Seaweed alkaline extract of the genus lessonia spp, method for the production thereof and use as a biostimulant, fertiliser and / or soil improver

Abstract

The present invention relates to a seaweed alkaline extract, in particular an alkaline extract of Lessonia spp, more specifically an alkaline extract of Lessonia berteroana with a pH equal to or greater than 11. The invention also relates to a method for the production thereof and its use as a biostimulant, fertiliser and / or soil improver.

Description

Qualification Alkaline extract of seaweed of the genus Lessonia spp, its production method and its use as a biostimulant, fertilizer and / or soil improver Summary The invention relates to an alkaline extract of seaweed, in particular an alkaline extract of Lessonia spp, more particularly an alkaline extract of Lessonia berteroana with a pH equal to or greater than 11, the method for its preparation and its use as a biostimulant, fertilizer and / or soil improver (Abstract). 1. Seaweed and its components. Extracts and extraction procedures Seaweed, also known as macroalgae, is a plant organism that grows in aquatic environments, mainly in the ocean. It is divided into three main classes according to its pigmentation: a) Brown, such as kelp and wakame, which are commercially recognized in the food and cosmetics industries as a source of alginates; b) Green, such as Chlorella and Spirulina, which are known as commercial sources of chlorophyll and proteins useful for making dietary supplements; and c) Red, such as Agarophyton chilensis (pelillo), which are a recognized source of carrageenan fiber, a food additive used as a thickener and stabilizer. [1] [2], [3] Brown algae, or Phaeophyceae, have been commercially exploited by various industries, including, among others, the food, cosmetics, and agribusiness sectors. In agribusiness, the use of complex compositions derived from their extracts is notable, which, depending on their components, achieve beneficial effects as biostimulants and / or fertilizers. In this regard, these compositions derived from brown algae extracts improve plant growth and resistance to adverse conditions, as well as their use as soil improvers. In every field, seaweed extracts exhibit obvious advantages over the use of raw material, including the concentration of its beneficial agents, allowing a smaller amount of the product to be used to obtain the same, or even greater, benefits than using raw algae. Another advantage associated with seaweed extracts corresponds to the conservation, durability and reproducibility of the desired effects, and versatility in application through the change of physical format for its application and / or dilution, which results in the optimization of storage space, among many other advantages. The composition of an algal extract is not a constant that depends solely on the raw material used, but is closely and inextricably linked to the extraction process. This allows us to understand, for example, the plurality of commercial presentations of A nodosum extracts and their different compositions such as Super fyfty prime®, Stimplex®, BM-86®, Bayfola Alga®, Atlantica fitomare®, and Basfoliar algae®, which ultimately lead to technical effects in different areas of agriculture. There are a variety of extraction processes for algal biomass content, and their selection leads to different compositions. Processes can be classified according to the dominant physical medium that allows for extraction. On the one hand, we find in the state of the art aqueous extraction, which is subdivided according to the pH at which the process is carried out. Organic phase extraction, increased temperature, pressure, assisted by sonication, and even processes that do not rely solely on physical means but also on enzymatic processes, and a combination of these, are also used.Likewise, each selection of extraction strategies must be accompanied by a correct selection of operational conditions and variables, such as the application time of the extractive measure and even its reversal, such as re-neutralization in processes that depend on pH, such that they allow the optimization of the desired components, and ultimately, the optimization of the technical effect sought. This application discloses and claims a novel a) process for extracting algal biomass, wherein the raw material is brown algae of the genus Lessonia spp., and the process comprises an alkaline extraction with a pH above 11, and b) the alkaline extract of Lessonia spp. resulting from this method. As demonstrated in the examples provided, this selection of variables and raw materials for the inventive method led to a product with unique qualities that advantageously affected plant germination processes through direct biostimulation, improved leaf and root content in plants, as well as productivity in fruit trees; and also improved the capacities of the treated soil, compared to control tests without stimulation and to other competing algae extracts present on the market. Figures text Figure 1. Controlled growth chamber and determination of root and leaf length, in 3 doses of alkaline extract of Lessonia spp. Figure 2. Summary of biostimulation assay, as a function of relative biostimulant potential. Dry matter production is graphed, both in leaves and roots, comparing the control (which is the baseline = 0), the best result obtained with market products, and two extractions with alkaline extract of Lessonia spp. Figure 3. Average increase in Beta-Glucosidase (BGLU) activity in soil treated with alkaline extract of Lessonia spp compared to the control as base 0, in plantations of treated fruit trees. Figure 4. Multispectral image analysis with NDVI index, indicates that the combined treatment of two products based on the alkaline extract of Lessonia spp, in a mixed