Composition comprising a protein hydrolyzate and a microalgae extract and its use as an agricultural biostimulant.

Abstract

The present invention relates to compositions comprising a protein hydrosol and a microalgae extract. The protein hydrolysate which is included in the compositions of the invention comes from the hydrolysis of animal proteins, in particular from the hydrolysis of pig farming co-products. The present invention also relates to the process for preparing these compositions, but also to the use of these compositions as an agricultural biostimulant. Figure for abstract: None

Description

Title of the invention: Composition comprising a protein hydrolyzate and a microalgae extract and its use as an agricultural biostimulant. FIELD OF THE INVENTION

[0001] The present invention relates to the agricultural field. It relates to compositions comprising a protein hydrosol and a microalgae extract. The protein hydrolyzate which is included in the compositions of the invention comes from the hydrolysis of animal proteins. The present invention also relates to the process for preparing these compositions, as well as to the use of these compositions as agricultural biostimulants. STATE OF THE ART

[0002] For many years, the application of chemical fertilizers has been a widely used agricultural practice in the development of intensive agriculture (Oui, L. et al., 2020; Gonzalez, M. et al., 2015). However, the continued use and abuse of these synthetic fertilizers have caused many negative effects related to the depletion of natural resources as well as the generation of greenhouse gases, water eutrophication, soil salinization, and food security issues and quality deterioration (Gonzalez, M. et al., 2015; Ashour, M. et al., 2021; Hassan, SM. et al., 2021). These chemicals also increase plant susceptibility to pathogens, altering the soil microbiome and influencing plant health, posing a significant threat to consumers (Colla, G. and Rouphael, Y., 2015).For this reason, sustainability in agricultural production is necessary both to meet consumer demand for healthy products and to try to eliminate or reduce the aforementioned problems (Gonzalez, M. et al., 2015; Hassan, SM. et al., 2021; Colla, G. and Rouphael, Y., 2015; Colla, G. et al., 2014).

[0003] Currently, the use of biostimulants is widespread in sustainable agriculture because they represent an alternative to chemical fertilizers (Avila-Pozo P. et la., 2022).

[0004] Biostimulants are natural preparations that have the potential to reduce the need for fertilizers with increased plant growth, even under water and abiotic stress. Current biostimulants are of different types namely microbial or non-microbial. They include active substances such as humic and fulvic acids, protein hydrolysates, seaweed extracts, beneficial fungi, bacteria and algae. These can be formulations with one or more components, although it can provide a positive action of different components. Biostimulants can be applied foliarly or to the soil. They can be hydrolysis products, fermented or extracted.

[0005] A number of biostimulants consisting of protein hydrolysates have been used (Colla, G. et al., 2014). These substances, which are generally obtained by enzymatic hydrolysis processes, are generally made up of low molecular weight peptides, amino acids, polysaccharides, etc., used in a wide variety of crops such as cereals, fruits and vegetables and have been shown to have a large number of positive effects on these crops, such as increased productivity and better quality (Ertani, A. et al., 2014; Tejada, M. et al., 2016; Tejada, M. et al., 2018; Rouphael Y. and Colla G., 2018).

[0006] According to Searchinger T., 2013 and Kapoore RV et al., 2021, the use of these biostimulants in agriculture has two objectives, namely to increase the supply and quality of food for the world population and to reduce the negative effect of agriculture on the environment and human health.

[0007] To find solutions to the challenge of improving the sustainability of agricultural systems by reducing the use of synthetic inorganic fertilizers, there is therefore still a real need to develop new compositions with a biostimulant effect. In particular, compositions that exhibit a biostimulant effect under both normal and water-stressed conditions would be of enormous scientific and agricultural interest. Statement of the invention

[0008] The present invention makes it possible to meet the needs set out above. The present inventors have in fact developed a new composition obtained from a protein hydrolyzate combined with a microalgae extract.

[0009] The inventors have notably shown that such a composition has a significant, even synergistic, biostimulating effect, making it possible to increase the growth and yield (in grains, fruits, etc.) of various plants useful in plant production. This new composition is all the more interesting since this advantageous effect is obtained both under “normal” crop irrigation conditions, but also under water stress conditions (lack of water or drought).

[0010] The inventors have in fact succeeded in obtaining a biostimulant whose composition in amino acids of interest presents a particularly advantageous peptide signature, and in particular concerning its tryptophan content which has a role as an auxiliary hormonal precursor in plants.

[0011] In addition, some of the components of this new composition with a biostimulant effect may in particular come from industrial co-products such as co-products from animal farming, particularly pig farming. The production of this biostimulant therefore constitutes an alternative of great environmental interest since the recovery of these co-products leads to their elimination, and therefore a reduction in their environmental impact.

[0012] Similarly, the energy of industrial installations can be used to meet the temperature requirements of microalgae cultivation. Thus, the production of microalgae from which the extract contained in this new composition is derived can in particular make it possible to reduce the energy bills of industrial production sites.

[0013] It is in this context that the present invention provides a novel composition comprising a protein hydrolysate of animal origin, preferably derived from the hydrolysis of co-products of pig farming, and a microalgae extract.

[0014] Preferably, this composition comprises a porcine bristle protein hydrolyzate and / or a porcine lung mucus protein hydrolyzate.

[0015] Preferably, this composition comprises a spirulina extract, and in particular a phycocyanin extract.

[0016] Advantageously, the composition according to the invention contains the quantities of amino acids as described in Table 2.

[0017] Preferably, the composition of the invention contains amino acids whose molecular weight distribution is 3-4% between 1000 Da and 3000 Da, 2.5-3% between 800 Da and 1000 Da, 68-72% between 204 Da and 800 Da and 23-25% less than 204 Da.

[0018] Preferably, the composition of the invention comprises 1-15% hydrolyzate and 1-5% microalgae extract, and optionally a support or vehicle acceptable in the agricultural field.

[0019] The present invention also relates to a process for preparing the above-mentioned composition comprising: - the supply of at least one protein hydrolysate of animal origin, preferably from the hydrolysis of co-products of pig farming, - the supply of a microalgae extract, and - their mixtures.

[0020] The present invention also relates to the use of such a composition as an agricultural biostimulant, and in particular under normal irrigation conditions or under water stress conditions.

