PL237993B1 - Biostimulator of plant growth and resistance - Google Patents

Description

The subject of the invention is a biostimulator of plant growth and resistance, containing a composition of biologically active free L-amino acids and bound into small-molecule peptides.

The current concept of agricultural development, the so-called Sustainable agriculture assumes a reduction in the use of chemicals in favor of resources of natural origin. A plant under the influence of stress can trigger defense reactions, so more and more attention is focused on inducing plant defense reactions. Increasingly, foliar biostimulants are used to improve the processes of plant growth and development and to reduce stress. Soil biostimulants improve the absorption of minerals from the substrate, accelerate the processes of transpiration and photosynthesis, and stimulate root growth. The effect of biostimulants on plants does not lie in the direct participation in the regulation of life processes, but in their influence on the plant's metabolism. Biostimulants improve the life processes taking place in plants without modifying their natural behavior. They have a positive effect on plant growth and the quality of the crops.

Known plant biostimulators are based on active substances of protein and mineral origin, and the active substances they contain may be synthetic or of natural origin: plant, animal and zoonotic. For practical reasons, it is recommended to use biostimulants, synthetic or of natural origin, together with plant protection products.

Amino acids constitute an extremely diverse group of compounds that perform many important functions in living organisms. They are the basic building blocks of plant cells, stimulate synthesis, accelerate the process of tissue reconstruction and take part in many physiological processes. Sequentially connected by peptide bonds to form peptides. Plants can synthesize amino acids from the basic elements oxygen, carbon and nitrogen taken from water, air and soil. This process is time consuming and requires a very large amount of energy. Providing plants with amino acids, in free and bound form, supports their development under abiotic stresses (environmental, nutritional, mechanical) as well as biotic stresses (pests and pathogenic organisms). Amino acids can be obtained in the process of chemical synthesis, chemical hydrolysis or enzymatic hydrolysis of animal proteins (e.g. keratin, collagen) or vegetable proteins (e.g. algae, corn, soybeans).

Preparations based on biologically active amino acids significantly improve plant resistance to stress thanks to osmoprotective compounds. For example, proline is an osmoprotective amino acid that has a complementary effect to other existing osmoprotectants, e.g. glycine.

Many of the preparations / fertilizers / biostimulants available on the market contain amino acids that can come from various sources and types of raw materials. The hydrolysis process produces amino acids and peptides that are soluble in aqueous solutions from insoluble proteins. Chemical hydrolysis, acidic or alkaline, is a high temperature process which produces the amino acids in the L - and D - forms. On the other hand, in living organisms, amino acids occur only in left-handed (biologically active) forms and only in such forms can they effectively nourish plants. Natural enzymes of plant, animal or microbiological origin are used in the enzymatic hydrolysis of proteins. The process is low-temperature and does not cause enantiomeric changes, and the enzymes used cut the protein chain in specific places in a repeatable manner. The product is free, undamaged and fully active molecules of L-amino acids and L-amino acids bound into small-molecule peptides. Depending on the protein raw material, as well as the specific role of the enzyme, the obtained hydrolysates differ in their amino acid composition.

There is a known biostimulator that increases the productivity of plants in the form of a preparation available under the trade name Natural CropSL by Natural Crop Poland Sp. z o. o. It is an enzymatic concentrate of sixteen most important plant L-amino acids, obtained in the enzymatic hydrolysis of collagen. The preparation contains nitrogen only in organic form in the amount of 9% by weight, L-amino acids> 50% by weight. % (of which free L - amino acids> 2 wt.%), organic carbon> 24.5 wt.%.

The commercially available Kaishi preparation by Sumi Agro Poland Sp. z o. o. is a product containing nineteen amino acids obtained in the process of enzymatic hydrolysis of proteins of purely vegetable origin. The preparation contains; 12.0 wt.% % free L-amino acids, nitrogen in total 2.0 wt.%, organic nitrogen 2.0 wt.%.

PL 237 993 B1

Another known plant stimulator based on amino acids being the product of enzymatic hydrolysis, commercially available under the name Aminoplant, is the preparation of Chemtura Europe Limited Sp. z o. o. The L-amino acid content in the preparation is 17.3 wt.%, short chain peptides 82.7 wt.%, total nitrogen 8.5 wt.%, organic matter 54.0 wt.%. The preparation contains eighteen L-amino acids, the largest of which is L-glycine, which plays an important role in the formation of plant cells and the synthesis of chlorophyll.

From the Polish patent application P-415595 a plant growth stimulator is known, obtained by combining the acid hydrolysis product with the alkaline hydrolysis product of natural substances containing keratin, into which minerals are incorporated. The stimulator obtained in this way contained up to 35 wt. % of biologically active L-amino acids, including at least 5 wt. biologically active L-amino acids capable of complexing microelements, with amino acid chelated forms of microelements constituting at least 0.01 wt.

