Animal amino acid-derived biostimulant from wool fiber and production method thereof

Abstract

The invention relates to an animal amino acid-derived biostimulant obtained by utilizing wool fiber waste generated from leather production for use in the field of agriculture, and the production method thereof. The biostimulant of the invention is produced by alkaline hydrolyzation method. Said biostimulant is used to support plant growth and development against abiotic stress factors, and to solve drought and salinity-induced problems, particularly in dry agriculture.

Description

[0001] ANIMAL AMINO ACID-DERIVED BIOSTIMULANT FROM WOOL FIBER AND PRODUCTION METHOD THEREOF

[0002] Technical Field of the Invention

[0003] The invention relates to an animal amino acid-derived biostimulant obtained by utilizing wool fiber waste generated from leather production for use in the field of agriculture and the production method thereof. The biostimulant of the invention is used to reduce the stress factor that plants encounter due to environmental factors that influence their growth and development, as well as to address issues caused by drought and salinity, especially in irrigated agriculture.

[0004] State of the Art

[0005] To thrive, plants need sunlight and CO2 from the air, and soil needs water, minerals, and other nutrients. As vegetation advances, the plant undergoes growth and development, resulting in the formation of distinct cells, tissues, and organs, each acquiring a unique morphology. It is known that a number of internal and external factors play a role together in order for normal growth and development to occur [1], The stress that plants experience from environmental factors affecting their growth and development is generally referred to as "abiotic stress". Abiotic stress is a condition that is induced by physical or chemical factors that limit the ability of plants to adapt to environmental conditions. These stress factors adversely affect the physiological and biochemical processes of plants and consequently can have negative effects on growth, development, yield, and general health. Water stress, one of the abiotic stress factors, exposes plants to stress in regions with inadequate or uneven water distribution. Over-irrigation also leads to this stress. Another factor is salinity, and high salt concentrations in the soil are also a source of stress for plants. This negatively affects the water and nutrient uptake of plants. In addition, high or low temperatures also cause stress for plants. Temperature changes negatively affect plant metabolism and fundamental processes such as photosynthesis. These abiotic stressors, such as drought, salinity, and temperature extremes, are currently causing massive crop losses globally. Plants face various abiotic stressors throughout their life cycle that negatively affect their growth and production. Globally, these stresses reduce annual crop yields by up to 70% and cause malnutrition as well as food shortages for the growing world population. Different strategies are implemented to overcome these problems and have been successful to some extent. One example is the exogenous use of plant growth promoters, which operate through various mechanisms to enhance plant growth and production under stressful conditions, and are used on a commercial level to achieve improved agricultural yields. However, since most of these are inorganic chemicals, they are toxic to plants and are not environmentally friendly products. Therefore, in order to avoid these losses, biostimulants are increasingly being integrated into production systems in order to increase and optimize production efficiency and improve / modify physiological processes in plants.

[0006] Yield and quality in agricultural production are negatively affected by reasons such as biotic and abiotic stress factors, input costs, climatic factors, adaptation of disease and pest agents, and restriction of fertilizer and pesticide applications to reduce production costs. On the other hand, the nutrient requirements of newly developed plant varieties are increasing. Additionally, increasing applications of fertilizers and pesticides, have resulted in residues found in both natural resources and agricultural plants, which negatively impacts quality and poses threats to the environment and public health. In addition, wrong fertilization and irrigation practices cause salinization and barrenness of soils [2], Moreover, we are facing global climate change, which is increasingly affecting agricultural production and is expected to cause serious yield losses in significant part of agricultural areas. Biostimulants are defined as any substance or microorganism applied to plants to enhance growth, nutrition, product quality, and yield, and to increase the resistance of plants to abiotic stress while being distinct from the nutritional content of the product and facilitating nutritional processes. Plant biostimulants are also referred to as products containing mixtures of such substances and / or microorganisms [3,4,5]. According to this definition, plant biostimulants are classified into following groups: (i) humic and fulvic acids, (ii) animal and plant-based protein hydrolysates, (iii) macroalgae seaweed extracts, and (iv) silicon along with beneficial microorganisms [4], Biostimulants are substances that can be applied to various plant parts including seeds, leaves, and soil, and some biostimulants also possess the ability to regulate soil structure [3]. In conclusion, biostimulants are natural products of plant, animal, or microorganism origin that, when applied in small quantities to plants (leaves or rhizosphere), stimulate natural processes that enhance growth, crop quality, nutrient utilization efficiency, and tolerance to abiotic stress. Therefore, the use of biostimulants allows for a reduced use of agrochemicals (especially fertilizers) in agriculture without compromising crop productivity and quality, while also providing protection against abiotic stress. The use of biostimulants does not have to directly provide nutrients to plants or target pathogens but instead regulates physiological processes, making it possible to increase plant growth and improve tolerance to abiotic stress [6].