[0021] The composition of the invention is preferably used as a biostimulant for large-scale crops, market gardening, viticulture, forestry, arboriculture and horticulture.

[0022] Finally, the present invention also relates to a kit for the preparation of a biostimulant which comprises on the one hand, at least one protein hydrolyzate of animal, in particular from the hydrolysis of co-products of pig farming and a microalgae extract or a mixture of these, on the other hand at least one support or vehicle acceptable in the agricultural field, and possibly instructions for use. DESCRIPTION OF FIGURES

[0023] Other characteristics, aims and advantages of the invention will emerge from the following description, which is purely illustrative and non-limiting, and which must be read in conjunction with the appended drawings in which:

[0024] [Fig.l] [Fig.l] represents, in the form of histograms, the results of grain yield measurement in rapeseed (g / plant), grown without treatment (MO) or treated with 2L / ha of microalgae extract (AEC 2L), 6L / ha of microalgae extract (AEC 6L), with 2L / ha of protein hydrolysate of pork co-products (CP 2L), 6L / ha of protein hydrolysate of pork co-products (CP 6L) or with the mixture of 6L / ha of microalgae extract and 6L / ha of protein hydrolysate of pork co-products (MA).

[0025] [Fig.2] [Fig.2] represents, in the form of histograms, the measurement results of the total dry matter quantity in rapeseed, grown under normal irrigation conditions, without treatment (MO) or treated with 2L / ha of microalgae extract (AEC 2L), 6L / ha of microalgae extract (AEC 6L), with 2L / ha of protein hydrolysate from pork co-products (CP 2L), 6L / ha of protein hydrolysate from pork co-products (CP 6L) or with the mixture of 6L / ha of microalgae extract and 6L / ha of protein hydrolysate from pork co-products (MA).

[0026] [Fig.3] [Fig.3] represents, in the form of histograms, the measurement results of the total dry matter quantity in rapeseed, grown under drought conditions, without treatment (MO) or treated with 2L / ha of microalgae extract (AEC 2L), 6L / ha of microalgae extract (AEC 6L), with 2L / ha of protein hydrolysate of pork co-products (CP 2L), 6L / ha of protein hydrolysate of pork co-products (CP 6L) or with the mixture of 6L / ha of microalgae extract and 6L / ha of protein hydrolysate of pork co-products (MA).

[0027] [Fig.4] [Fig.4] represents, in the form of histograms, the effect of a composition according to the invention applied at several doses (BST 1.5L / ha, BST 3 L / Ha, BST 6L / Ha), to tomato cultivation, in comparison with that of a reference product (IDROGENA ENERGY; 3L / Ha) or with an untreated sample (TNT), during three successive harvests.

[0028] [Fig.5] [Fig.5] represents, in the form of histograms, the effect of a composition according to the invention applied at several doses (BST 1.5L / ha, BST 3 L / Ha, BST 6L / Ha), on the production yield of tomatoes, in comparison with that of a reference product (IDROGENA ENERGY; 3L / Ha), during three successive harvests.

[0029] [Fig.6] [Fig.6] represents, in the form of histograms, the effect of a composition according to the invention applied at several doses (BST 1.5L / ha, BST 3 L / Ha, BST 6L / Ha), on the number of fruits harvested (tomatoes), in comparison with that of a reference product (IDROGENA ENERGY; 3L / Ha) or with an untreated sample (TNT), during three successive harvests.

[0030] [Fig.7] [Fig.7] represents, in the form of histograms, the effect of a composition according to the invention applied at several doses (BST 1.5L / ha, BST 3 L / Ha, BST 6L / Ha), on the quality of the harvest evaluated by measuring the sugar content of the harvested fruits (tomatoes) according to the BRIX scale, in comparison with that of a reference product (TRAINER EDEN STRIM; 3L / Ha) or with an untreated sample (TNT).

[0031] [Fig.8] [Fig.8] represents, in the form of histograms, the effect of a composition according to the invention applied at several doses (BST 1.5L / ha, BST 3 L / Ha, BST 6L / Ha), on the physiology of the plant evaluated by measuring the chlorophyll content of tomato plants, in comparison with that of a reference product (GOACTIV; 100ml diluted in 100 L of water applied per hectare) or with an untreated sample (TNT).

[0032] [Fig.9] [Fig.9] represents, in the form of histograms, the effect of a composition according to the invention applied at several doses (BST 1.5L / ha, BST 3 L / Ha, BST 6L / Ha), on the physiology of the plant evaluated by measuring the induction of flowering on tomato plants, in comparison with that of a reference product (GOACTIV; 100ml diluted in 100 L of water applied per hectare) or with an untreated sample (TNT), during two successive harvests.

[0033] [Fig. 10] [Fig. 10] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the physiology of the vine evaluated by measuring the percentage of flowers, in comparison with that of a reference product (VIVAFLOR; 2L / ha) or with an untreated sample (TNT).

[0034] [Fig. 11] [Fig. 11] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the production of the vine evaluated by measuring the yield in tonnes of grapes harvested per hectare, in comparison with that of a reference product (VIVAFLOR; 2L / ha) or with an untreated sample (TNT).

[0035] [Fig. 12] [Fig. 12] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the production of the vine evaluated by measuring the number of grape bunches harvested per plot (47 m2), in comparison with that of a reference product (VIVAFLOR; 2L / ha) or with an untreated sample (TNT).

[0036] [Fig. 13] [Fig. 13] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the quality of the harvest of the vine evaluated by measuring malic acid in the harvested grapes, in comparison with that of a reference product (VIVAFLOR; 2L / ha) or with an untreated sample (TNT).

[0037] [Fig. 14] [Fig. 14] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the quality of the vine harvest evaluated by measuring tartaric acid in the harvested grapes, in comparison with that of a reference product (VIVAFLOR; 2L / ha) or with an untreated sample (TNT).

[0038] [Fig. 15] [Fig. 15] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the quality of the vine harvest evaluated by measuring the degree of alcohol in the harvested grapes, in comparison with that of a reference product (VIVAFLOR; 2L / ha) or with an untreated sample (TNT).

[0039] [Fig. 16] [Fig. 16] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the quality of the vine harvest evaluated by measuring the potassium in the harvested grapes, in comparison with that of a reference product (VIVAFLOR; 2L / ha) or with an untreated sample (TNT).

