RU2845351C1 - Method of producing plant growth biostimulants, phytohormones, antistress agents from seaweed - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: disclosed is a method of processing seaweed to obtain an extract containing complex polysaccharides, phytohormones, vitamins, polyphenols, organic acids, antimicrobial and anti-stress agents, plant growth stimulants, which includes washing seaweed of salts and impurities and mechanical grinding – mechanical activation. Method also includes enzymatic hydrolysis using enzymes from a range of amylase, protease, pectinase, cellulase, alginate lyase, betagluconase in acid medium with acetate or citrate buffer, chemically inactivating enzymes, stabilizing and correcting pH with liquid potassium or sodium peat humate, and enriching the obtained extract with humic substances.

EFFECT: invention provides stimulation of growth processes of plants and reduction of negative effect of abiotic stress factors.

6 cl

Description

The invention relates to agriculture, to the technology of processing seaweeds and concerns a method for obtaining complex polysaccharides, phytohormones, vitamins, polyphenols, organic acids, antimicrobial and anti-stress agents, plant growth stimulants, and other bioactive substances of agronomic importance from seaweeds in the form of an extract enhanced with humic substances from peat. The seaweed extract obtained by mild enzymatic acid hydrolysis with an acetate or citrate buffer and enriched with liquid potassium or sodium humate represents a new class of agricultural resources, expanding the range of methods and techniques for efficient agricultural production to increase the productivity of crops and improve the quality of the harvest.

The final product made from seaweed in combination with potassium or sodium peat humate is rich in phytohormones and plant growth regulators, namely auxins, gibberellins, cytokinins, abscisic acid, ethylene, betaine and polyamines. A significant number of scientific works in the world have been devoted to the study of the influence of biological activity of various components of seaweed and humic substances on plant development. Auxin is known for its positive effect on the development of the root system. Cytokinins trigger rapid cell division. Thus, they promote the growth and development of roots, flowers and fruits. Gibberellins stimulate germination and support the development of stems and flowers. Betaines stimulate the synthesis of chlorophyll.

The benefits of using seaweed extract and humic substances are often associated with increased plant resistance to abiotic stress. The main abiotic factors that negatively affect crop yields are drought, extreme temperatures, mineral deficiency, and salinization.

Scientific research conducted in recent years has revealed a number of positive effects of using bioactive compounds of seaweed and peat. Very often, adverse abiotic factors cause oxidative stress resulting from excessive formation of reactive oxygen species (ROS). They act as factors damaging DNA, lipids, carbohydrates and proteins, and also disrupt signal transmission in cells. In response, antioxidant defense mechanisms are launched in the plant. Numerous studies show that bioactive compounds have the ability to reduce the level of abiotic stress in plants. For example, it has been shown that polysaccharides extracted from algae increase plant resistance to chloride salinity. In addition, under salinity conditions, the addition of polysaccharides reduced the degree of oxidative damage to plants by reducing membrane permeability and lipid peroxidation, as well as by increasing the activity of superoxide dismutase (SOD), guaiacol peroxidase and catalase (CAT), which participate in the neutralization of ROS. An increase in SOD activity (by 47 and 181%, respectively) was noted in turf grass as a result of monthly application of seaweed extract and humic substances. This was accompanied by an increase in the rate of photosynthesis and led to an improvement in the quality of the lawn. Similar effects were noted when studying the effect of foliar feeding with seaweed extract and liquid humate on turf grass during long intervals between waterings and under salinity conditions. In addition, resistance to drought and salinity was attributed to cumulative effects, in particular, higher photochemical activity associated with the mineral composition of bioactive compounds and growth regulators such as cytokinins and ABA, enhanced root growth and changes in its morphology, accumulation of non-structural carbohydrates, which improved metabolism and osmotic regulation. Accumulation of proline was also noted. Therefore, seaweed extract together with humic substances are capable of acting as regulators of plant responses to adverse factors.

Extracted from algae biomass without changing their chemical structure and molecular weight distribution, carrageenans and alginates contain a wide range of organic compounds, including several common amino acids, including aspartic and glutamic acids, alanine. Alginic acid, laminaran and mannitol make up almost half of the total carbohydrate content in the final product. Seaweed also contains a large set of vitamins that can be used by plants. They contain vitamins C, B1 (thiamine), B2 (riboflavin), B3 (nicotinic acid), B12, D3, E, K, pantothenic, folic and folinic acids. Vitamin A is absent, but its precursors, carotene and fucoxanthin, are present.

