fungicide

Abstract

The present invention relates to a composition for use as a fungicide comprising a hydrolysate of keratin-containing material, as well as a method for this purpose.

Description

[0001] FUNGICIDE

[0002] TECHNICAL FIELD

[0003] The invention relates to a composition for use as a fungicide.

[0004] PRIOR ART

[0005] The agricultural sector faces significant challenges in the area of plant health, particularly the control of fungal diseases. Fungal diseases in plants, such as downy mildew, powdery mildew, white rust, clubroot, leaf spot disease, Fusarium head blight, and Sclerotinia rot, have a significant negative effect on the yield and quality of agricultural crops. These diseases are not only harmful to the crops themselves but can also cause economic losses for farmers and disrupt the food supply chain.

[0006] Traditionally, chemical fungicides have been used to combat such fungal diseases. However, the use of these chemical agents poses several problems. Firstly, many chemical fungicides can leave residues on crops, leading to potential health risks for consumers. Secondly, there is increasing concern about the environmental effects of these chemical agents, including the possibility of soil and water contamination.

[0007] Moreover, many fungi have developed resistance to traditional fungicides, reducing the effectiveness of these products. This leads to a vicious cycle where increasingly higher doses or stronger chemical formulations are needed to achieve the same level of disease control, which in turn leads to increased environmental risks and health concerns.

[0008] In recent years, there has been growing interest in developing alternative methods to combat fungal diseases. These alternatives focus not only on directly killing or suppressing fungi but also on enhancing the natural resistance of the plants themselves against these diseases. There is a growing need for biological and environmentally friendly solutions that are not only effective in combating diseases but also safe for consumers and the environment.

[0009] Gaidau Carmen et al. describes a keratin hydrolysate from wool. Due to the hardness of wool, wool keratin hydrolysate is often less soluble and has a molecular composition that may be less suitable for applications requiring high biological availability, such as a fungicide. Simon Demetra et al. describes protein hydrolysates. JPH02131530 describes a growth accelerator for seaweed obtained by adding phosphate salt, nitrate salt, organic acid, and saccharides to a solution mainly consisting of sulfur-containing amino acids. US20200383352 discloses a keratin hydrolysate. US20090269308 describes the combination of keratin hydrolysate and live fungi as an insecticide.

[0010] In this context, there is a clear need for innovative approaches in combating fungal diseases in agriculture. These approaches must not only be effective and safe but also sustainable and economically feasible for farmers.

[0011] SUMMARY OF THE INVENTION

[0012] The invention relates to a composition comprising a hydrolysate of keratin-containing material for use as a fungicide according to claim 1. This hydrolysate offers an efficient solution in the fight against fungal diseases, minimizing the use of traditional chemical fungicides. By utilizing keratin-containing material, especially poultry feathers, this invention contributes to increasing sustainability in the agriculture and food industry and promoting a circular economy.

[0013] The composition offers multiple benefits, including reducing the ecological footprint by using renewable sources and increasing safety for both farmers and consumers by avoiding toxic chemical residues. Moreover, the composition shows potential in enhancing the natural resistance of plants, thereby improving the overall health and resilience of the crops.

[0014] It has been found that the use of a hydrolysate according to the invention in plants results in a significant stimulation of glutathione metabolism. This increased glutathione metabolism contributes to improved plant resistance by modulating genes involved in the production of antimicrobial and antifungal components. Moreover, glutathione plays an important role in the synthesis of lignin and other cell wall-strengthening substances. This strengthened cell structure effectively prevents the penetration of fungi and inhibits further spread of pathogens, thereby better protecting plants against infections.

[0015] Furthermore, the administration of a hydrolysate according to the invention to plants leads to an upregulation of sucrose synthase (SUSY) within 24 hours. This enzyme is important for both cell wall reinforcement and the regulation of metabolic signaling functions associated with plant defense. The strengthening of the cell wall acts as a physical barrier against fungal pathogens, while the signaling function of sucrose synthase triggers defense mechanisms that enable the plant to adequately respond to biotic stress. Within 2 hours of uptake of a hydrolysate according to the invention in plants, an upregulation of defensins is also observed. Defensins are proteins that act as powerful antimicrobial agents by forming pores in the cell membranes of fungi and inducing the production of reactive oxygen species (ROS), leading to cell death. Additionally, a hydrolysate according to the invention may contain cationic keratin peptides with a molecular weight of more than 2500 Daltons, which have a direct effect on fungi by interacting with lipids and membrane proteins in the cell membranes. This interaction disrupts the membrane structure, resulting in a delay in fungal growth and an enhancement of plant resistance.