[0007] Protein-based biostimulants (PBB) are mainly mixtures of peptides and amino acids and most PBB products are derived from protein-rich plant and animal derived substances that have been enzymatically or chemically treated or subjected to thermal hydrolysis [7], For this reason, these products are often referred to as protein hydrolyzates (PHs). Peptides contain free essential and non-essential amino acids in different amounts depending on the protein source, the processing methods used, and the degree of hydrolysis. These active components (peptides and amino acids) in PHs contribute to increased uptake of beneficial elements into plant tissues through leaves or roots [8]. However, in the European Union (EU) there are restrictions on the use of PBBs derived from animal by-products; animal-derived products can only be used as feedstock for biostimulants at the end of the production chain. Furthermore, under current EU regulations, biostimulants obtained from animal-derived products cannot be applied directly to the edible parts of the plant and the maximum concentration of heavy metals likely to be present must be either undetectable or contain no heavy metals at all [4,9], PHs containing peptides and free amino acids as main substance, are gaining importance as plant biostimulants due to their potential to enhance germination, seedling growth, plant growth, fruit and vegetable quality as well as crop productivity, especially under environmental stress conditions [4], Some amino acids (such as proline) found in PHs are not only effective in salt and drought stress, but also provide plants with resistance to heavy metal stress. Also, the chelating and complexing activities of certain amino acids and peptides enhance the mobility of micronutrients, improve nutrient availability, and facilitate uptake by roots through chelation. Furthermore, some nitrogen compounds, including the amino acids glycine, betaine and proline, provide antioxidant activity by scavenging free radicals, which contributes to the reduction of environmental stress [3]. PHs are also known to increase microbial biomass and activity, soil respiration, and soil fertility in general. Finally, the safety of animal-derived PHs has been tested and no genotoxicity, ecotoxicity, or phytotoxicity has been reported [5]. Plant-derived biostimulants are organic or inorganic compounds used to enhance plant growth, development and productivity. These biostimulants are usually given to plants as seed, foliar, or root applications. In the present art, hormones regulating the growth of plants are used as biostimulants. For example, plant growth hormones such as cytokinins, auxins, and gibberellins promote plant growth and increase their ability to cope with stress. Fulvic and humic acid improve soil structure, enhance nutrient absorption and promote plant growth, as well as improving soil fertility by increasing the activity of microorganisms in the soil. Microorganisms such as nitrogen-fixing, phosphate solubilizing bacteria promote plant growth through better absorption of plant nutrients. Furthermore, some plant extracts strengthen the defense system of the plants and increase their resistance to diseases. However, some plant-derived biostimulants are more expensive than chemical fertilizers, which can create additional costs for agricultural enterprises and increase production costs. Furthermore, some biostimulants raise concerns about their environmental impact, as some biostimulants, such as seaweed extracts, are harvested from marine ecosystems and excessive harvesting from such sources can upset the environmental balance.

[0008] The limitations and shortcomings of the state of the art solutions necessitate the development of a plant biostimulant due to factors such as abiotic stressors adversely impacting plant growth and development, the toxic effects and residual presence of inorganic chemicals used to mitigate abiotic stressors in soil, plants and, food crops, the low concentrations of amino acids and nitrogen in existing plant biostimulants, the high costs associated with plant-based biostimulants, their negative impact on the ecosystem, the diminishing effectiveness of certain biostimulants over time due to inducing plant dependency, and the challenges faced by microbial biostimulants in maintaining viability in commercial formulations while failing to obtain the expected effects in their inoculated environments. Additionally, there is a need for compatibility with chemical fertilizers and plant protection products, alongside the adverse impact of excessive humic compound application, causing a decline in root growth and development due to lipid peroxidation.

[0009] Summary and Objects of the Invention

[0010] The invention describes an animal amino acid-derived biostimulant obtained by utilizing wool fiber waste generated from leather production for use in the field of agriculture and the production method thereof. The biostimulant of the invention is used to reduce the stress factor that plants encounter due to environmental factors that influence their growth and development, as well as to address issues caused by drought and salinity, especially in irrigated agriculture.

[0011] The object of the invention is to provide a plant biostimulant that provides a rapid effect on the growth mechanism in the development of plants and provides a response in the shortest time. The biostimulant of the invention has a high content of animal amino acids and thus has a rapid effect on plant growth.

