, because they advantageously reduce oxidation reactions in plants, which helps protect them, ensure their growth safely and optimally, and increase their lifespan.

Claims (10)

Biostimulation and/or biocontrol composition for plants, said composition comprising compounds of interest from at least one algae and a DES type solvent used for the extraction of said compounds of interest, characterized in that said algae is chosen from the group consisting of laminaria, ulva, fucales, gigartinales and their mixtures. The composition of claim 1, wherein said algae is a macroalga. Composition according to claim 2, wherein said macroalga is selected from Ascophyllum nodosum, Mastocarpus stellatus, Chondrus crispus, Ulva lactuca, Fuccus serratus, Fucus vesiculosus, Laminaria digitata and mixtures thereof. A composition according to any one of claims 1 to 3, wherein said DES type solvent is NaDES. The composition of claim 4, wherein said NaDES is selected from DES Proline/Fructose/Glycerol (1:1:1), DES Urea/Proline (1:1), DES Betaine/D-glucose (1:1), DES Betaine/D-Sorbitol (1:1), DES Lactic Acid/Glycerol (1:1), DES Glycine/Arginine/Sorbitol (1:1:3) and DES Lactic Acid/Dodecanol (1:1). Composition according to any one of claims 1 to 5, wherein said compounds of interest are phytohormones, polyphenols, sugars and sugar derivatives comprising monosaccharides, oligosaccharides and polysaccharides, organic acids, amino acids, mycosporine like amino acid (MMA), digeneasides and/or hydrophobic compounds comprising carotenoids and chlorophylls. Use of the composition defined according to any one of claims 1 to 6 in agriculture for the biostimulation and/or biocontrol of plants. Use according to claim 7, comprising the application of said composition to the soil, or to a seed of a plant, or directly to a plant. Process for obtaining a composition defined according to any one of claims 1 to 6, comprising the following steps:
- harvesting at least one algae;
- possibly, drying of the harvested algae;
- possibly, grinding of the harvested seaweed, whether dry or fresh;
- bringing said harvested algae into contact with a DES type solvent. The method of claim 9, further comprising the steps of:
- filtration of the algae residues after said contacting of said algae with said DES type solvent, to obtain an extract;
- vacuum filtration of said extract, or on a cartridge, or on a membrane, or by means of a filter press, to obtain a final extract;
- recovery of said final extract in liquid form, said extract corresponding to said biostimulation and/or biocontrol composition for plants.