[0016] In a second aspect, the invention relates to a method for producing the composition. This method emphasizes innovation in the processing of waste materials and promotes a sustainable production method that is both economically and ecologically advantageous. The optional step of enzymatic hydrolysis offers a refinement in the production process, which can lead to even higher efficiency and effectiveness of the final product.

[0017] In a following aspect, the invention relates to the use of the composition for the treatment of fungal diseases in plants, particularly agricultural crops. This aspect enhances the potential of the invention to have a significant impact on modern agricultural practices by contributing to the development of sustainable, environmentally friendly, and safe agricultural methods.

[0018] DETAILED DESCRIPTION

[0019] Unless otherwise defined, all terms used in the description of the invention, including technical and scientific terms, have the meaning as commonly understood by a person skilled in the art to which the invention pertains. For a better understanding of the description of the invention, the following terms are explained explicitly.

[0020] The term "hydrolysate of keratin-containing material" refers to a product obtained by breaking down keratin-containing material, such as feathers, bone marrow, and other organic material, through hydrolysis, whereby larger molecules are converted into smaller ones.

[0021] The term "biocides" refers to chemical or biological substances used to kill or neutralize harmful organisms such as pests, bacteria, and fungi. The term "poultry feathers" refers to the feathers derived from poultry, such as chickens, ducks, and geese.

[0022] The term "agricultural crops" refers to plants cultivated for food production, fibers, or other agricultural purposes, such as vegetables, grains, and fruit trees.

[0023] The term "fungal diseases" refers to diseases in plants caused by fungi, which can cause various symptoms such as discoloration, leaf spots, rot, and death of plant tissue.

[0024] The term "wt%" is synonymous with "m%" and refers to weight percent, a measure of the concentration of a particular ingredient in a mixture, expressed as a percentage of the total weight of the composition.

[0025] The term "thermal hydrolysis" refers to a method of breaking down chemical compounds using heat.

[0026] The term "enzymatic hydrolysis" refers to a process in which enzymes are used to break down chemical compounds, resulting in the formation of a hydrolysate.

[0027] In this document, "a", "an" and "the" refer to both the singular and the plural, unless the context presupposes otherwise. For example, "a segment" means one or more than one segment.

[0028] The terms "comprise", "comprising", "consist of", "consisting of", "provided with", "have", "having", "include", "including", "contain", "containing" are synonyms and are inclusive or open terms that indicate the presence of what follows, and which do not exclude or prevent the presence of other components, characteristics, elements, members, steps, as known from or disclosed in the prior art.

[0029] Quoting numeric intervals by the endpoints includes all integers, fractions, and / or real numbers between the endpoints, including those endpoints.

[0030] In a first aspect, the invention relates to a composition comprising a hydrolysate of keratin-containing material, preferably keratin-containing slaughterhouse waste. The term "keratin-containing material" means material, preferably natural material, which consists of at least 10 m% keratin, preferably at least 20 m%, more preferably at least 30 m%, even more preferably at least 40 m%, even more preferably at least 50 m%, even more preferably at least 60 m%, even more preferably at least 70 m%, and most preferably at least 80 m%. The keratin may comprise alpha-keratin, beta-keratin, or a mixture thereof. Beta-keratin is advantageous. Preferably, the keratin-containing material is derived from birds, more preferably from chickens. The keratin-containing material can be present in any form, for example, but not limited to, powder form.

[0031] A hydrolysate of keratin-containing material is, for example, but not limited to, a hydrolysate of feathers, a hydrolysate of hair, a hydrolysate of hides, a hydrolysate of horny tissue, or a mixture thereof. Preferably, the hydrolysate of keratin-containing material is chosen from the list of: a hydrolysate of feathers, a hydrolysate of hair, a hydrolysate of hides, a hydrolysate of horny tissue, a hydrolysate of by-products from the textile industry, or a mixture thereof, more preferably chosen from the list of: hydrolysate of feathers, hydrolysate of hair, or a mixture thereof. Particularly preferably, the hydrolysate of keratin-containing material is a hydrolysate of feathers, also referred to as feather hydrolysate. By-products from the textile industry include textile rags of animal origin, old woolen clothing, down residues, or other keratin-containing material.