strategy of soil and foliar application, produced significant changes in the greenness and volume of leaves. Figure 5. C represents the control replicates, while D represents the results obtained by applying the alkaline extract of Lessonia spp. Where it is observed that the alkaline extract reduces both the EC and the presence of N-NO3. Figure 6. Comparison of the change in soil pH over the baseline, the control and the alkaline extract of Lessonia spp, medium 30 and 60 days after application (DAD) of the extract. Figure 7. Images show root development in lettuce seedlings, A is the control and B is the application of the alkaline extract formulation of Lessonia spp with microelements applied foliarly. Figure 8. Abundance of harvest visually observed between control A and the protocol comprising alkaline extract of Lessonia spp. Figure 9. Shows that roots treated with alkaline extract of Lessonia spp outperformed the control with stronger, more abundant roots and greater exploration depth. Details of the invention: Generalities The commercial components of seaweed are varied, varying in content across macroalgae families and also within species and subspecies, with a certain degree of seasonal variation within the same organism. Their components can be generically grouped into the following categories: • Protein: Seaweed can contain up to 60% protein by dry weight, making it a valuable source of plant protein. • Fibers: Seaweed fibers are composed primarily of polysaccharides such as alginate, carrageenan, and agarose. Alginate is a water-soluble fiber extracted primarily from brown algae, such as kelp. It is used in a variety of industries as a thickener, gelling agent, and stabilizer. Alginate is a polysaccharide composed of 0-D-mannuronic acid (M) and αL-guluronic acid (G), which are randomly distributed within the polymer chain structure. Additionally, alginate can present monomer sequences of M and G residues, as well as alternating residues, thereby obtaining M blocks and G blocks (homopolymeric), and MG blocks (heteropolymeric). The percentage ratio of the distribution of M and G blocks depends on the species from which the alginate is obtained.Likewise, parameters such as geographical location, habitat, and species determine the distribution of these groups, having an effect on the physicochemical properties according to the M / G ratio of the alginate obtained. [4] Other relevant polysaccharides are carrageenan and agarose, extracted mainly from red algae and used in the food and cosmetics industries. • Minerals: Some seaweeds, such as kelp, are an excellent source of essential minerals such as iodine, calcium, iron, and magnesium. • Vitamins: They contain a variety of vitamins, including B vitamins (B1, B2, B3, B6, B12), vitamin C and vitamin E. • Lipids: Lipids are present in small amounts ranging from 1 to 5% of the dry matter of algae. However, the majority of lipids are polyunsaturated fatty acids (JÜ-3 and (JÜ-6). Red and brown algae predominantly contain the 20-carbon polyunsaturated fatty acids eicosapentaenoic acid (EPA, CÜ-3, C 20:5) and arachidonic acid (AA, CÜ-6, C 20:4)[l]. Bioactive Compounds: This generic definition includes: o Polyphenols: Seaweeds, particularly red algae, are known for their polyphenol content. These compounds have antioxidant properties, such as phycobiliproteins and phlorotannins. o Bioactive polysaccharides identified in seaweed extracts are galactans (carrageenans and agarans) and floridean starch, ulvans, starch, lignin and cellulose, laminarin and fucoidan [5]. Fucoidan is known as an elicitor of the local defense response and a priming agent in systemic plant defense [6]. Alginates and their oligoderivatives act in stimulating plant growth, development and resistance [7]. o Chlorophyll: Green algae, such as chlorella and spirulina, are rich in chlorophyll, the pigment responsible for their green color. Chlorophyll has been studied for its potential antioxidant and detoxifying properties.o Unusual secondary metabolites, such as sesquiterpenes, diterpenes, meroterpenoids, phlorotannins and spheroids. Some compounds belonging to these families exhibit varied biological activities such as antibacterial, antioxidant among others.[8]. Seaweed and agroindustry Macroalgae, due to their unique properties, have significant potential for agribusiness. Below are some of the ways in which seaweeds relate to agribusiness: Biostimulants and Fertilizers: Seaweed extracts contain bioactive compounds that can stimulate plant growth, increase disease resistance, and improve crop quality. Seaweed biostimulants can also help plants withstand stressful conditions such as drought and soil salinity. Soil Enhancers: Macroalgae can improve soil structure, increasing its water and nutrient retention capacity and promoting beneficial microbial activity. Seaweed residues are also often used as organic amendments to improve soil fertility. Pest and Disease Control: Some seaweed extracts have natural repellent and pesticide properties that can be used in agriculture to control plant pests and diseases in a more sustainable and organic way. Macroalgae extracts Macroalgae extracts are a valuable tool in agriculture, being used as biostimulants, fertilizers, and soil improvers. These extracts have the potential to significantly improve plant growth, strengthen their resistance to disease, and enhance crop quality. Numerous scientific studies support the use of seaweed extracts in various conditions, both normal and stressful.[3] Macroalgae extracts in agriculture Macroalgae-derived biostimulants represent the fastest-growing biostimulant industry. [9] They have been successfully used to prevent and / or recover from the damaging effects of heat and frost, and have demonstrated improved growth and survival under drought and salinity conditions.