[0040] [Fig. 17] [Fig. 17] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the quality of the vine harvest evaluated by the measurement of assimilable nitrogen, in comparison with that of a reference product (VIVAFLOR; 2L / ha) or with an untreated sample (TNT).

[0041] [Fig. 18] [Fig. 18] represents, in the form of histograms, the effect of a composition according to the invention applied at 6L / Ha, on the production of apples evaluated by measuring the quantity of apples harvested from two trees (Kg / 100 Kg fruits), in comparison with that of a reference product (PHYLGREEN; 300 ml / 1000L) or with an untreated sample (TNT). DETAILED DESCRIPTION OF THE INVENTION Definitions

[0042] By "livestock co-products" also called "livestock by-products" is meant a co-product that is created during a livestock process and at the same time as the main product of livestock. In the case of animal livestock co-products, these are the parts of the animal that are not intended for human consumption. For example, co-products of pig farming are testicles, bristles (i.e. hair), lobes, spleen, heart, lungs, lung mucus, trachea, blood and cruor (i.e. the solid part of the blood).

[0043] By "protein hydrolysate" or "protein hydrolysate" or "peptone" is meant a mixture of amino acids and peptides derived from proteins. Protein hydrolysis corresponds to the cleavage of the peptide bonds between the amino acids resulting in a protein hydrolysate. Protein hydrolysates are mainly produced by enzymatic and / or chemical hydrolysis (with strong or alkaline acids) of animal or vegetable proteins. In the context of the present invention, it is an animal protein hydrolysate, and preferably an animal protein hydrolysate except a hydrolysate derived from chicken feathers.

[0044] By "components" or "constituents" is meant the active substances of the composition according to the invention, that is to say those which give it its biostimulating effect on plant production.

[0045] In the present application, the ranges of values ​​are indicated in abbreviated form, in order to avoid having to set out in detail and describe each of the values ​​in the range. Any suitable value within the interval may be chosen, as appropriate, as the upper value, lower value or end of the interval. For example, a range of 0.1-1.0 represents the terminal values ​​of 0.1 and 1.0, as well as the intermediate values ​​of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges within the range 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.

[0046] The words "comprises," "include," and "comprising" shall be construed inclusively. Similarly, the terms "include," "including," and "or" shall all be construed as inclusive. All such terms, however, shall be construed as encompassing exclusive embodiments that may also be designated by words such as "consist of."

[0047] "Proteins" are macromolecules present in all living cells. They are mainly made up of carbon (C), hydrogen (H), oxygen (O) and nitrogen (N). They perform a multitude of biological functions and are composed of peptides, themselves composed of amino acids.

[0048] The term "amino acid" refers to a molecule containing both an amine group and a carboxyl group. In some embodiments, the amino acids are α-, β-, γ-, or δ-amino acids, including their stereoisomers and racemates.

[0049] By "biostimulant" is meant both the biostimulating action and the composition which has this action in the agricultural field. The biostimulating action provides a positive effect on crop performance (physiology, production, yield, harvest qualities, etc.), and more particularly under conditions of environmental stress. Biostimulants are fertilizers which stimulate the nutrition process of plants independently of the nutrients they contain, with the sole aim of improving one or more of the following characteristics: of plants or their rhizosphere: the efficiency of nutrient use, tolerance to abiotic stress, qualitative characteristics, and the availability of nutrients confined in the soil and the rhizosphere (definition of the European Commission from European Regulation 2019 / 1009 published in the Official Journal in June 2019 and which will be applicable in July 2022).

[0050] By "agricultural" or "agricultural field" is meant here everything relating to plant production, and in particular working the land and the production of plants. Thus, the agricultural field / field / specialty / activity includes, consists essentially of, consists of or is a field / field / specialty / activity which relates to plant production (i.e. the field devoted to or relating to working the land and the production of plants). The agricultural composition includes, consists essentially of, consists of or is therefore a composition adapted for and / or a composition for working the land and the production of plants. The agricultural use includes, consists essentially of, consists of or is therefore a use adapted for and / or in working the land and the production of plants.

[0051] By "plant production" or "plant production" we mean everything relating to large-scale crops, market gardening, viticulture, forestry, arboriculture and horticulture.

[0052] In this context, "field crops" include cereal crops (wheat, barley, corn, oats, etc.), oilseed crops (rapeseed, sunflower) and protein crops (fava beans and peas), beet cultivation (sugar and fodder) and potato cultivation. "Market gardening" concerns the production of fresh vegetables grown in greenhouses or in open fields. "Arboriculture" concerns the cultivation of perennial fruit trees and the cultivation of small shrub-type fruits. "Horticulture" can cover all specialized agricultural orientations: market gardening, arboriculture, floriculture and nursery. However, the term "horticulture" is restricted here to floriculture (or floral horticulture), ornamental horticulture and nursery activities.

[0053] By "vehicle" or "support" is meant any substance of a composition other than the active component(s) thereof. The addition of a support to a composition is intended to give it a given stability, consistency, form, dissolution property(ies), targeting property(ies), preservation property(ies), half-life, bioavailability, taste, color, aesthetic property(ies) and / or texture, or other physical, chemical, biological or taste characteristics. A support is therefore defined more by its use than by a particular chemical composition, said use resulting in particular from its physicochemical properties which make it suitable for fulfilling its role as a support. The support may in particular be a stabilizing agent, a preservative, an antioxidant, a texturizing agent or texture agent, a binder, an emulsifier, a colorant, a pigment, a corrosion inhibitor, a polymerization inhibitor, an anti-caking agent, a surfactant (which may also be called an emulsifier, wetting product, dispersant, emulsifier), an antifoaming agent, an anti-leaching agent, a thickener, a solubilizer, a humectant, a desiccant, an antiozonant agent, an anti-UV agent (photoprotector, UV absorber), a heat stabilizer, an antifreeze agent, a flame retardant, an adhesion promoter, a solvent, a diluent, an antistatic agent, a buffer additive, an olfactory tracer, an agent combining any of these characteristics.The support preferably has no negative interaction, particularly chemical, with the components of the composition which contains them, that is to say that the support does not induce any reduction in the desired activity / action / effect of these active substances, whether before or after application / administration on the plants to be treated. The support may also be neutral with respect to the components, that is to say that it does not affect in any way the activity / action / effect of these components. The support is preferably non-toxic (in particular for the plants to be treated, in particular cereal crops, market garden crops, vines, fruit trees. The support is preferably essentially of natural origin. The support is preferably essentially biodegradable. More preferably, the support is essentially biosourced. In a particularly preferred manner, the support is essentially biosourced and essentially biodegradable.