[0032] In a further preferred embodiment, the hydrolysate is a hydrolysate of keratin- containing slaughterhouse waste, preferably poultry slaughterhouse waste. In a further preferred embodiment, the keratin-containing slaughterhouse waste is chosen from the list of feathers, hair, horny tissue, hides, preferably chosen from the list of hair, feathers, or a combination thereof. The use of keratin-containing slaughterhouse waste as a source for making a fungicide contributes to the reuse of waste material that would otherwise be discarded. In an embodiment, the poultry slaughterhouse waste may consist of feathers, skin, organs, bones, or a combination thereof. These materials are rich in keratin, a protein that can result in bioactive peptides after hydrolysis.

[0033] The hydrolysate can contribute to the improvement of the antioxidative capabilities of the plant. This can lead to better control of ROS levels in plant cells, resulting in increased protection against damage caused by fungal infections. In an embodiment, the hydrolysate can be applied to the leaf surface of the plant, where it can act as a barrier against fungal spores.

[0034] The hydrolysate may potentially function as a less toxic fungicide, as it is a product naturally present in the environment compared to traditional, synthetic fungicides that often leave harmful chemical residues in the soil. Additionally, the use of such a natural product can contribute to the reduction of synthetic pesticide use, which is advantageous for the environment. In an embodiment, the hydrolysate can be combined with other natural ingredients to enhance its effectiveness. These ingredients may include, but are not limited to, natural oils, herbal extracts, or other natural pesticides.

[0035] It is important to note that the specific properties of the hydrolysate, such as the degree of hydrolysis, the concentration of bioactive peptides, and the specific composition of the hydrolysate, may vary depending on the specific source of the keratin-containing slaughterhouse waste, the specific hydrolysis method used, and other factors.

[0036] In a preferred embodiment, the composition does not include biocides. The absence of biocides in the composition ensures a significantly reduced environmental impact, as biocides often contain harmful substances that can lead to environmental pollution. The inventors found that the hydrolysate itself possesses sufficient fungicidal activity, eliminating the need for other biocides in the composition.

[0037] Chemical biocides are often used to kill or control harmful organisms. They are often used in agricultural and horticultural products to combat pests and diseases. While they are effective in combating these problems, they can also be harmful to the environment. They can contaminate the soil, pollute the water, and be harmful to non-target organisms such as bees and other pollinators. By creating a composition that does not contain additional biocides, the potential environmental impact caused by the use of these substances is reduced.

[0038] The composition according to the invention can be used to prevent or reduce the spread of fungal diseases on plants. This is achieved by applying the composition via the leaves to fruits or crops. The composition comprises a hydrolysate that can not only affect the fungi but also strengthen the plant to be more resistant to these diseases.

[0039] The hydrolysate in the composition can be obtained from keratin-containing material through thermal hydrolysis, optionally in combination with enzymatic hydrolysis. Through hydrolysis, the hydrolysate comprises peptides rich in certain amino acids such as cysteine. These peptides can serve as building blocks for glutathione, which plays an important role in the mechanisms that control ROS levels in plants. This can increase the plant's resistance to fungi.

[0040] These peptides have a dual function. On one hand, they affect the fungi that cause plant diseases, reducing the spread of these diseases. On the other hand, they strengthen the plant itself, making it more resistant to these diseases. This is an important advantage of the invention, as it not only addresses the symptoms of the disease but also increases the plant's resistance.

[0041] In a preferred embodiment, the composition comprises sulfoxide derivatives of sulfide and cysteine. The inventors found that these sulfoxide derivatives possess antifungal activity against certain Fusarium fungi. Fusarium fungi are responsible for various plant diseases and can cause significant damage to crops. These derivatives thus also contribute to the plant's resistance to fungi.

[0042] In a preferred embodiment, the composition comprises sulfur derivatives chosen from the list of dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, thiosulfonates, and thiosulfinates, preferably thiosulfonates and thiosulfinates. The inventors found that thiosulfonates and thiosulfinates contribute to the antifungal activity of the composition. Thiosulfinates, such as allicin, have strong antimicrobial properties, including antifungal activity. Thiosulfinates can react with various sulfur-containing functional groups of fungal enzymes, inhibiting the activity of these enzymes. This can lead to a disruption of metabolic processes within the fungal cells, ultimately resulting in the death of the fungal cells. Thiosulfonates can work by disrupting the integrity of the cell wall or cell membrane of fungi, leading to cell damage and death. Moreover, thiosulfonates, like thiosulfinate, can disrupt enzymatic processes within the fungal cells.