As studies show, alginates not only affect the properties of the soil, but also promote the growth of beneficial fungi. Thus, alginate oligosaccharides produced as a result of the enzymatic breakdown of alginic acid have a strong stimulating effect on the growth of hyphae and the elongation of arbuscular mycorrhizal (AM) fungi. It has been established that both red and brown and green algae contain compounds that play a positive role in the development of mycorrhiza in higher plants.

Seaweed extract and liquid peat humate contain more than 60 microelements and nutrients. First of all, these are potassium, nitrogen, phosphorus, iodine, manganese, molybdenum and boron. The chelated form allows nutrients to be easily absorbed.

Some compounds derived from seaweeds have antibiotic properties and contain antimicrobial agents, the use of which protects plants from diseases and pests, in particular from root rot and mosaic disease.

The positive effect of peat humates on increasing crop yields, improving the quality of agricultural products, plant resistance to diseases and adverse environmental impacts, and the efficiency of applying complex mineral fertilizers has been studied for a long time. Peat humates, due to their simpler chemical structure (unlike humic acids), are easily absorbed by plants, are highly soluble in water, and are quickly included in metabolic processes at the cellular level. This leads to the activation of metabolism, improves the permeability of cell membranes, and facilitates the penetration of nutrients (including minerals) into cells.

The multifunctional structure of humates has determined their versatile action on plants and soil. Humates affect plants directly and indirectly. The direct effect is expressed in the increased activity of respiratory enzymes, protein and carbohydrate synthesis, activation of metabolic processes, and increased penetration of nutrients through the pores and membranes of plant cells. Humates slow down the release of water by cells and simultaneously maintain the appropriate degree of hydration in them, increase the chlorophyll content, productivity of photosynthesis and transpiration, which leads to accelerated growth and development of plants. The indirect effect is expressed in their influence on the formation of soil structure, activation of microflora, improvement of water-physical properties of soil, thermal regime, increase in the coefficient of use of mineral fertilizers, binding of toxic agents by forming very strong high-molecular complex compounds.

Humates and seaweed extract allow to obtain a harvest in the most effective and least expensive way: by maximum use of internal reserves of plants, full disclosure of their biopotential. Biological activity of extracted compounds causes active synthesis of enzymes and metabolism. This leads to an increase in the amount of available nutrients, to an increase in the content of proteins, vitamins, sugars, amino acids. The taste and nutritional qualities of plant products are improved.

The combined use of humates and bioactive compounds obtained from seaweeds helps to activate the immune system, the plant more successfully resists sudden changes in temperature and humidity, and the incidence of diseases decreases. In field and vegetation experiments, when treating plants with pesticides mixed with humates, a decrease in the inhibitory effect of pesticides on crops and a decrease in the accumulation of pesticides in the final crop products were revealed. In addition, according to the results of scientific research, humates with seaweed extract suppress the reproduction of some types of pathogenic microorganisms.

The combined use of humates and bioactive compounds obtained from seaweeds leads to stimulation of plant growth and development, increased absorption of nutrients from the soil, therefore, in this case, high efficiency of application of complex mineral fertilizers is achieved even with a reduction in their dosages.

A method [1] for obtaining bioactive compounds from seaweed is known, according to which seaweed is subjected to chloroform treatment of air-dried raw materials crushed to a particle size of 0.03-0.20 mm with a moisture content of 9 wt.% in a Soxhlet apparatus for 8 hours. Then the defatted seaweed residue is dried at 40°C, crushed to a fraction of less than 0.01 mm and extracted with acetone at 50°C in two stages for 60 min with a water module of 1:20 with constant stirring. The resulting acetone extract is concentrated on a rotary evaporator to a thick state and distilled water is added at a rate of 50 ml per 100 g of seaweed to obtain an aqueous polyphenol extract.

The disadvantage of this method is the aggressive extraction of polyphenols, which destroys other agronomically valuable bioactive compounds.

A method [2] for producing fertilizers from sea plants is known, according to which algae are fermented at a temperature of 15÷40°C for 24 to 240 hours. Then they are infused in water with a weight ratio of solid phase to water from 1:50 to 1:3.

The disadvantage of this method is the length of the process and ineffective extraction of bioactive compounds.

A method is known [3] for obtaining a product from brown seaweed, according to which the raw material is first soaked in water at a temperature of 35÷40°C for 12 hours, then washed, the algal mass is acidically treated with solutions of organic acids, the algal mass is washed, alkaline hydrolysis in an aqueous extract is carried out, followed by continuous homogenization, neutralization with food acids, pasteurization, while after soaking the raw material in water, the algal mass is separated, washed and squeezed out, and the aqueous extract is filtered, after acid treatment, the acid extract is additionally separated and neutralized to a pH of 5.5÷7.5, followed by purification from the formed salts, before alkaline hydrolysis, the washed algal mass is mixed with purified neutralized acid and filtered aqueous extracts, the homogenate is neutralized to a pH of 4.0÷7.5, after which the mixture is cooled and subjected to enzymatic hydrolysis. Enzymatic hydrolysis is carried out using enzymes of the hydrolase class.