[0054] By "agriculturally acceptable support" is meant a support suitable for agricultural use, and in particular for plant production. In particular, it is meant a support capable of being incorporated into a composition for agricultural use. Advantageously, the agriculturally acceptable support does not induce any reduction in the desired phytosanitary activity / action / effect of the components of the composition of the invention. The agriculturally acceptable support is preferably non-toxic (in particular for the plants to be treated, in particular cereal crops, market garden crops, vines, fruit trees). The support is preferably of natural origin. The support is preferably essentially biodegradable. The support is more preferably essentially biosourced. The support is preferably biodegradable and / or biosourced.

[0055] By "sprayable solution" is meant a solution that can be projected in fine liquid droplets or in powder form. Advantageously, the sprayable solution is in suspension, emulsion or solution in a gaseous medium, preferably in an inert gas. Advantageously, the sprayable solution is presented in the form of a sprayer, vaporizer, aerosol, or spray.

[0056] By "spraying" is meant the projection of a solution in fine liquid droplets, in the form of an aerosol, in the form of a vapor, or in the form of a powder, onto an object, preferably a crop, in particular cereal crops, market garden crops, vines, fruit trees. Description of the invention

[0057] According to a first aspect, the present invention relates to a composition comprising a protein hydrolyzate of animal origin, preferably derived from the hydrolysis of co-products of pig farming, and a microalgae extract.

[0058] The protein hydrolyzate may be obtained by any hydrolysis process well known to those skilled in the art, and in particular by chemical hydrolysis or by enzymatic hydrolysis. For example, the protein hydrolyzate of the composition according to the invention may correspond to CAS number 100085-61-8.

[0059] The composition according to the invention may comprise, in addition to the microalgae extract, two animal protein hydrolysates. They may be obtained by the same type of hydrolysis process or by different hydrolysis processes, and preferably they are obtained by different hydrolysis processes. Advantageously, when the composition comprises two animal protein hydrolysates, one is obtained by chemical hydrolysis and the other by enzymatic hydrolysis. Thus, a particularly preferred composition comprises on the one hand a chemical hydrolysate and an enzymatic hydrolysate of animal proteins and on the other hand an aqueous extract of microalgae.

[0060] Preferably, the composition according to the invention comprises, in addition to the microalgae extract, a pig bristle protein hydrolyzate and / or a pig lung mucus protein hydrolyzate. More preferably, it comprises a pig bristle protein hydrolyzate and a pig lung mucus protein hydrolyzate. A particularly preferred composition according to the invention comprises a protein hydrolyzate derived from the chemical hydrolysis of pig bristles and a protein hydrolyzate derived from the enzymatic hydrolysis of pig lung mucus.

[0061] Preferably, the microalgae extract contained in the composition according to the invention is a spirulina extract, and in particular a phycocyanin extract. For example, the microalgae extract of the composition according to the invention may correspond to CAS number 20298-86-6.

[0062] The microalgae extract contained in the composition according to the invention ideally has significant antioxidant activity. This activity can be measured by any method known in the field by the person skilled in the art, and in particular by spectrophotometry, in particular at 620 nm and 652 nm. Preferably, the microalgae extract has an antioxidant activity of at least 70 measured according to the ORAC method (notably described by Agregan R. et al., 2018) and more preferably an antioxidant activity in a range from 70 to 100 according to the ORAC method, or equivalent if this activity is measured by another method. The antioxidant activity of the microalgae extract contained in the composition according to the invention may in particular range from 60 to 90, and advantageously, it has an antioxidant activity ranging from 73 to 76, preferably from 74 to 75, of approximately 75 according to the ORAC measurement method, and in particular of 74.5.

[0063] Preferably, the constituents of the composition of the invention contain the amounts of amino acids which are indicated in the following table 1.

[0064] [Tables 1] Amino acids Protein hydrolysate (mg / 100g protein) Microalgae extract (mg / 100g extract) glutamic acid 10-20, preferably 12-16 20-32, preferably 25-30 glycine 5-10, preferably 7-9 17-25, preferably 20-23 aspartic acid 6-12, preferably 8-10 20-32, preferably 20-25 leucine 6-12, preferably 8-10 20-32, preferably 25-30 alanine 3-10, preferably 4-7 20-30, preferably 20-25 lysine 3-10, preferably 4-8 3-10, preferably 4-7 proline 3-10, preferably 4-7 8-15, preferably 9-12 valine 3-10, preferably 4-7 6-15, preferably 8-10 arginine 4-12, preferably 5-10 20-32, preferably 25-28 phenylalanine 2-6, preferably 4-7 17-25, preferably 20-24 serine 3-10, preferably 4-9 14-20, preferably 15-18 threonine 2-6, preferably 3-5 12-18, preferably 14-17 histidine 1-4, preferably 1,5-3 3-10, preferably 4-6 tyrosine 2-6, preferably 3-5 25-35, preferably 27-32 isoleucine 2-6, preferably 3-5 20-32,preferably 25-28 cysteine ​​1-8, preferably 1.5-6.5 2-10, preferably 3-6 methionine 0.5-4, preferably 1-3 9-15, preferably 11-14 tryptophan 0-3, preferably 0.5-2.5 1-6, preferably 2-4 ,

[0065] The composition according to the present invention has a peptide signature of great interest due to the presence of a combination of amino acids leading to specific effects which play a very important role for metabolism or constitution of proteins of particular interest to the plant. In particular, we can note the significant presence of alanine, aspartic acid, glutamic acid, involved in improving the assimilation of nutrients, but also leucine, cysteine, valine for the improvement of energy production, or proline, and serine which are involved in signaling in the event of stress. Furthermore, it has been shown that, on apple trees, tryptophan and glycine, alone or in combination, significantly increase the height and diameter of the aerial part, the leaf area, the chlorophyll content, the percentage of fruits and the yield as well as the physical and chemical characteristics of the fruits, and also the mineral composition of the leaves (Mosa et al., 2021).