Polish patent application P-415593 discloses a plant growth and development stimulator based on protein hydrolysates from raw materials of keratin origin (poultry feathers, ground hooves, bristles), in which biologically active amino acids come from independently conducted acid and alkaline chemical hydrolysis processes. The obtained plant stimulator contained up to 35 wt. biologically active L-amino acids.

The European patent EP 2 752 399 B1 describes a method of obtaining an enzyme extract from sampling waste for use as a bio-fertilizer and supplement for animals. The product is obtained in the process of chemical (alkaline) hydrolysis followed by enzymatic hydrolysis, carried out in the presence of a proteolytic enzyme of microorganisms. The product obtained had a carbohydrate content of 40 to 45 wt.%, A nitrogen content of 7 to 8 wt.%, And the processing amino acid was histidine.

Appropriate selection of enzymes in the enzymatic hydrolysis of proteins allows to obtain hydrolysates with specific properties, which was described, among others, by in the Polish patent specification PL 196531 B1, for which the method of obtaining a peptide preparation with antibacterial activity from milk thistle endosperm is known. The preparation is obtained in the process of enzymatic hydrolysis carried out in the presence of proteolytic enzymes. The resulting hydrolyzate is separated into fractions of different molecular weight, with only selected fractions showing antibacterial activity.

The aim of the invention was to develop a biostimulator that stimulates the growth and resistance of plants to abiotic and biotic stress.

Conducting research on the processing of milk thistle seeds (also known as fruits), it was unexpectedly found that milk thistle seeds, including cake, hulled seeds (endosperm) and husks, subjected to enzymatic hydrolysis with the use of proteolytic enzymes, are a source of active substances that stimulate growth and plant resistance. The raw materials used were by-products of the food and herbal industries.

Biostimulant of plant development and resistance, in the form of dried protein hydrolyzate of natural raw material, obtained in the process of enzymatic hydrolysis in the presence of proteolytic enzymes, trypsin or pancreatin, containing carbohydrates, fats, amino acids and possibly other components naturally occurring in the natural raw material subjected to hydrolysis, according to the invention is characterized by in that it is a dried protein hydrolyzate of milk thistle seeds, which contains from 30 to 80% by weight of biologically active L-amino acids, in free form and bound into small-molecule peptides having from 2.0 to 6.5 amino acid residues in the molecule, wherein the total nitrogen content of the hydrolyzate is from 7.0 to 14.0% by weight.

The biostimulant is preferably a milk thistle seed protein hydrolyzate which is a by-product of the food or herbal industry, most preferably cake, hulled seed / endosperm or husk expeller.

Among the substances naturally occurring in milk thistle seeds that may be present in the hydrolyzate, there are, among others, biologically active compounds from the group of polyphenols, polyunsaturated acids, vitamins, macro- and microelements, plant hormones.

The biostimulator with the composition according to the invention is obtained in a method known from the state of the art, consisting in the enzymatic hydrolysis of milk thistle seed proteins. The raw material for hydrolysis was pre-ground and / or degreased, and then treated with proteol enzymes

These are preferably trypsins or pancreatins (a mixture containing trypsin, chymotrypsin, lipase, amylase, fat). The process was carried out in an aqueous environment, at a temperature of 35-45 ° C, for 3-6 hours, maintaining the pH at 7.0-8.0. After the enzyme inactivation, the obtained hydrolyzate solution was either spray-dried at a temperature of up to 140 ° C or freeze-dried. The obtained biostimulator was an amorphous sediment with a color ranging from brown to beige.

The starting material for obtaining the hydrolyzate may be by-products formed after various types of milk thistle seed treatment from the food or herbal industry, which are preferably: cake (a by-product of cold pressing oil from milk thistle seeds), hulled seeds, called endosperm (residue from the process of dehulling, i.e. removing the husk of milk thistle seeds, which is used to obtain silymarin), pomace of the husk after silymarin extraction. This is a great advantage of the invention as it allows for the efficient management of the said waste.

The plant stimulant according to the invention is characterized by a high content of amino acids, free and bound, with the L conformation of up to 80% by weight, especially containing the amino acid residues of aspartic and glutamic acid, serine, glycine, proline, alanine, arginine, valine, lysine, threonine, leucine and isoleucine. The L conformation is the natural enantiomeric form of the proteins found in plants and therefore non-toxic and active.

The biostimulator according to the invention is applied to plants in the form of an aqueous solution with a concentration of 0.1 to 5% by weight. It is intended for foliar application and / or soil application in agricultural and horticultural crops in order to stimulate development, increase plant tolerance to abiotic and biotic stress, as well as to regenerate them faster after stress relief has passed.