[0043] In another or further preferred embodiment, the composition comprises precursors that lead to a higher concentration of glucosinolates in the Brassicaceae family or a higher allicin concentration in the Amaryllis family with antifungal activity. This increases the effectiveness of the composition in preventing or reducing the spread of fungal diseases on plants.

[0044] The composition according to the invention offers an environmentally friendly alternative to traditional biocides. It can be effective in preventing or reducing the spread of fungal diseases on plants while reducing the potential environmental impact caused by the use of biocides.

[0045] In a preferred embodiment, the keratin-containing material mainly comprises poultry feathers. Poultry feathers are an important source of keratin, a protein that is the main component of hair, nails, and feathers. The current embodiment offers a sustainable solution to this problem by using these feathers as a raw material for the production of a hydrolysate with antifungal properties. This composition can be obtained by thermal hydrolysis, optionally in combination with enzymatic hydrolysis, of poultry feathers. This process results in the production of peptides rich in certain amino acids, such as cysteine, known for their antifungal properties. These peptides can function as bioactive substances that increase the resistance of plants to fungi or as building blocks for the production of other antifungal substances.

[0046] The use of a composition mainly derived from poultry feathers thus not only promotes sustainability by contributing to waste reduction and efficient reuse of raw materials but also has significant benefits for agriculture. It can increase the resistance of plants to fungi and can contribute to the protection of crops against various plant diseases. This can lead to higher yields and better quality of the crops, ultimately contributing to food security.

[0047] In a preferred embodiment, the invention relates to a composition for use as a fungicide on plants. In a preferred embodiment, the plants are agricultural crops. The hydrolysate has the potential ability to reduce fungal diseases on these crops, which can lead to increased harvest production.

[0048] In a further preferred embodiment, the agricultural crops are chosen from the list of: Apiaceae (Carrot family), Fabaceae (Legume family), Amaryllidaceae (Amaryllis family), Asteraceae (Aster family), Amaranthaceae (Amaranth family), Brassicaceae (Mustard family), Cucurbitaceae (Cucumber family), Solanaceae (Nightshade family), Vitaceae (Grape family), Poaceae (Grass family), with a preference for Vitaceae.

[0049] In a further preferred embodiment, the agricultural crops are chosen from the list of: carrots, beans, onions, lettuce, celery, spinach, kale, herbs, cucumber, melons, squash, zucchini, parsnip, beet, potato, bitter melon, tomato, pepper, Brussels sprouts, cabbage, grapes, soy, grains, corn, and rutabagas. But it should be clear that other plants and crops can serve equally well as a substrate for the fungicide.

[0050] In another or further preferred embodiment, the invention relates to a composition for use as a fungicide on plant foliage, preferably plant foliage of agricultural crops. This ensures a direct and effective treatment, as the active substances can be quickly absorbed by the plant and perform their function. This is an improvement over traditional fungicides that are applied to the ground and take time to be absorbed by the plant. It is important to note that although the composition is particularly suitable for use on agricultural crops, it can also be applied to other types of plants, such as fruit trees, to offer similar benefits. The precise amount of the composition to be applied may vary depending on the specific plant species and prevailing environmental conditions.

[0051] The invention relates to a composition that can be used as a fungicide against various fungal diseases, including but not limited to downy mildew, powdery mildew, white rust, clubroot, leaf spot disease, Fusarium head blight, Sclerotinia rot, southern wilt, yellow rust, and gray mold.

[0052] In another or further preferred embodiment, the composition is a composition for use as a fungicide against fungal diseases caused by fungi. These fungi are preferably chosen from a list that comprises: Phytophthora species, Aphanomyces species, Erysiphales, Albugo species, Plasmodiophora brassicae, Alternaria species, Fusarium species, Sclerotinia sclerotiorum, Sclerotium rolfsii, Puccinia species, and Botrytis cinerea. It is important to note that this list is not exhaustive and that other fungi may also be considered.

[0053] In an embodiment, the composition is in the form of a powder, a liquid, a gel, a spray, or another suitable medium.