[0066] Thus, preferably, the composition according to the present invention advantageously contains the quantities of amino acids which are mentioned in Table 2 below.

[0067] [Tables2] Amino Acids Composition according to the invention (mg / 100g of composition) TRYPTOPHAN 8-20, preferably 10-15 Aspartic acid 250-300, preferably 270-290 Threonine 120-150, preferably 125-145 Serine 160-200, preferably 170-190 Glutamic acid 500-700, preferably 525-650 Proline 150-200, preferably 165-195 Glycine 230-300, preferably 240-280 Alanine 180-240, preferably 200-230 Valine 160-230, preferably 185-215 Cysteine ​​30-80, preferably 40-70 Methionine 20-60, preferably 30-50 Isoleucine 100-180, preferably 130-160 Leucine 200-320, preferably 230-300 Tyrosine 60-140, preferably 10-80-120 Phenylalanine 100-140, preferably 115-135 Lysine 10-50, preferably 15-35 Histidine 40-80, preferably 45-65 Arginine 230-300, preferably 240-290

[0068] Preferably, the composition according to the invention contains amino acids whose molecular weight distribution is 3-4% between 1000 Da and 3000 Da, 2.5-3% between 800 Da and 1000 Da, 68-72% between 204 Da and 800 Da and 23-30% less than 204 Da. More preferably, it contains amino acids whose molecular weight distribution is 3.2-3.4% between 1000 Da and 3000 Da, 2.5-2.8% between 800 Da and 1000 Da, 69-70% between 204 Da and 800 Da and 24-25% less than 204 Da.

[0069] According to a particularly preferred embodiment of the present invention, the composition comprises 1-15% of hydrolyzate, preferably 10-15%, and very particularly approximately 13%, and 1-5% of microalgae extract, preferably 2-4%, in particular spirulina extract, and preferably a spirulina extract containing 1.5 to 2g / L of phycocyanin, and optionally a support or vehicle acceptable in the agricultural field.

[0070] The composition according to the invention may be in a liquid form or in a solid form (such as powder, granules, tablets). Preferably, the composition is in a liquid form, preferably in the form of an aqueous solution.

[0071] According to a second aspect, the present invention relates to a process for preparing the composition according to the invention as described above, characterized in that it comprises: - the supply of at least one protein hydrolysate of animal origin, in particular from the hydrolysis of co-products of pig farming, - the supply of a microalgae extract, and - their mixtures.

[0072] The animal protein hydrolysate can be obtained according to any hydrolysis method well known in the field by the person skilled in the art, in particular by enzymatic hydrolysis, for example in the presence of one or more proteases (pepsin, papain, pancreatin, subtilisin, etc.), or by chemical hydrolysis, for example with one or more strong or alkaline acids.

[0073] The preparation process according to the invention may in particular comprise the provision of a protein hydrolysate corresponding to CAS number 100085-61-8.

[0074] Preferably, the preparation method comprises providing a chemical hydrolyzate and an enzymatic hydrolyzate. The chemical hydrolyzate is preferably derived from pig bristles. The enzymatic hydrolyzate is preferably derived from pig lung mucus. The microalgae extract is preferably an aqueous extract, in particular an aqueous extract of spirulina. The method may in particular comprise providing a microalgae extract corresponding to CAS number 20298-86-6.

[0075] Advantageously, the process for preparing the composition according to the invention comprises: - the provision of a chemical hydrolysate of pig bristles, preferably obtained by acid thermal hydrolysis, and / or the provision of an enzymatic hydrolysate of pig lung mucus, preferably obtained using one or more proteases - the provision of a spirulina extract, preferably an aqueous extract, and in particular an aqueous extract of phycocyanin; and - mixtures thereof.

[0076] Preferably, the method for preparing the composition of the invention comprises the preparation of a spirulina extract obtained by extraction without any solvent other than water, in particular from dry spirulina. Optionally, this preparation step is followed by a filtration step. The spirulina extract can also be prepared in advance and then frozen until use.

[0077] According to a third aspect, the present invention relates to the use of the composition described above as an agricultural biostimulant.

[0078] The composition according to the invention can be used as an agricultural biostimulant under normal irrigation conditions or under water stress conditions.

[0079] The composition of the invention can be used as a biostimulant on any type of plant crops. Preferably, it is used for field crops, market gardening, viticulture, forestry, arboriculture and horticulture. More preferably, it is used as a biostimulant on cereal crops, market garden crops, vines, fruit trees. Advantageously, the composition of the invention is used as a biostimulant for the cultivation of rapeseed, tomatoes, vines and apple trees.

[0080] The biostimulant action of the composition according to the present invention can be demonstrated according to conventional methods, well known to those skilled in the art, and which will be chosen according to the relevant parameters to be taken into account for this purpose. They will depend on the type of crop concerned, for example the measurement of the production yield based for example on the number of grains, fruits, the measurement of growth (foliar or root extension, induction of flowering), the assessment of the quality of the harvests (fruits, grains).

[0081] The composition in fact has a biostimulant action regardless of the form in which it is presented. Thus, the composition of the invention can be applied to crops in liquid form, or in solid form, and in particular in powder form. When the composition is applied in liquid form, it can be presented in liquid form directly ready for use, or to be diluted from a concentrated form.

[0082] When the composition is applied in liquid form, it can also be presented in solid form and can then be diluted / dissolved / put into solution / solubilized in a suitable solvent before use (for example extemporaneously, or for short, medium or long term storage before use).

[0083] According to one embodiment, the composition is a liquid composition, preferably in the form of an aqueous solution, preferably in the form of a sprayable aqueous solution, more preferably in the form of an aerosol. The liquid composition may in particular be in a concentrated form (which may for example be diluted before use, for example 20 times concentrated, or 15 times, 10 times, 8 times, 5 times, 4 times, 3 times or even 2 times concentrated) or in a ready-to-use form.

[0084] According to one embodiment, the composition is a liquid composition obtained by diluting (and / or dissolving and / or dissolving and / or solubilizing) the composition in a solid form in an appropriate solvent, preferably according to an appropriate dilution factor, in particular so as to obtain the amino acid contents as described in Table 2.