The subject of the invention is illustrated in the examples showing the composition of active substances in the form of L-amino acids, free and bound in small-molecule peptides, with a biostimulating effect on the development and resistance of plants, obtained from various parts of milk thistle seeds, including expeller, husked fruit or husks. .

P r z k ł a d 1

780 g of water was added to 130 g of ground milk thistle seed cake and, while stirring, the pH of the suspension was adjusted to 7.2 using a 3% sodium hydroxide solution. The temperature of the reaction mixture was brought to 40 ° C, then 1 wt.% Was added. trypsin in relation to the amount of protein contained in milk thistle cake. The reaction was run for 3 hours maintaining the above conditions. The enzyme was then deactivated by heating the reaction mixture to 80 ° C. The hydrolyzate was filtered off and freeze dried. The obtained hydrolyzate was characterized by a total nitrogen content of 9.9% by weight, an average number of amino acid groups in a peptide molecule of 6.1. The total content of L-amino acids was 42.61% by weight. The amino acid composition (aminogram) of the hydrolyzate is presented in Table 1, and its effectiveness - in Table 6.

PL 237 993 Β1

Table 1. Amino acid composition of the hydrolyzate of milk thistle cake.

Amino acids The total content of L-amino acids in the hydrolyzate * [wt.%] Aspartic acid 4.88 Glutamic acid 8.71 Serine 2.20 Glycine 2.45 Histidine 1.05 Arginine 4.04 Threonine 1.57 Alanine 1.94 Proline 2.10 Tyrosine 1.47 Valine 2.43 Methionine 0.69 Cysteine 0.42 Isoleucine 1.91 Leucine 2.97 Phenylalanine 1.80 Lysine 1.98

* The total content of L-amino acids in the hydrolyzate of milk thistle cake is 42.61% by weight.

Example 2

Using the procedure described in Example 1, but the hydrolysis process was carried out for 6 hours, a hydrolyzate was obtained from decorticated milk thistle seeds, which had a total nitrogen content of 7.5% by weight, with an average number of amino acid groups in the peptide molecule of 3.0. The total content of L-amino acids was 34.88% by weight. The amino acid composition of the hydrolyzate is presented in Table 2, and its effectiveness - in Table 6.

PL 237 993 Β1

Table 2. Amino acid composition of the hydrolyzate from decorticated milk thistle seeds.

Amino acids The total content of L-amino acids in the hydrolyzate [wt.%] Aspartic acid 3.70 Glutamic acid 7.90 Serine 1.67 Glycine 2.11 Histidine 0.75 Arginine 3.30 Threonine 1.28 Alanine 1.46 Proline 1.52 Tyrosine 1.34 Valine 2.03 Methionine 0.66 Cysteine 0.34 Isoleucine 1.60 Leucine 2.36 Phenylalanine 1.31 Lysine 1.55

* The total content of Z-amino acids in the hydrolyzate of decorticated milk thistle seeds is 34.88% by weight.

Example 3

Using the procedure described in Example 1, a hydrolyzate was obtained from defatted, decorticated milk thistle seeds, which had a total nitrogen content of 13.3% by weight, an average number of amino acid groups in a peptide molecule of 6.4. The total content of l-amino acids was 74.29% by weight. The amino acid composition of the hydrolyzate is presented in Table 3, and its effectiveness in - Table 6.

PL 237 993 Β1

Table 3. Amino acid composition of the hydrolyzate from defatted decorticated milk thistle seeds.

Amino acids The total content of L-amino acids in the hydrolyzate * J [wt.%] Aspartic acid 8.45 1 Glutamic acid 15.24 Serine 3.72 Glycine 4.15 Histidine 1.75 Arginine 8.00 Threonine 2.73 Alanine 3.09 Proline 3.46 Tyrosine 2.83 Valine 4.13 Methionine 1.27 Cysteine 0.81 Isoleucine 3.34 Leucine 4.92 Phenylalanine 3.06 Lysine 3.34

* The total content of Z-amino acids in the hydrolyzate from defatted, decorticated milk thistle seeds is 74.29% by weight.

Example 4

780 g of water was added to 130 g of defatted, decorticated milk thistle seeds, and the pH of the suspension was adjusted to 7.4 with stirring using a 3% sodium hydroxide solution. The temperature of the reaction mixture was brought to 38 ° C, then 1 wt.% Was added. pancreatin in relation to the amount of protein contained in defatted, decorticated milk thistle seeds. The reaction was run for 3 hours maintaining the above conditions. The enzyme was then deactivated by heating the reaction mixture to 80 ° C. The hydrolyzate was filtered off and spray dried. The obtained hydrolyzate was characterized by a total nitrogen content of 12.7% by weight, an average number of amino acid groups in a peptide molecule of 5.3. The total content of L-amino acids was 50.00% by weight. The amino acid composition of the hydrolyzate is presented in Table 4, and its effectiveness - in Table 6.