[0010] Application of algal extracts reduced relative water loss and promoted plant recovery from short-term (4 to 10 days) drought stress in Arabidopsis, tomato, soybean, and wheat.[9],

[0011] ,

[0012]

[0012]

[0013]

[0014] These studies also observed a reduction in leaf wilting and a significant increase in plant growth. Chickpea plants treated with seaweed extracts before exposure to salinity stress showed a significant increase in vegetative growth compared to their control, and are salt-stressed plants when not treated with seaweed extracts.

[0015] Seaweed extracts also improved the growth of rice and wheat grown under saline conditions.

[0016] ,

[0017] The use of hydrogels, such as alginate, as soil improvers is widely known. Their improvement mechanism is closely related to their three-dimensional networks formed by cross-linked polymers mediated by calcium ions, which can retain a significant amount of water due to their high content of hydrophilic functional groups. This cross-linked material not only reduces irrigation water loss but also functions as a slow releaser of fertilizing agents and cations essential for plant development, such as potassium. Additionally, the positive influence of macroalgae extracts containing alginate on the microorganisms present in the plant's rhizosphere has been reported.

[0018] Extraction methods In the industry, there are multiple processes that allow the extraction of general and / or bioactive components from macroalgae for the production of relevant agroindustrial products. These processes These include aqueous extractions, which are performed at different pH levels, solvent extractions, and extractions assisted by physical adjuvants such as pressure, microwaves, ultrasound, and supercritical fluids. A recent trend in extraction methods includes the use of enzymes and, in some cases, the combination of the aforementioned techniques to maximize the yield of beneficial compounds. Each process seeks to improve the yield in obtaining and preserving certain components of macroalgae, allowing for the enhancement of the technical effect of said agent. In fact, while aqueous-neutral extraction (also known as dry water extraction) allows for efficient polyphenol recovery, alkaline aqueous extraction limits its yield because it modifies these components. On the other hand, acidic aqueous extraction degrades polyphenols until they are eliminated. The need to establish a known relationship between the desired analyte and the method employed is not limited to the example of polyphenols. Alginate is a component of macroalgae of high commercial value; it is very slightly soluble in its polymerized form. It becomes soluble as a result of both an acidic and alkaline environment, which allows its depolymerization.This step is not qualitative, and both the pH value and the time to which the raw material is subjected lead to different degrees of depolymerization and hydrolysis seen as a continuum in the reduction of molecular weight. Alkaline extraction is the most reported industrial process in the production of extracts from A. nocfosum

[0019] -

[0021] . The protocol, initially proposed by Milton in 1952, includes the steps of obtaining algal biomass, sol ubi lization using alkaline solutions, such as NaOH, KOH, CaCO3, Na2CO3 or K2CO3, sometimes accompanied by heating to temperatures between 70 Q and 100°C, and in certain cases, the application of pressure between 275 and 827 kPa. After solubilization is complete, the extract is neutralized using citric or phosphoric acid. This process results in the decomposition of the insoluble polysaccharide fiber chains into smaller, soluble units.