[0054] In a particular preferred embodiment, the composition comprises the hydrolysate of keratin-containing material, preferably the hydrolysate of keratin-containing slaughterhouse waste, in an amount of at least 50 wt%, preferably at least 55 wt%, more preferably at least 55 wt%, even more preferably at least 60 wt%, even more preferably at least 65 wt%, even more preferably at least 70 wt%, even more preferably at least 75 wt%, even more preferably at least 80 wt%, even more preferably at least 85 wt%, even more preferably at least 90 wt%, even more preferably at least 95 wt%, even more preferably at least 99 wt%. The use of keratin-containing slaughterhouse waste as a source for the hydrolysate contributes to the reduction of waste production in the poultry industry, resulting in a more sustainable and environmentally friendly practice.

[0055] In a particular preferred embodiment, the composition comprises the hydrolysate of keratin-containing material, preferably the hydrolysate of keratin-containing slaughterhouse waste, in an amount of at least 50 wt% of the total amount of dry matter of the composition, preferably at least 55 wt%, more preferably at least 55 wt%, even more preferably at least 60 wt%, even more preferably at least 65 wt%, even more preferably at least 70 wt%, even more preferably at least 75 wt%, even more preferably at least 80 wt%, even more preferably at least 85 wt%, even more preferably at least 90 wt%, even more preferably at least 95 wt%, even more preferably at least 99 wt%. The use of keratin-containing slaughterhouse waste as a source for the hydrolysate contributes to the reduction of waste production in the poultry industry, resulting in a more sustainable and environmentally friendly practice.

[0056] In a further particular preferred embodiment, the composition consists of: a hydrolysate of keratin-containing material, preferably a hydrolysate of keratin- containing slaughterhouse waste, in an amount of at least 95 wt%;

[0057] - additives or impurities, in an amount of up to 5 wt%, preferably up to 4 wt%, more preferably up to 3 wt%, even more preferably up to 2 wt%, even more preferably up to 1 wt%, even more preferably up to 0.5 wt%.

[0058] In a further particular preferred embodiment, the composition consists of: a hydrolysate of keratin-containing material, preferably a hydrolysate of keratin- containing slaughterhouse waste, in an amount of at least 95 wt% of the total amount of dry matter of the composition;

[0059] - additives or impurities, in an amount of up to 5 wt% of the total amount of dry matter of the composition, preferably up to 4 wt%, more preferably up to 3 wt%, even more preferably up to 2 wt%, even more preferably up to 1 wt%, even more preferably up to 0.5 wt%.

[0060] In a further particular preferred embodiment, the composition mainly consists of a hydrolysate of keratin-containing material, preferably a hydrolysate of keratin- containing slaughterhouse waste. In this case, the invention relates to a hydrolysate of keratin-containing material, preferably a hydrolysate of keratin-containing slaughterhouse waste, for use as a fungicide. Keratin-containing slaughterhouse waste, which would otherwise be a waste product, is used in an environmentally friendly way to contribute to the prevention or reduction of fungal diseases.

[0061] In an embodiment, the composition also comprises other components that contribute to its effectiveness. These may include, but are not limited to, other antifungal agents, plant-strengthening agents, or other components that promote the health and resistance of the plant. In a preferred embodiment, the composition comprises more than 80 wt% keratin and / or derivatives thereof on the total amount of dry matter of the composition, preferably more than 85 wt%, even more preferably more than 90 wt%, even more preferably more than 95 wt%. Keratin derivatives are chemical compounds derived from keratin. Keratin derivatives can be obtained by subjecting keratin to various processes, such as hydrolysis, resulting in smaller proteins and peptide chains.

[0062] According to an embodiment, the total amount when applying to crops is at least 5 L / hectare, preferably 10 L / hectare, more preferably 15 L / hectare, more preferably 20 L / hectare, and most preferably 25 L / hectare and at most 50 L / hectare, preferably 45 L / hectare, more preferably 40 L / hectare, and most preferably 35 L / hectare. As understood by one skilled in the art, this amount is expressed per year and is preferably reapplied at least annually. The composition is preferably applied in a total amount between 10 and 50 L / hectare per year, preferably between 15 and 45 L / hectare per year, more preferably between 20 and 40 L / hectare per year, and most preferably between 25 and 35 L / hectare per year.

[0063] According to an embodiment, the total amount applied to crops is at least 0.25 kg dry matter / hectare, preferably 5 kg dry matter / hectare, more preferably 8 kg dry matter / hectare, and most preferably 10 kg dry matter / hectare and at most 25 kg dry matter / hectare, preferably 20 kg dry matter / hectare, more preferably 18 kg dry matter / hectare, and most preferably 15 kg dry matter / hectare. As understood by one skilled in the art, this amount is expressed per year and is preferably reapplied at least annually. The composition is applied in a preferred embodiment in a total amount of between 0.25 and 25 kg dry matter / hectare per year, preferably between 5 and 20 kg dry matter / hectare per year, more preferably between 8 and 18 kg dry matter / hectare per year, and most preferably between 10 and 15 kg dry matter / hectare per year.