PL 237 993 Β1

Table 4. Amino acid composition of the hydrolyzate from defatted, decorticated milk thistle seeds.

Amino acids Total content of Z-amino acids in the hydrolyzate [wt.%] Aspartic acid Glutamic acid 6.50 11.41 Serine 2.15 Glycine 1.85 Histidine 1.10 Arginine 2.59 Threonine 5.77 Alanine 2.02 Proline 2.16 Tyrosine 2.15 Valine 2.54 Methionine 0.25 Cysteine 0.24 Isoleucine 2.09 Leucine 3.05 Phenylalanine and _____... ... .....________________________ ____________, ____ 2.00

* The total content of i-amino acids in the hydrolyzate from defatted, decorticated milk thistle seeds is 50.00% by weight.

Example 5

Using the procedure described in Example 1, a hydrolyzate was obtained from ground pomace of milk thistle seeds, which had a total nitrogen content of 8.8% by weight, and an average number of amino acid groups in the peptide molecule 5.6. The total content of l-amino acids was 38.37% by weight. The amino acid composition of the hydrolyzate is presented in Table 5, and the effectiveness of action - in Table 6.

PL 237 993 Β1

Table 5. Amino acid composition of the hydrolyzate from milk thistle seed expeller.

Amino acids The total content of L-amino acids in the hydrolyzate * [wt.%] Aspartic acid 4.73 Glutamic acid 8.41 Seiyn 1.93 Glycine 2.08 Histidine 0.82 Arginine 3.08 Threonine 1.56 Alanine 1.68 Proline 1.91 Tyrosine 1.29 Valine 2.28 Methionine 0.60 Cysteine 0.39 Isoleucine 1.69 Leucine 2.58 Phenylalanine 1.69 Lysine 1.65

* The total content of L-amino acids in the hydrolyzate of milk thistle seed husks is 38.37% by weight.

PL 237 993 Β1

Table 6. Assessment of the effect of an aqueous solution of the hydrolyzate on the development of disease symptoms of powdery mildew of roses cultivar Rosa der Einheit grown under cover (initial infection = 1).

Biostimulator The degree of plant infestation after 2 sprayings after 4 sprayings Control 2 5 3% hydrolyzate solution according to example 1 0 0 1% hydrolyzate solution according to example 2 0 1 3% hydrolyzate solution according to example 2 0 0 3% hydrolyzate solution according to example 3 0 0 3% hydrolyzate solution according to example 4 0 0 1% hydrolyzate solution according to example 5 2 5 3% hydrolyzate solution according to example 5 1 2 5% hydrolyzate solution according to example 5 0 0

The scale of the infestation:

- no symptoms,

- up to 1% of the surface of shoots / leaves covered with mycelium,

- 1.1 to 5% of the shoot / leaf area covered with mycelium,

- from 5.1 to 10% of the shoot / leaf area covered with mycelium,

- from 10.1 to 20% of the shoot / leaf area covered with mycelium,

- more than 20% of shoots / leaves area covered with mycelium.

The research confirmed that the use of an aqueous solution of milk thistle seed protein hydrolyzate on roses infected with powdery mildew increases their resistance and ability to regenerate, stimulates physiological processes, while favorably influencing flowering. Optimal stimulation of plant growth and resistance was achieved with a concentration of the hydrolyzate solutions of 3 wt.%.

Claims (3)

1. Biostimulant of plant development and resistance, in the form of dried protein hydrolyzate of natural raw material, obtained in the process of enzymatic hydrolysis in the presence of proteolytic enzymes, trypsin or pancreatin, containing carbohydrates, fats, amino acids and possibly other components naturally occurring in the natural raw material subjected to hydrolysis, characterized by that it is a dried protein hydrolyzate of milk thistle seeds, which contains from 30 to 80% by weight of water-soluble biologically active L-amino acids, in free form and bound into small-molecule peptides having from 2.0 to 6.5 amino acid residues in the molecule, with the total nitrogen content of the hydrolyzate is from 7.0 to 14.0% by weight. 2. A biostimulator according to claim 1 The process of claim 1, characterized in that it is a hydrolyzate of milk thistle seed proteins, which are a by-product of the food or herbal industry. 3. A biostimulator according to claim 1 The process of claim 2, characterized in that it is a protein hydrolyzate of cake, hulled seed / endosperm or husk cake.