[0012] Another object of the invention is to obtain a plant biostimulant with an environmentally friendly approach and low cost. The biostimulant of the invention is obtained from wool fiber produced as waste in leather production. In this way, waste materials are recycled by being used as inputs in biostimulant production, and also production can be realized at a low cost.

[0013] Detailed Description of the Invention

[0014] The invention relates to an animal amino acid-derived biostimulant obtained by utilizing wool fiber waste generated from leather production for use in the field of agriculture, and the production method thereof. The biostimulant of the invention can be applied to plants from seed, soil, and leaves. Said biostimulant is used in the elimination of the abiotic stress factors experienced by plants due to environmental factors arising in their growth and development, in the solution of abiotic stress problems of the plants caused by drought and salinity, especially in irrigated agriculture.

[0015] The biostimulant of the invention comprises 10-15 wt.% animal-derived free amino acids, bound amino acids, macro- and micropeptides of animal-derived keratin protein, and water. In an embodiment of the invention, said biostimulant comprises 11% free amino acids. The biostimulant of the invention containing animal amino acids comprises 14 different free form amino acids (GLU; glutamic acid, SER; sericin, HIS; histamine, GLY; glycine, ALA; alanine, TYR; tyrosine, CYS; cysteine, VAL; valine, MET; methionine, RHE; phenylalanine, ILE; isoleucine, LEU; leucine, LYS; lysine, PRO; proline). The free amino acids with the highest ratios in this composition are PRO (1-5 wt.%, optimum 2.5%), GLY (0.75-4 wt.%, optimum 2.5%), ALA (0.5-3 wt.%, optimum 2.5%) and GLU (0.25-3 wt.%, optimum 1 .5%).

[0016] The biostimulant of the invention is obtained by hydrolyzing the raw material (wool) in protein structure under high temperature and chemical effect (alkali). After the completion of the hydrolyzation period, the product brought to a neutral pH value is filtered with filter paper and then the number of amino acids contained therein is analyzed using instrumental methods (HPLC device).

[0017] The production method of the plant biostimulant of the invention comprises the process steps of: i. cleaning, washing, drying, and preparing waste wool fibers for hydrolysis, ii. preparing and delivering sodium hydroxide (NaOH) to the hydrolyzation tanks together with the waste wool fibers, iii. performing the hydrolyzation process of waste wool fiber and bringing it to room temperature, iv. filtering the hydrolyzate with filter paper, v. adjusting the filtrate to a neutral pH value (pH:7), vi. re-filtering the hydrolyzate with filter paper.

[0018] In an embodiment of the invention, the production method of the plant biostimulant of the invention comprises the process steps of: i. cleaning, washing, drying, and preparing 5- 50 grams of waste wool fibers for hydrolysis, ii. preparing and delivering 0.5- 2.5 N sodium hydroxide (NaOH) to the hydrolysis tanks together with the waste wool fibers and bringing it to room temperature, iii. performing hydrolyzation process at a process temperature of 70-100°C for a process time of 5-24 hours, iv. filtering the hydrolyzate with filter paper, v. adjusting the filtrate to a neutral pH value (pH:7), vi. re-filtering the hydrolyzate with filter paper. In an embodiment of the invention, the production method of the plant biostimulant of the invention comprises the process steps of: i. cleaning, washing, drying, and preparing 15 grams of waste wool fibers for hydrolysis, ii. preparing and delivering 1 .5 N sodium hydroxide (NaOH) to the hydrolysis tanks together with the waste wool fibers and bringing it to room temperature, iii. performing hydrolyzation process at a process temperature of 90°C for a process time of 10 hours, iv. filtering the hydrolyzate with filter paper, v. adjusting the filtrate to a neutral pH value (pH:7), vi. re-filtering the hydrolyzate with filter paper.

[0019] Industrial Applicability of the Invention

[0020] The invention relates to an animal amino acid-derived biostimulant obtained by utilizing wool fiber waste generated from leather production for use in the field of agriculture, particularly dry agriculture, and to a production method thereof, and is industrially applicable.

[0021] The invention is not limited to the above descriptions and the person skilled in the art can readily present other different embodiments of the invention. These should be considered within the protection scope of the invention claimed by the claims.

[0022] REFERENCES

[0023] [1] Kumlay, A. M., & Eryigit, T. (2011, June 30). Bitkilerde Buyume ve Geli§meyi Duzenleyici Maddeler: Bitki Hormonlan. Journal of the Institute of Science and Technology.

[0024] [2] Kutman, Bahar Yildiz. Tarimsal Biyoteknoloji. Biyoteknoloji agma Ho§ Geldinizl, 179.