[0020] As previously described, alkaline treatments have an impact on the polyphenol content achieved in the final product extract, leading to a particular hydroxylation pattern of the original polyphenol.

[0019] The well-known commercial biostimulants Maxicrop® in the United States, Seasol® in Australia and Acadian® in Canada are the result of different alkaline extraction processes of A. nodosum. Other examples of extraction methods from algal material are: • The simplest in technical terms are the aqueous-neutral or also known as water-based extractions. As its name indicates, this is a process that essentially involves contacting dried seaweed flour with water, raising the temperature and mixing for a period of time, to finally separate the aqueous phase from the solid waste by, for example, filtration.

[0020] These extracts have been reported to be rich in agents that exhibit phytohormone-like activity.

[0022] • Acid aqueous extractions comprise the steps of hydrating the algal flour, and then treating it with sulfuric acid or hydrochloric acid at 40-50 °C for 30 min.

[0020] This method removes complex phenolic compounds and increases the depolymerization of polysaccharides.

[0023] This method is generally used for the extraction of sulfated polysaccharides containing fucose.

[0023] Sulfated algal polysaccharides are a class of bioactive compounds in algal extracts that promote plant growth.

[0024] They purified fucoidans from A. nodosum by acid hydrolysis. AZAL5® is a commercially available biostimulant manufactured from A. nodosum, which is extracted by acid hydrolysis.

[0025] Patents comprising this method are described CN112479782A. In general, the aforementioned are the dominant technical methods in the area in the field of agriculture. However, the list of extraction methods is extensive and includes other physically assisted extraction methods such as microwaves to extract fucoidan, sodium alginate and phenolic compounds from A. nodosum

[0026] ; Ultrasound to isolate bioactive compounds from A. nodosum and Laminaria hyperborea CN104447008A; Pressurization that raises the temperature of the solvents facilitating the extraction of bioactive compounds.

[0027] (Enzyme-assisted, this method takes advantage of the fact that sometimes the goal is not to preserve bioactive principles but to degrade them for a more efficient incorporation into the receiving system.In this framework various carbohydrate degrading enzymes and proteases such as Viscozyme, Cellucast, Termamyl, Ultraflo, carrageenan, agarase, xylanase, Kojizyme, Neutrase, Alcalase and Umamizyme are commonly used for the extraction of bioactive compounds from seaweed.

[0028] The application of hydrolytic enzymes converts water insoluble chemical components of the selected algal biomass into water soluble products, thus eliminating the problem of water solubility of bioactive compounds seen in Wijesinghe

[0028] and CN112661543B. In short, macroalgae extracts are complex chemical compositions whose components and quantities depend on the species of macroalgae used as raw material and the process used for their extraction. Differential effect as a biostimulant, fertilizer or soil improver of seaweed extracts depending on the procedure. Goñi et al. compared the composition and effects of two Ascophyllum nodosum-derived biostimulants on Arabidopsis thaliana. The effect on plant mRNA profile induced by macroalgae extracts obtained using two different methods (ANE A and ANE B) was compared. ANE A was manufactured using a patented process at >100 °C at neutral pH, while ANE B was manufactured using a patented process at >100 °C at alkaline pH. Notably, despite starting with identical starting material, the differences in the processing they underwent resulted in significantly different compositions. For example, a 4-fold difference in the levels of determined polyphenols was determined. The level of uranic acids (representing the processed alginate content) was also significantly different between the two extracts, being 40% higher in ANE B than in ANE A.Furthermore, the concentrations of fucoidan, laminarin and mannitol in ANE A were found to be 15–33% higher than in ANE B. Consistently, notable differences in the biostimulation effects on plants were observed, as seen in the parameters of plant height and the number of leaves in each rosette.