[0064] The term "dry matter" refers to the total of all solid substances (organic and inorganic) present in a composition after all water or other volatile substances have been removed, for example, by evaporation.

[0065] According to an embodiment, the amount is applied at different times with a period between two applications of at least 1 week, preferably 1 month. According to an embodiment, the amount is applied to a plant at 2, 3, 4 or 5 different times. According to an embodiment, 4 times between 5 and 10 L / hectare is applied during a year. According to an embodiment, 3 times between 8 and 12 L / hectare is applied during a year. According to an embodiment, 4 times between 1 and 5 kg dry matter / hectare is applied during a year. According to an embodiment, 3 times between 2 and 6 L / hectare is applied during a year.

[0066] The hydrolysate, as discussed above, comprises peptides, preferably in an amount of 60-95 wt%. According to a preferred embodiment, more than 40 wt% of the peptides in the hydrolysate have a molecular mass lower than 2500 Daltons, preferably more than 50 wt%, more preferably more than 70 wt%, even more preferably more than 80 wt%. According to a preferred embodiment, more than 20 wt% of the peptides in the hydrolysate have a molecular mass lower than 1500 Daltons, preferably more than 30 wt%, more preferably 50 wt%, even more preferably more than 60 wt%.

[0067] According to an embodiment, 40-99 wt% of the peptides in the hydrolysate have a molecular mass lower than 2500 Daltons, preferably 50-99 wt%, more preferably 70- 99 wt%, even more preferably 80-99 wt%. According to another or further embodiment, 20-80 wt% of the peptides in the hydrolysate have a molecular mass lower than 1500 Daltons, preferably 30-70 wt%, more preferably 40-60 wt%, even more preferably 45- 55 wt%.

[0068] These peptides with a molecular mass lower than 1500 Daltons preferably include at least 1 cysteine amino acid.

[0069] In a particular preferred embodiment, the hydrolysate comprises peptides with a molecular mass lower than 1500 Daltons and that include at least 1 cysteine amino acid, in an amount of at least 30 wt%, preferably at least 50 wt%.

[0070] The inventors found that these smaller peptides indirectly boost plants for the production of defensins against fungi.

[0071] According to an embodiment, up to 50 wt% of the peptides in the hydrolysate have a molecular mass between 1500 and 5000 Daltons, preferably up to 40 wt%, more preferably up to 30 wt%, even more preferably up to 20 wt%. The inventors found that these peptides are less advantageous for the fungicidal activity of the hydrolysate.

[0072] According to a further or other preferred embodiment, more than 30 wt% of the peptides in the hydrolysate have a molecular mass higher than 5000 Daltons, preferably more than 40 wt%, more preferably 50 wt%, even more preferably more than 60 wt%. According to another or further embodiment, 20-80 wt% of the peptides in the hydrolysate have a molecular mass higher than 5000 Daltons, preferably 30-70 wt%, more preferably 40-60 wt%, even more preferably 45-55 wt%.

[0073] These peptides with a molecular mass higher than 5000 Daltons are preferably rich in cysteine. According to an embodiment, 2-15 wt% of the amino acids of the peptides with a molecular mass higher than 5000 Daltons in the hydrolysate are cysteine, preferably 2-10 wt%.

[0074] In a particular preferred embodiment, the hydrolysate comprises peptides with a molecular mass higher than 5000 Daltons in an amount of at least 30 wt%, preferably at least 50 wt%, where 2-15 wt% of the amino acids of the peptides with a molecular mass higher than 5000 Daltons are cysteine, preferably 2-10 wt%.

[0075] The inventors found that these large peptides rich in cysteine, likely according to the principle of a "cystine knot motif" found in defensins / cyclotides, are responsible for a direct effect against the fungi.

[0076] In a particular preferred embodiment, the hydrolysate comprises peptides with a molecular mass lower than 1500 Daltons, in an amount of at least 30 wt%, preferably at least 50 wt%, wherein the peptides with a molecular mass lower than 1500 Daltons include at least 1 cysteine amino acid; and peptides with a molecular mass higher than 5000 Daltons in an amount of at least 30 wt%, preferably at least 50 wt%, wherein 2-15 wt% of the amino acids of the peptides with a molecular mass higher than 5000 Daltons are cysteine, preferably 2-10 wt%.