[0085] The solvents suitable / appropriate for diluting / dissolving / putting into solution / solubilizing the concentrated liquid composition and / or the solid composition may for example be chosen from aqueous solvents, and preferably water.

[0086] A person skilled in the art knows how to determine the appropriate dilution factors in order to arrive at a liquid composition having amino acid contents as described in Table 2, from a concentrated liquid composition or a solid composition.

[0087] In the context of its use as a biostimulant, the composition of the invention can be applied by deposit, in particular by dripping and / or spraying and / or vaporization and / or projection, for example directly onto the growing soil or onto the plants, in particular via a foliar and / or root application. Thus, the composition can be deposited at the foot of the crops and / or sprayed and / or vaporized and / or projected onto the crops.

[0088] The method of application will also be adapted to the type of crops. According to a preferred embodiment, the use is characterized in that the composition is applied to the crops by spraying and / or vaporization and / or projection.

[0089] The quantity and number of applications and their frequency will be adapted according to the type of crop concerned. For example, the composition of the invention can be used in quantities ranging from 1 to 10L / ha, ranging from 1.5 to 6L / ha, or even ranging from 3 to 5L / ha, in one or more applications more or less spaced out over time.

[0090] Furthermore, the use of the composition of the invention will also be adapted to the stages of development or growth of the culture concerned, for example the use is characterized in that the composition is applied to the crops before and / or after harvesting said crops. The person skilled in the art will be able to adapt all of the criteria of use, which include the method of application, the number, the frequency and the stage(s) of development or growth of the crop concerned.

[0091] In the context of its use, the composition of the invention can also be integrated into a more complex formulation, in particular a fertilizer, a phytosanitary product or any other composition useful for plant production. Preferably, the composition of the invention is integrated into a fertilizer composition.

[0092] According to yet another aspect, the present invention relates to a kit for the preparation of a biostimulant, said kit comprising: - either at least one protein hydrolysate of animal origin, preferably from the hydrolysis of co-products of pig farming and a microalgae extract, or a mixture of these, - at least one support or vehicle acceptable in the agricultural sector, - possibly a user manual.

[0093] Preferably, the kit according to the invention comprises: - either a hydrolyzate of pig bristle protein and / or a hydrolyzate of pig lung mucus protein and a microalgae extract, preferably a spirulina extract, or a mixture thereof, - at least one support or vehicle acceptable in the agricultural sector, - possibly a user manual.

[0094] Advantageously, the kit according to the invention comprises: - either a pork bristle protein hydrolyzate, a pork lung mucus protein hydrolyzate, and a microalgae extract, preferably a spirulina extract, or a mixture of these, - at least one support or vehicle acceptable in the agricultural sector, - possibly a user manual.

[0095] All the preferred embodiments described above concerning the composition of the invention, and its components, as well as the support or vehicle acceptable in the agricultural field apply mutatis mutandis to the kit of the invention.

[0096] The present invention also relates to the use of a kit as defined above for stimulating the growth and / or production of crops, preferably large-scale crops, market gardening, viticulture, forestry, arboriculture and horticulture.

[0097] The present invention further relates to a method for stimulating the growth and / or production of crops, comprising the application of a composition such as defined above; in which the crops are preferably field crops, market gardening, viticulture, forestry, arboriculture and horticulture.

[0098] The present invention also relates to a method for stimulating the growth and / or production of crops, comprising the following steps: a. preparation of a composition as defined above, comprising for example the dilution of the concentrated composition as defined above with a suitable solvent, and / or the implementation of the kit as defined above); and b. applying the composition obtained in step a) to the crop concerned.

[0099] Advantageously, the composition is applied extemporaneously to proceed to step b).

[0100] Likewise, all preferred embodiments described above regarding the composition of the invention, and its components, the agriculturally acceptable carrier or vehicle as well as the kit, apply mutatis mutandis to these other aspects of the invention. EXAMPLES Example 1 - Composition according to the invention

[0101] A protein hydrolyzate corresponding to CAS number 100085-61-8 was mixed with a microalgae extract corresponding to CAS number 20298-86-6 so as to obtain an example of a composition of the invention.

[0102] The protein hydrolyzate and the microalgae extract were added one by one to a cleaned and dried tank, containing the desired quantity of water with continuous stirring, so as to obtain a composition comprising 13% hydrolyzate combined with 3% microalgae extract.

[0103] The temperature during this process was 15 - 35 °C.

[0104] The mixture was then passed through a Dyno-Mill grinder until a particle size conforming to the requirement (corresponding to approximately 20 micrometers) was obtained, in a tank equipped with a turbine mixer.

[0105] Characterization of a composition according to the invention

[0106] The composition thus obtained contains the mixture of amino acids which is mentioned in Table 3 below.

[0107] [Tables3] Amino Acids Composition according to the invention (mg / 100g of composition) TRYPTOPHAN 11.2 Aspartic acid 282.5 Threonine 137.5 Serine 187.5 Glutamic acid 555 Proline 182.5 Glycine 260 Alanine 210 Valine 205 Cysteine ​​50 Methionine 37.5 Isoleucine 145 Leucine 260 Tyrosine 100 Phenylalanine 120 Lysine 20 Histidine 57.5 Arginine 270

[0108] The distribution of molecular weights (MW) of the amino acids present in the composition of the invention is summarized in Table 4 below.

[0109] [Tables4] Distribution (%) PM in Dalton (Da) 3.3 3,000>PM>l 000 2.6 1,000>PM>800 69.7 800>PM>204 24.4 204>PM Example 2 - Biostimulant effect

[0110] The biostimulant effect of the composition prepared according to Example 1 was tested in field trials on different types of crops, in comparison with other biostimulants. Effect on rapeseed

[0111] One of the crops that was targeted to evaluate the biostimulation effect is rapeseed (Brassica napus Lp which is an annual dicotyledonous herbaceous plant of the family Brassicaceae (formerly cruciferous). This plant is characterized by its yellow flowers, the processed seeds of which are used for animal and human food, as well as for the production of biofuel. It can also be used as fodder for livestock. It is one of the main sources of edible vegetable oil in Europe, since rapeseed contains around 45 to 50% oil and 18 to 22% protein. In France, the crop represents approximately 1.1 million hectares for an average yield of 35 q / ha, depending on annual climatic conditions. Rapeseed is the most widely cultivated oilseed in France and is the leading source of oil and protein-rich materials of French origin.