[0029] . Another example of the differential biostimulant effects of macroalgae extracts achieved by different extraction methods is described by Fauziatul Fitriyah et al. In this study, the composition and effects of three biostimulants derived from the macroalga S. sargassum obtained by different methods (alkaline pH 9, neutral pH 9, and acidic pH 2 extraction) were compared, resulting in different phytohormone contents in each extract and a different germination and growth profile in a maize model. In this work, the researchers conclude that Sargassum spp. extracts obtained by acidic, alkaline, and water-based (neutral) extraction methods improved shoot and root growth of maize plants during the early growth stage. Among these, the acidic extract showed the greatest growth promotion among other extracts with the lowest phytohormone contents.

[0030] Apparently, the selection criteria for macroalgae extraction methods combine a delicate balance between variables and operational conditions that are extreme enough to maximize the extractive efficiency, but at the same time gentle enough so that the extraction does not degrade the components, known or unknown, responsible for the biostimulant / fertilizer / soil-improving action. As evidenced in the background presented above, macroalgae extracts are complex and determined by both the raw material and the extraction method used, resulting in different technical results. Given the growth of the macroalgae-derived biostimulant, fertilizer, and soil improver industry, there is a need to explore alternative extracts with improved properties. This search can be driven by an alternative raw material or a novel extraction method, or a combination of these variables. Selection of variables of the invention Raw material: The inventors of this application, after exploring the field of biostimulants / fertilizers and / or soil improvers derived from macroalgae extracts, chose the genus Lessiona spp. as their raw material. This genus belongs to the Lessoniaceae family, within the group of brown Phaeophyceae. These plants are known for their ability to form shrub-like structures on the ocean floor. They have a characteristic morphology comprising branched stems and elongated, laminated leaves resembling fern fronds. These leaves are often serrated or have wavy edges. They are found in marine environments, in cold and temperate waters, especially in coastal and rocky areas. Their rational selection, without limiting the amazement to its unexpected technical consequences, was based on: I. Remarkable compositional qualities in terms of total protein and total crude fiber have been described in macroalgae of the genus Lessonia spp.

[0031]

[0032] This fact is known to the flour industry comprising ground whole material from macroalgae of the species Lessonia spp useful as fertilizers, and are commercially available from a plurality of companies, for example, in the presentations of "Algaex" from (https: / / algaex.pe / en / raw-materials / ) and "Gracias algas" from (https: / / gracias-algas.com / ). Moreover, in the field of patent applications, this species has been suggested as an alternative to other species of the brown macroalgae genus in classifications in the field of biostimulants / fertilizers and / or soil improvers such as, for example: W02020254407A1 (family member of CL2021003384A1), where Lessonia spp is presented as an alternative to Laminarias spp, and through an acid extraction that results in an acid extract of Laminarias spp, and W02016007987A1, where Lessonia spp is presented as an alternative among other possibilities (paragraph

[0012] ) to the preferred Laminaria hyperborea or Laminaria digitata, and through an alkaline extraction that results in an alkaline extract of macroalgae. II. There are known antecedents of the use of neutral aqueous extracts of Lessonia nigrescens and Lessonia flavicans that significantly increased enzymatic activities in the soil treated with said extracts, such as invertase, urease, proteinase and phosphatase compared to untreated soils.

[0033] III. The effect of two different solvent-extracted polysaccharides from Lessonia nigrescens (LNP) has also been described, which promoted plant growth, decreased membrane lipid peroxidation, increased chlorophyll content, enhanced antioxidant activities, and coordinated intracellular ion efflux and compartmentalization. They also induced plant resistance to salt stress.

[0034] Extraction method: IV. The rationale for selecting the process consisted of exploring ranges of steps typically discouraged by their degradative nature with respect to known biostimulants. In particular, this one describes an aqueous extraction process mediated by extreme alkalization, ensuring a pH above 11 throughout the process. Under a counterintuitive rationale in the sense of providing a method that includes mild variables and conditions to extract the potential components of Lessonias spp. that the prior art has suggested to understand, the invented process was executed under frankly degradative conditions. In particular, an extremely alkaline step where the addition of the alkalizing agent is adjusted to achieve a pH of 14 at room temperature for at least 12 hours, much more extreme than the moderate conditions commonly used in the prior art that limit the pH range between 8-10.5 and sometimes even end the process with neutralization. The rationale used, without limiting the scope of the surprising effects evidenced by the inventors, was to maximize the degradative performance of alginate to its minimum soluble elemental blocks, even at the expense of degrading other components. biostimulants, such as phytohormones, polyphenols, carotenoids and other useful secondary metabolites.