The disadvantage of this method is the duration of each stage and the complexity of the process, when acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis with multiple washing and neutralization at each stage are used sequentially to obtain the final product.

The closest to the claimed invention is the method [4] for obtaining an extract of seaweeds by enzymatic hydrolysis in an aqueous medium with the sequential use of three different enzymes (cellulase, glucoamylase, protease). The method allows obtaining bioactive substances from seaweeds, including phytohormones, polysaccharides and amino acids, but is not without drawbacks. The method of enzymatic hydrolysis in an aqueous medium described in the prototype requires a long time to carry out, since it takes place in three successive stages (12-96 hours - the first, 6-25 hours - the second, 6-48 hours - the third). In addition, the inactivation of enzymes is carried out by heating the extract to 100 ° C at each stage, which can lead to the destruction of complex organic compounds and vitamins.

The presented method solves the problem of increasing the safe extraction of agronomically useful bioactive compounds from seaweeds without changing their chemical structure and molecular mass distribution, and offers a time-efficient technology under optimal pH and temperature conditions.

The method is carried out as follows.

To obtain complex polysaccharides, phytohormones, vitamins, polyphenols, organic acids, antimicrobial and anti-stress agents, plant growth stimulants, and other bioactive substances of agronomic importance from seaweeds in the form of an extract enhanced with humic substances from peat, the following mass species of seaweeds from the Black and Azov Seas are used: phyllophora, porphyra, callithamnion, ceranium, a total of about 270 species of multicellular green, brown, and red bottom algae. The collected seaweeds, including storm discharges, are thoroughly washed with fresh water to remove salts, sand, stones, and other impurities. The washed seaweeds are mechanically crushed (mechanically activated) to a fine state to facilitate and accelerate the subsequent extraction of bioactive substances. The process of enzymatic hydrolysis using enzymes (amylase, protease, pectinase, cellulase, alginate lyase, beta-glucanase) in a mild acidic medium with acetate or citrate buffer with strict adherence to the acid-base balance, pH 4-6, and temperature conditions of 40°-60°C with constant stirring for 6-12 hours is used to extract primary and secondary nutrients, microelements, vitamins, phytohormones, anti-stress agents and other bioactive compounds. After extraction, the resulting solution is drained and filtered to remove large particles and sediment. To inactivate metalloprotease enzymes, preventing further destruction of bioactive compounds in the final product, sodium fluoride or EDTA in low concentrations is used instead of heating and pasteurization, which is considered safe in agricultural products. To adjust the pH and stabilize the extract, liquid peat humate of potassium or sodium is used instead of an alkali solution, which allows not only to stabilize the solution, obtain the desired pH (5.5 ÷ 7.0), but also to additionally enrich the final product with humic substances to increase crop productivity and improve crop quality.

An example of the implementation of the claimed method for obtaining complex polysaccharides, phytohormones, vitamins, polyphenols, organic acids, antimicrobial and anti-stress agents, plant growth stimulants, and other bioactive substances of agronomic importance from seaweeds in the form of an extract enhanced with humic substances from peat.

Washed, crushed, mechanically activated seaweed is placed in the reactor. A 0.1% aqueous solution of hydrochloric (acetic, citric, malic, oxalic) acid is poured in, an acetate or citrate buffer is added to maintain the pH at 4.0 ÷ 6.0 and heated to 40 ° ÷ 60 ° C with constant stirring. An enzyme preparation (amylase, protease, pectinase, cellulase, alginate lyase, beta glucanase) is added as a solution or powder and the required pH and temperature are maintained for 6-8 hours to ensure enzymatic hydrolysis of the seaweed. After hydrolysis is complete, the resulting solution is drained, filtered and chemically inactivated with a solution of EDTA or sodium fluoride. Stabilization of the solution, pH correction and enrichment with humic compounds is carried out by using liquid peat humate of potassium or sodium.

Below are the characteristics of biologically active substances in the resulting final product:

Nutrients

1. Essential Nutrients

Nitrogen (N): about 1.0%. Necessary for the overall growth and development of plants, nitrogen promotes the growth of leaves and stems.

Phosphorus (P): about 0.5%. Important for root development, flowering and fruiting.

Potassium (K): about 1.0%. Necessary for disease resistance, water regulation and overall plant viability.