[0112] The effect of the aqueous extract of phycocyanin from the microalgae spirulina (indicated AEC), protein hydrolysates from pork co-products (indicated CP), as well as the mixture of these components (indicated MA) was tested by spraying, and compared to that obtained on control plants (indicated MO). All plants (treated or not) received the same organic fertilization (organic fertilizer HUMIVAL NPK 5-5-2, supplier FERTIVAL, applied at a dose of 1 ton / hectare.

[0113] The grain yield under “normal” irrigation conditions, as well as the quantity of total dry matter of the plant, under “normal” (indicated irrigated) or drought conditions, were measured on the cultivated plants (treated or not) after applications made at the 5-9 leaf stage, then at the flower bud stage, then at flowering.

[0114] The results are shown in Figures 1 to 3. They demonstrate that the components (microalgae extract or protein hydrolysates), as well as their mixture, act positively on rapeseed grain yield ([Fig. 1]). In addition, these results show that the components as well as their mixture act positively on the total weight of the rapeseed plant, which demonstrates a favorable effect on plant growth, whether under normal irrigation conditions (sufficient water - [Fig.2]) or when the plants are subjected to water stress ([Fig.3]). Effect on tomato

[0115] The biostimulant effect of the composition prepared according to Example 1 (BST) was tested, by spraying at several doses, during a field experiment on tomato crops (in Bulgaria, France and Spain). During these tests, different parameters were measured: production (fresh weight / plot, number of fruits), harvest quality (sugar content of the fruits), as well as plant physiology (chlorophyll content, induction of flowering).

[0116] The sugar content was measured according to the Brix scale, which determines, in degrees Brix (°B or °Bx), the fraction of sucrose in a liquid. One degree Brix is ​​equivalent to 1 gram of sucrose per 100 grams of solution.

[0117] The chlorophyll content was measured using a SPAD 502 PLUS clamp-on CHLOROPHYLL METER (supplier SDEC France), which instantly measures the chlorophyll content of plant leaves on a scale of 0.0 to 99.9 (SPAD index).

[0118] Flowering induction was measured by scoring the % of flowers on a scale of 0 to 100%, at the flowering stage corresponding to 10% flowers, and the second flowering at the stage of 50% flowers.

[0119] The results of these tests are shown in Figures 4 to 9.

[0120] These results highlight a dose effect of growth stimulation. statistically significant, and which stands out in terms of effectiveness compared to crops treated with a fertilizer currently used (IDROGENA ENERGY).

[0121] All of these results show that the application of a composition according to the invention to a market garden crop has a positive impact on its growth, its production and the quality of the fruits obtained. Effect on the vine

[0122] The biostimulant effect of the composition prepared according to Example 1 (BST) was tested, by spraying at several doses, on the cultivation of vines in France and the associated wine production, on an Auxerrois grape variety, Pedro Ximenez, merlot.

[0123] During these tests, different parameters were measured: the physiology of the plant (induction of flowering) and production (number of fruits and different criteria of harvest quality).

[0124] Flowering induction was measured by scoring the % of flowers on a scale of 0 to 100%.

[0125] The malic acid content of the harvested grapes was measured by reflectometry using the reflectoquant® system marketed under ref. 1.16128.0001 by Merck Millipore. Malic acid (malate) is oxidized to oxalacetate by NAD under the catalytic action of malate dehydrogenase. The NADH thus produced reduces, in the presence of diaphorase, a tetrazolium salt to a red formazan which is measured by reflectometry.

[0126] The tartaric acid content of the harvested grapes was measured using a special wine photometer for measuring tartaric acid, HI83748-02, according to the supplier's recommendations (HANNA Instruments).

[0127] The alcohol content of the harvested grapes was measured with an OENO Cale mustimeter, according to the supplier's recommendations (Laboratoires Dujardin-Salleron), with which the probable alcohol obtained after complete fermentation is estimated; the calculations being based on the assumption that 16.83g of sugar per liter of must produce 1% volume of alcohol.

[0128] The results obtained with the use of the composition at the dose of 6L / Ha are shown in Figures 10 to 15.

[0129] Among the other criteria of the quality of the harvest, potassium and assimilable nitrogen were also measured to assess the effect of the composition of the invention (Figures 16 and 17). Indeed, potassium acts in the migration and storage of sugars in the fruits. Potassium is a factor for the growth of the vine involved in photosynthesis and for the yield with the neutralization of the organic acids formed. It was measured by ICP AES (iCAP 6000, Thermo), after wet mineralization (analysis method accredited ISO 17025). Assimilable nitrogen is also a factor for the growth of the vine and associated with good vinification of the musts. It was measured by the formalin method, also called the Sôrensen method (MASNEUF and DUBOURDIEU, 1999). Effect on the apple tree

[0130] The biostimulant effect of the composition prepared according to Example 1 (BST) was also tested by spraying using the dose of 6L / Ha, on Spanish apple trees (FUJI, IDARED, PINK LADY) and the associated tree production was evaluated.

[0131] The results obtained are shown in [Fig. 18].

[0132] All of these results demonstrate the advantageous biostimulant effect provided by the composition according to the invention on various crops, whether cereal, market garden, viticultural or arboricultural. REFERENCES

[0133] Agregan R.et al., Medicines 2018, 5, 33; doi:10.3390 / medicines5020033

[0134] Ashour, M.; Hassan, SM; Elshobary, ME; Ammar, GAG; Gaber, A.; Alsanie, WF; Mansour, AT; El-Shenody, R. Impact of commercial biostimulant liquid seaweed extract (TAM) and its bioactive molecules on the growth and antioxidant activities of chili pepper (Capsicum annuum). Plants 2021,10,1045.

[0135] Avila-Pozo P, Parrado J, Caballero P, Tejada M. Use of a Biostimulant Obtained from Slaughterhouse Sludge in a Greenhouse Tomato Crop Horticulturae 2022, 8, 622. https: / / doi.org / 10.3390 / horticulturae8070622

[0136] Colla, G.; Rouphael, Y. Biostimulants in agriculture. Sci. Hortique. 2015,196,1-2.