[0025] [3] Kulahta§, B., & Qokuysal, B. (2016, December 30). Biyostimulanlann Siniflandmlmasi ve Turkiye’Deki Durumu. Qukurova Tarim ve Gida Bilimleri Dergisi.

[0026] [4] Rouphael Y and Colla G (2020) Editorial: Biostimulants in Agriculture. Front. Plant Sci. 11 :40. doi: 10.3389 / fpls.2020.00040

[0027] [5] Oner, Muge.Misir (Zea mays. L.) bitkisinin gimlenme ve fide dbnemlerinde uygulanan kitosanm fizyolojik ve morfolojik ozellikler uzerine etkisi. 2023. Master's Thesis.

[0028] [6] Jolayemi, O. L., Malik, A. H., Ekblad, T., Fredlund, K., Olsson, M. E., & Johansson, E. (2022, December 18). Protein-based Biostimulants to enhance plant growth -state-of- the-art and future direction with sugar beet as an example.

[0029] [7] Nardi, S.; Pizzeghello, D.; Schiavon, M.; Ertani, A. Plant biostimulants: Physiological responses induced by protein hydrolyzed-based products and humic substances in plant metabolism. Sci. Agric. 2016, 73, 18-23.

[0030] [8] Colla, G.; Rouphael, Y.; Lucini, L.; Canaguier, R.; Stefanoni, W.; Fiorillo, A.; Cardarelli, M. Protein hydrolyzate-based biostimulants: Origin, biological activity and application methods. In Proceedings of the II World Congress on the Use of Biostimulants in Agriculture, Florence, Italy, 16-19 November 2015; p. 1148.

[0031] [9] The European Commission. Commission Implementing Regulation (EU) No 354 / 2014 of 8 April 2014. Official Journal of the European Union. 2014.

Claims

CLAIMS1. A plant biostimulant, characterized in that it comprises 10-15 wt.% free amino acids, bound amino acids, macro- and micropeptides of animal-derived keratin protein, and water.

2. A plant biostimulant according to claim 1 , characterized in that it comprises 11 wt.% free amino acids.

3. A plant biostimulant according to claim 1 or 2, characterized in that said free amino acid is glutamic acid, sericin, histamine, glycine, alanine, tyrosine, cysteine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, and proline.

4. A plant biostimulant according to claim 3, characterized in that it comprises 1 -5 wt.% proline, 0.75-4 wt.% glycine, 0.5-3 wt.% alanine, and 0.25-3 wt.% glutamic acid.

5. A plant biostimulant according to claim 4, characterized in that it comprises 2.5 wt.% proline, 2.5 wt.% glycine, 2.5 wt.% alanine, and 1 .5 wt.% glutamic acid.

6. A production method of a plant biostimulant, characterized in that it comprises the process steps of: i. cleaning, washing, drying, and preparing waste wool fibers for hydrolysis, ii. preparing and delivering sodium hydroxide (NaOH) to the hydrolyzation tanks together with the waste wool fibers, iii. performing the hydrolyzation process of waste wool fiber and bringing it to room temperature, iv. filtering the hydrolyzate with filter paper, v. adjusting the filtrate to a neutral pH value (pH:7), vi. re-filtering the hydrolyzate with filter paper.

7. A method according to claim 6, characterized in that it comprises the process steps of:i. cleaning, washing, drying, and preparing 5- 50 grams of waste wool fibers for hydrolysis, ii. preparing and delivering 0.5- 2.5 N sodium hydroxide (NaOH) to the hydrolysis tanks together with the waste wool fibers and bringing it to room temperature, iii. performing hydrolyzation process at a process temperature of 70-100°C for a process time of 5-24 hours, iv. filtering the hydrolyzate with filter paper, v. adjusting the filtrate to a neutral pH value (pH:7), vi. re-filtering the hydrolyzate with filter paper.

8. The method according to claim 7, characterized in that it comprises the process steps of; i. cleaning, washing, drying, and preparing 15 grams of waste wool fibers for hydrolysis, ii. preparing and delivering 1.5 N sodium hydroxide (NaOH) to the hydrolysis tanks together with the waste wool fibers and bringing it to room temperature, iii. performing hydrolyzation process at a process temperature of 90°C for a process time of 10 hours, iv. filtering the hydrolyzate with filter paper, v. adjusting the filtrate to a neutral pH value (pH:7), vi. re-filtering the hydrolyzate with filter paper.

9. The plant biostimulant produced by a method according to any one of claims 6-8.

10. A plant biostimulant according to claim 9 for use in dry agriculture.