2. Minor nutrients.

Calcium (Ca): about 0.3%. Maintains the structure of the cell wall and improves root development.

Magnesium (Mg): about 0.2%. Necessary for photosynthesis and enzyme activation.

Sulfur (S): about 0.1%. Important for protein synthesis and enzyme function.

3. Microelements

Iron (Fe): about 0.05%. Crucial for chlorophyll production and overall plant health.

Manganese (Mn): about 0.02%. Supports photosynthesis and enzyme function.

Zinc (Zn): about 0.02%. Important for the production of growth hormone and enzyme activity.

Copper (Cu): about 0.005%. Necessary for photosynthesis and enzyme function.

Boron (B): about 0.005%. Important for cell wall formation and reproductive development.

Molybdenum (Mo): about 0.002%. Supports nitrogen fixation and enzyme function.

Vitamins and organic compounds

1. Vitamins

B vitamin complex: includes B1 (thiamine), B2 (riboflavin), B6 (pyridoxine) and B12 (cobalamin). These vitamins play a role in plant metabolism and stress resistance.

2. Organic acids.

Alginate: helps improve soil structure and moisture retention.

Amino Acids: Includes various amino acids such as glycine, alanine and glutamic acid, which support plant growth and stress resistance.

Phytohormones

1. Auxins. Indole-3-acetic acid (IAA): promotes the development and elongation of roots by influencing cell division and growth.

2. Cytokinins. Kinetin and zeatin: stimulate cell division and promote shoot growth, improving the overall health of plants and increasing yields.

3. Gibberellins. Gibberellic acid (GA): promotes stem elongation and increases germination.

4. Abscisic acid. Abscisic acid (ABA): helps combat stress and regulates water consumption in plants.

Other biologically active compounds

1. Beta-glucans. Strengthens plant defense mechanisms and increases resistance to diseases.

2. Polysaccharides: Support soil health and plant growth by improving nutrient absorption and moisture retention.

Final specifications.

pH level: usually maintained between 5.5 and 6.0.

Colour: Dark brown to greenish liquid.

Odor: Faint, characteristic seaweed odor.

Consistency: Clear or slightly cloudy liquid, depending on concentration and composition.

List of documents cited in the search report:

Erokhin V.E. Preliminary data on the study of algal gibberellins.

Erokhin V.E., Churilov Yu.S. Prospects for the use of Black Sea algae for obtaining lectins.

V.A. Lukyanov, A.I. Stifeev. Applied aspects of using microalgae in agrocenosis.

T.G. Shibaeva, E.G. Sherudilo, A.F. Titov. Seaweed extracts as plant biostimulants.

Yurkevich M.G., Sidorova V.A., Dubrovina I.A. Effect of application of extracts of brown seaweed Fucus on soil fertility and plant productivity.

Metting V., Rayburn W.R., Raynand R.A. Algae and agriculture.

RU 2741634 C1, 24.07.2020.

RU 2161599 C2, 10.06.1998.

RU 2790975 C1, 08.02.2022.

CN112538511A, 26.01.2021.

Claims (6)

1. A method for processing seaweed to obtain an extract containing complex polysaccharides, phytohormones, vitamins, polyphenols, organic acids, antimicrobial and anti-stress agents, plant growth stimulants, characterized by the fact that it includes washing seaweed from salts and impurities, mechanical grinding - mechanical activation, enzymatic hydrolysis using enzymes from the series amylase, protease, pectinase, cellulase, alginate lyase, beta glucanase in an acidic medium with an acetate or citrate buffer, chemical inactivation of enzymes, stabilization and pH adjustment with liquid peat humate of potassium or sodium, enrichment of the obtained extract with humic substances. 2. The method according to paragraph 1, characterized in that before extraction the algae are cleaned of sea salt with fresh water. 3. The method according to claim 1, characterized in that the enzymatic hydrolysis is carried out using enzymes selected from the series amylase, protease, pectinase, cellulase, alginate lyase, beta-glucanase in an acidic medium, in a 0.1% aqueous solution of hydrochloric acid selected from the series acetic, citric, malic, oxalic. 4. The method according to claim 1, characterized in that the enzymatic hydrolysis is carried out using an acetate or citrate buffer to establish and maintain the pH level throughout the entire time of extraction of bioactive compounds from seaweed. 5. The method according to item 1, characterized in that the inactivation of enzymes occurs chemically using sodium fluoride or EDTA. 6. The method according to item 1, characterized in that the pH adjustment and stabilization of the final product occurs with the help of liquid alkaline peat humate of potassium or sodium, while simultaneously enriching the seaweed extract with humic compounds useful for plants.