[0137] Colla, G.; Rouphael, Y.; Canaguier, R.; Svecova, E.; Cardarelli, M. Action Biostimulant of plant-based protein hydrolysates produced by enzymatic hydrolysis. Front. Plant Sci. 2014,5,448.

[0138] Ertani, A.; Pizzaghello, D.; Francioso, O.; Sambo, P.; Sanchez-Cortes, S.; Nardi, S. Capsium chinensis L. growth and nutraceutical properties are enhanced by long-term biostimulants: chemical and metabolomic approaches. Front. Plant Sci. 2014,5,1-2.

[0139] Gonzalez, M.; Cea, M.; Médine, J.; Gonzalez, A.; Dïez, M.; Cartes, P.; Montréal, C.; Naria, R. Evaluation of biodegradable polymers as encapsulating agents for the development of a controlled-release urea fertilizer using biochar as a carrier material. Sci. Environ. 2015,505,446-453.

[0140] Hassan, SM; Ashour, M.; Soliman, AAE; Hassanien, HA; Alsanie, WF; Gaber, A.; EIShobary, ME The potential of a novel commercial seaweed extract to boost the morpho-agronomic and bioactive properties of Eruca vesicaria (L.) Cav. Sustainable Development 2021,13,4485.

[0141] Kapoore RV, Wodd EE, Llewellyn CA, Algae biostimulants: A critical look at microalgal biostimulants for sustainable agricultural practices. Biotechnol Adv. 2021, 49, 107754.

[0142] MASNEUF I. and DUBOURDIEU D., 1999. Assimilable nitrogen: interest of its dosage by formol titration; study of some parameters at the origin of the variations of its content in musts. Rev. Œnol., n°93, 31-32.

[0143] Mosa W FA, Ali HM, Abdelsalam NR The utilization of tryptophan and glycine amino acids as safe alternatives to chemical fertilizers in apple orchards. Environ Sci Pollut Res Int 2O21.january;28(2): 1983-1991

[0144] Yes, L.; Zhao, X.; Bao, E.; Li, J.; Zou, Z.; Cao, K. Bio-organic fertilizer with reduced chemical fertilization rates improves soil fertility and enhances tomato yield and quality. Sci. Rep. 2020,10,177.

[0145] Rouphael Y; Colla G. Synergistic biostimulatory action: designing the next generation of plant biostimulants for sustainable agriculture. Frontiers in Plant science. 2018,9,1655.doi: 10.3389 / fpls.2018.01655

[0146] Searchinger T. The great balancing CCT: Installment 1 of creating a sustainable food future, World resources Institute: Washington DC, USA. 2O13.pp 1-15.

[0147] Tejada, M.; Rodrîguez-Morgado, B.; Gomez, I.; Franco-Andreu, L.; Bemtez, C.; Parrado, J. Use of biofertilizers obtained from sewage sludge on maize yield. EUR. J.Agron. 2016,78,13-19.

[0148] Tejada, M.; Rodriguez-Morgado, B.; Paneque, P.; Parrado, J. Effects of foliar fertilization of a biostimulant obtained from chicken feathers on corn yield. EUR. EAgron. 2018,96,54-59.

Claims

1.

2.

3. Claims Composition comprising a microalgae extract, a pork bristle protein hydrolyzate and a pork lung mucus protein hydrolyzate. Composition according to claim 1, characterized in that the microalgae extract is a spirulina extract, preferably a phycocyanin extract. Composition according to either of claims 1 and 2, characterized in that it contains the following quantities of amino acids. Amino Acids Composition according to the invention (mg / 100g of composition) TRYPTOPHAN 8-20, preferably 10-15 Aspartic acid 250-300, preferably 270-290 Threonine 120-150, preferably 125-145 Serine 160-200, preferably 170-190 Glutamic acid 500-700, preferably 525-650 Proline 150-200, preferably 165-195 Glycine 230-300, preferably 240-280 Alanine 180-240, preferably 200-230 Valine 160-230, preferably 185-215 Cysteine ​​30-80, preferably 40-70 Methionine 20-60, preferably 30-50 Isoleucine 100-180, preferably 130-160 Leucine 200-320, preferably 230-300 Tyrosine 60-140, preferably 10-80-120 Phenylalanine 100-140, preferably 115-135 Lysine 10-50, preferably 15-35 Histidine 40-80, preferably 45-65 Arginine 230-300, preferably 240-290 Composition according to any one of claims 1 to 3, characterized in that it contains amino acids of which

4. molecular weight distribution is 3-4% between 1000 Da and 3000 Da, 2.5-3% between 800 Da and 1000 Da, 68-72% between 204 Da and 800 Da and 23-25% below 204 Da.

5. Composition according to any one of claims 1 to 4 comprising 1-15% hydrolyzate and 1-5% microalgae extract, preferably spirulina extract, and optionally a support or vehicle acceptable in the agricultural field.

6. A method of preparing a composition according to any one of claims 1 to 5, characterized in that it comprises: - providing a hydrolyzate of pig bristles and a hydrolyzate of pig lung mucus, - providing a microalgae extract, and - mixtures thereof.

7. Preparation process according to claim 6, characterized in that it comprises: - providing a chemical hydrolyzate of pig bristles and providing an enzymatic hydrolyzate of pig lung mucus, - providing a spirulina extract, preferably an aqueous extract; and - mixtures thereof.

8. Use of a composition according to any one of claims 1 to 5 as an agricultural biostimulant.

9. Use of a composition according to claim 8, under normal irrigation conditions or under water stress conditions.

10. Use of a composition according to any one of claims 8 and 9, for large-scale crops, market gardening, viticulture, forestry, arboriculture and horticulture.

11. Use of a composition according to any one of claims 8 to 10, characterized in that said composition is applied by spraying onto the crop, preferably in quantities ranging from 1 to 100 / ha.

12. Kit comprising: - at least one hydrolyzate of pork bristle protein, one hydrolyzate of pork lung mucus protein and one microalgae extract, in particular one spirulina extract, or a mixture thereof; - at least one support or vehicle acceptable in the agricultural sector, - possibly instructions for use, for the preparation of a biostimulant.

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