[0077] In a second aspect, the invention relates to a method for producing a composition according to the first aspect.

[0078] In a preferred embodiment, the method comprises the steps of: a. providing keratin-containing material comprising at least water; b. thermally hydrolyzing the keratin-containing material, whereby a hydrolysate of keratin-containing material is obtained.

[0079] The hydrolysis time of the keratin-containing material is advantageously at least 3 minutes, preferably at least 4 minutes, and most preferably at least 5 minutes. The hydrolysis time of the keratin-containing material is advantageously at most 25 minutes, preferably at most 20, preferably at most 19 minutes, more preferably at most 18 minutes, even more preferably at most 17 minutes, more preferably at most 16 minutes, and most preferably at most 15 minutes.

[0080] During this process, a wide range of moderately condensed organic sulfur derivatives can be formed. Examples include dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, thiosulfonates, thiosulfinates.

[0081] In an embodiment, the obtained hydrolysate is treated with peroxides. This deodorizes the hydrolysate and also increases the concentration of the aforementioned substances.

[0082] Advantageously, the keratin-containing slaughterhouse waste is hydrolyzed at an elevated pressure of at least 3 bar, preferably at least 4 bar, more preferably at least 5 bar, and most preferably at least 6 bar. Preferably, the keratin-containing material is hydrolyzed at an elevated pressure of at most 12 bar, preferably at most 11 bar, more preferably at most 10 bar, and most preferably at most 9 bar.

[0083] Advantageously, the keratin-containing material is hydrolyzed at a temperature of at least 135°C, preferably at least 145°C, more preferably at least 155°C, even more preferably at least 165°C, and most preferably at least 175°C. Preferably, the keratin- containing material is hydrolyzed at a temperature of at most 245°C, preferably at most 235°C, more preferably at most 225°C, even more preferably at most 215°C, more preferably at most 205°C, and most preferably at most 195°C. Good results were observed when the keratin-containing material was hydrolyzed at a temperature range of 160°C to 205°C.

[0084] Advantageously, the hydrolysis of the keratin-containing material of the present invention is carried out in at least one reactor, thereby forming a hydrolysate.

[0085] Providing keratin-containing material can be achieved, for example, by collecting waste from poultry slaughterhouses. This waste may consist of feathers, organs, bones, and other parts of the animal not intended for human consumption. The water content of this waste can vary but is generally high, contributing to the weight and volume of the waste. Therefore, the method may further include a step of pressing at least part of the water out of the keratin-containing material.

[0086] It should be clear to one skilled in the art that the keratin-containing material can arrive at the conversion facility in various forms and states. The keratin-containing material may still be dirty or may have been pre-washed. It may also already be partially ground or chopped.

[0087] Pressing the water out of the keratin-containing material can be performed using various techniques, such as mechanical pressing or centrifugation. The purpose of this step is to reduce the volume and weight of the waste and to increase the concentration of useful components in the waste. In a further embodiment, the pressed water can be treated to recover useful components, or it can be purified and reused.

[0088] During the step of thermal hydrolyzing, the proteins in the keratin-containing material are broken down into smaller peptides and amino acids under the influence of heat and water. These smaller molecules act as bioactive peptides that can contribute to the resistance of plants against fungi.

[0089] In a further embodiment, the hydrolysate of keratin-containing material can be further treated to increase the concentration of certain useful components or to remove unwanted components. For example, the hydrolysate can be filtered to remove solid particles, or it can be centrifuged to separate heavier components. It can also be treated with enzymes to further promote the breakdown of proteins into peptides and amino acids, or it can be treated with acids or bases to adjust the pH.

[0090] In a further embodiment, in step b, an enzymatic hydrolysis or an acid hydrolysis, preferably an enzymatic hydrolysis, is also performed on the keratin-containing material.

[0091] The enzymatic hydrolysis of keratin-containing material can be performed using various types of enzymes, such as pepsin, trypsin, pancreatin, keratinase, and papain, where the choice of enzyme may depend on the specific type of keratin-containing material used. It is important to note that enzymatic hydrolysis not only results in the formation of antifungal peptides but also in the formation of other useful products, such as amino acids and fatty acids, which can help plants to better withstand various types of stress factors.