[0033] Additionally, a rationally sought advantage was to take advantage of the known effect of increasing solubility as a function of extreme pHs, and thus achieve a product optimized for current agricultural disposal processes such as foliar spraying and fertigation. V. The selection of the alkalizing agent, in particular the counterion, was not random; KOH was chosen so that the alkaline extract of Lessonia spp would serve as a slow-release agent of the potassium ion (K), essential for the development of plants and their rhizosphere. EXAMPLES The following examples illustrate embodiments within the scope of the invention, but do not constitute a limitation on the scope of the subject matter and processes of the invention. EXAMPLE 1 Preparation of alkaline extract of Lessonia spp Lessonia spp. macroalgae meal: Macroalgae of the Lessonia spp. species, particularly Lessonia berteroana biomass, were collected off the Chilean coast, from Coquimbo to northern Chile, according to quotas set by a panel of experts and monitored by SERNAPESCA. The collected material was dried to a moisture content of 7% and ground. Alkaline extraction of Lessonia spp on a laboratory scale 1. A solution of KOH in water was prepared, 2. The seaweed flour was added slowly and continuously to a final concentration of 10% (w / v) under continuous stirring at a rate of 35 revolutions per minute. The pH was assessed, and KOH was added to adjust to pH 13.54. 3. The composition was stirred for 12 hours at room temperature. 4. Stirring was stopped, and the solids were separated by centrifugation at 13,000 rpm xlO. 5. The soluble phase corresponding to the alkaline potassium extract of Lessonia spp was separated. 6. The soluble phase of the alkaline extract of Lessonia berteroana was packaged at 10% (w / v) pH 13.54. Modification of the process for industrial scale The separation step of suspended solids was carried out by decantation for 24 hours until the soluble phase was translucent. Example 2 Characterization of the alkaline extract of Lessonia spp. The extract was stained, and 400 MHz nuclear magnetic resonance (NMR) analysis was performed using Ultrashield (Bruker) equipment. A signal characteristic of chlorophyll hydrocarbon chains was detected at 1.8 ppm; this was confirmed by thin-plate chromatography. The resonance identified high-intensity signals in the aliphatic region, and no signals were observed in the aromatic region (between 7 and 9 ppm) or in the aldehyde region. The final alginate content was determined by alcoholic precipitation and subsequent drying at 40°C, giving an alginate concentration in the alkaline extract of Lessonia spp. of 0.01 g / mL. The sugar content relative to the alginate mass was greater than 95%, with the remaining 4% being substances such as glycosylated proteins and phenols. An intrinsic density of 174.5 was determined for the extract in an Oswald viscometer which, using the Mark-Houwink relationship, allows an average weight of 100,000 g / mol to be deduced, which is an expected value at the lower limit of the alginate degradation process. The third-party analytical laboratory analyzed the alkaline extract of Lessonia spp exhibiting the following qualities: pH 11.8 Density 1.0904 g / ml Organic Carbon 2.9% (w / w) Total Nitrogen less than 0.5% (w / w) Potassium 5.16% (w / w) K2O Fulvic acid 9.33% (w / w) Humic acid 0.63% (w / w) Total humic extract 9.96% (w / w) Heavy metals (Ar, Cd, Hg and Pb) less than 0.5% (w / w) TOTAL AMINO ACID PROFILE ASPARTIC ACID 0.043 g / 100 g GLUTAMIC ACID 0.128 g / 100 ALANINE 0.071 g / 100 g TYROSINE 0.026 g / 100 g VALINE 0.054 g / 100 g METHIONINE <0.103 g / 100 g LEUCINE 0.125 g / 100 TOTAL CYSTEINE 0.459 g / 100 g HYDROXYPROLINE, SERINE, GLYCINE, <0.01 g / 100 g HISTIDINE, ARGININE, TAURINE, <0.01 g / 100 g THREONINE, PROLINE, ISOLEUCINE, <0.01 g / 100 g PHENYLALANINE, LYSINE, TRYPTOPHANE <0.01 g / 100 g TOTAL PROTEI NA 0.550 g / 100g Example 3 Biostimulant use of alkaline extract of Lessonia spp on plant model 3-1 Biostimulation study in hybrid corn seeds (Agriservice Laboratory) The effect of alkaline extract of Lessonia spp on the germination and early growth activity of hybrid corn seeds was tested. Equivalent procedures have been previously described.

[0030] Corn seeds were treated with water as a CONTROL, alkaline extract of Lessonia spp and nine other commercial extracts derived from macroalgae and humic acids available in the market. Corn seeds were germinated under controlled conditions individually in test tubes, and inert structures similar to "beads" were used as a substrate to support the roots, to facilitate the detachment and cleaning of the same roots and thus obtain an objective result. All germinated seeds were cultivated for the following 30 days to observe the dry weight of the sprout and root.The dosage concentrations used were the optimal commercial ones recommended by the manufacturers, with 5 repetitions each, and in the case of our product, dosages were made with dilutions of 0.1%, 0.5% and 1%. The biostimulant effect of the alkaline extract of Lessonia spp, resulted in an increase in the mass of dry matter in leaves (202%) and roots (145%) compared to the untreated CONTROL group, and higher than other market reference products from 100% leaves to 45% in roots. Results are summarized graphically in figures 1 and 2. 3-2 Field trials on mandarin and avocado in OVALLE + plum and cherry in MARIA PINTO (Farmer Manuel Contador + Agrícola Zunino) Objective: Validation and optimization of the product as a biological activator by measuring the activity of the beta-glucosidase enzyme in four crops and two agricultural areas, with and without the application of other amendments. Result: The concentrations at which the product produces significant changes in the activity of the Beta-Glucosidase enzyme in the soil are determined, as a form of correlation with the microbiological activity, with responses higher than the control between 15% and 72%. Results are summarized graphically in Figure 3. 3-3 Essay (responsible) Study of the extension of the photosynthetic capacity of the leaf, using NDVI images in Autum Crisp table grapes (Advisor Hernan Mena at Exportadora Fomagri SA) Objective To validate a dosage strategy to extend the photosynthetic activity of leaves. Result Treatment with a joint application of alkaline extract formulations of Lessonia spp, was superior to the control treatment with the usual field applications and with less products, extending the photosynthetic activity and increasing the amount of Fe in the leaf. Going from an NDVI Index of 0.75 in the control to 0.85 with the alkaline extract treatment of Lessonia spp. Results are summarized graphically in Figure 4. 3-4 Essay (responsible) Essay in Cerezos (Advisor Jordi Casas) Objective: Field test, which correlates soil and foliar doses of the different products with soil quality parameters up to commercial quality. Result Although it is under development and ends in April 2024. Initial data showed that it has a positive and significant effect on microbiological activity, on the presence of carbon in the soil and on the correlation between EC and N-NO3. It was confirmed that the addition of the alkaline extract of Lessonia spp. in the arrangement used to achieve the beneficial effects did not change the soil pH. The results are summarized graphically in Figures 5 and 6, and Table 1. Table 1. Summary of the averages obtained between the control and the alkaline extract 3-5 Trial (responsible) Trial on Vegetable Seedbeds. (Agrícola Nuovo Cultivo) Objective To confirm that the alkaline extract of Lessonia spp constitutes a balanced growth alternative in vegetables. Result: It was visually confirmed that the alkaline extract of Lessonia spp. produced significant and positive changes in the roots, replacing a rooting agent and an amino acid in the program. It produced more and better roots than the normal program. Results are summarized graphically in Figure 7. 3-6 Field trial with lettuce. (Agrícola Dos Marías) Objective To confirm that the alkaline extract of Lessonia spp constitutes a balanced growth alternative in Vegetables. Result: It was visually confirmed that the alkaline extract of Lessonia spp. produced significant and positive changes in the roots, replacing a rooting agent and an amino acid in the program. It produced more and better roots than the normal program. Results are summarized graphically in Figure 8 and Table 2. Table 2 shows the summary of the average weight, quantity of plants and dry matter where the alkaline algae extract stands out, achieving 10.2 tons of clean lettuce over the control and reaching a higher quality that translates into a higher sales price. 3-7 Trial (responsible) Trial on blueberries (Agrícola HUNE) Objective To see the behavior of the roots in Blueberry in the ground. Result There was a significant increase in the presence of deeper and more abundant roots, seeing a greater abundance of roots in plants treated with alkaline extract of Lessonia spp compared to the control. Results are summarized graphically in Figure 9. Literature [1] L. Misurcová, "Chemical Composition of Seaweeds," in Handbook of Marine Macroalgae, Wiley, 2011, pp. 171-192. doi: 10.1002 / 9781119977087.ch7. [2] JS Sangha et al., “Carrageenans, sulphated polysaccharides of red seaweeds, differentially affect Arabidopsis thaliana resistance to Trichoplusia ni (Cabbage Looper),” PLoS One, vol. 6, no. 10, 2011, doi: 10.1371 / journal. pose.0026834. [3] W. Khan, R. Zhai, A. Souleimanov, A. T. Critchley, D. L. Smith, and B. Prithiviraj, “Commercial Extract of Ascophyllum nodosum Improves Root Colonization of Alfalfa by Its Bacterial Symbiont Sinorhizobium meliloti,” Commun Soil Sci Plant Anal, vol. 43, no. 18, 2012, doi: 10.1080 / 00103624.2012.708079. [4] Y. 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Claims

CLAIMS 1. An alkaline extract of algae Characterized because said algae belongs to the genus Lessonia spp.

2. An alkaline seaweed extract according to claim 1, characterized in that the composition has a pH greater than 11.

3. An alkaline extract of algae according to any of the preceding claims, characterized in that said Lessonia is selected from Lessonia berteroana.

4. An alkaline extract of algae according to any of the preceding claims, characterized in that it comprises 10% (w / v) of Lessonia berteroana.

5. A process for the preparation of an alkaline extract of algae of the genus Lessonia spp., characterized in that it comprises the steps of: a. Preparing a KOH solution. b. Slowly and continuously adding Lessonia flour to a final concentration of 10% (w / v) under continuous stirring. c. Adjusting the pH with KOH to pH 13.

54. c. Stirring the composition for 12 hours at room temperature. d. Stop stirring and discard the suspended solids. e. Separation of the soluble phase.

6. The process according to the preceding claim, CHARACTERIZED in that during stirring it comprises the supplementary addition of KOH to reach a pH of 13.54 7. Use of an alkaline extract of Lessonia spp CHARACTERIZED because it serves as a biostimulant / fertilizer / soil improver.

8. Use of the alkaline extract of Lessonia spp according to the previous claim, CHARACTERIZED in that it serves as a plant biostimulant.

9. Use of the alkaline extract of Lessonia spp according to claim 8 CHARACTERIZED in that the plant is corn, increasing the mass of roots and green parts.

10. Use of the alkaline extract of Lessonia spp according to claim 8 CHARACTERIZED in that the plant is grape, increasing the Fe content in the leaves.

11. Use of the alkaline extract of Lessonia spp according to claim 8 CHARACTERIZED in that the plant is vegetables, increasing the volume and mass of the roots.

12. Use of the alkaline extract of Lessonia spp according to claim 8 CHARACTERIZED in that the plant is blueberries, increasing the abundance and depth of the roots.

13. Use of the alkaline extract of Lessonia spp according to claim 7, CHARACTERIZED in that it serves as a soil improver and / or fertilizer, evaluated by the increase in the activity of the Beta-Glucosidase enzyme in the soil.

14. Use of the alkaline extract of Lessonia spp according to claim 7, CHARACTERIZED in that it serves as a soil improver and / or fertilizer, evaluated by the increase in the content of carbon and total nitrates in the soil.

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