[0092] This combination of enzymatic and thermal hydrolysis can result in a higher yield of antifungal peptides, which can lead to better protection of the plants against fungal threats. In a third aspect, the invention relates to a method for treating fungal diseases in plants, comprising applying a composition according to the first aspect to plants, preferably agricultural crops.

[0093] When applying the composition to plants, the plants can absorb and metabolize these amino acids and peptides into their innate immune system. This leads to increased resistance to diseases, particularly fungal diseases. However, it is also possible that the plants develop increased resistance to other diseases, thanks to the amino acids and peptides in the composition.

[0094] In another embodiment of the invention, the composition can be applied to various types of plants, including but not limited to fruit crops, vegetable crops, grains, etc. The composition can be applied in various ways, for example, by spraying, soil injection, etc., preferably the composition is applied to the plant foliage.

[0095] It is clear that the invention offers many advantages compared to traditional methods for treating fungal diseases. By using a natural product such as keratin hydrolysate, the resistance of plants to diseases can be increased, while simultaneously improving soil fertility and nutrient concentration in the soil. Moreover, the invention offers an alternative application for an otherwise wasted product, which can lead to an optimization of resources in the agriculture and poultry industry.

[0096] In another aspect, the invention relates to a method for regulating the glutathione metabolism in a plant, whereby a composition as described herein is applied to the plant.

[0097] It has been found that the use of a hydrolysate according to the invention in plants results in a significant stimulation of glutathione metabolism. This increased glutathione metabolism contributes to improved plant resistance by modulating genes involved in the production of antimicrobial and antifungal components. Moreover, glutathione plays an important role in the synthesis of lignin and other cell wall-strengthening substances. This strengthened cell structure effectively prevents the penetration of fungi and inhibits further spread of pathogens, thereby better protecting plants against infections.

[0098] In another aspect, the invention relates to a method for regulating sucrose synthase (SUSY) in a plant, whereby a composition as described herein is applied to the plant. Administration of a hydrolysate according to the invention to plants leads to an upregulation of sucrose synthase (SUSY) within 24 hours. This enzyme is important for both cell wall reinforcement and the regulation of metabolic signaling functions associated with plant defense. The strengthening of the cell wall acts as a physical barrier against fungal pathogens, while the signaling function of sucrose synthase triggers defense mechanisms that enable the plant to adequately respond to biotic stress.

[0099] In another aspect, the invention relates to a method for regulating defensins in a plant, whereby a composition as described herein is applied to the plant.

[0100] Within 2 hours of uptake of a hydrolysate according to the invention in plants, an upregulation of defensins is observed. Defensins are proteins that act as powerful antimicrobial agents by forming pores in the cell membranes of fungi and inducing the production of reactive oxygen species (ROS), leading to cell death. Additionally, a hydrolysate according to the invention may contain cationic keratin peptides with a molecular weight of more than 2500 Daltons, which have a direct effect on fungi by interacting with lipids and membrane proteins in the cell membranes. This interaction disrupts the membrane structure, resulting in a delay in fungal growth and an enhancement of plant resistance.

[0101] The invention is described below with non-limiting examples that illustrate the invention and are not intended or to be interpreted to limit the scope of the invention.

[0102] EXAMPLES

[0103] Example 1: In vitro Antifungal Activity Tests

[0104] Testing the antifungal activity of the hydrolysate against various fungal pathogens.

[0105] Fungal Cultures: Pathogens used are Fusarium oxysporum, Phytophthora infestans, and Botrytis cinerea.

[0106] Hydrolysate Treatment: Different concentrations of the hydrolysate (1%, 5%, 10% w / v) are prepared in sterile water.

[0107] Inoculation: Fungal spores are inoculated in Petri dishes with PDA medium and treated with the hydrolysate solutions.

[0108] Incubation: Dishes are incubated at 25°C and fungal growth is observed daily. Growth Inhibition Assessment: The diameter of fungal colonies is measured and compared with controls without hydrolysate.

[0109] Results Significant inhibition of fungal growth will be observed at concentrations of 5% and 10% hydrolysate, with the strongest inhibition at 10%.

[0110] Example 2: Plant Bioassays in Growing Conditions Investigating how the hydrolysate affects plant resistance to fungal diseases.

[0111] Plant Material: Young seedlings of tomatoes and cucumbers are used.

[0112] - Treatment: Plants are sprayed with a 5% hydrolysate solution.

[0113] - Infection: Three days after treatment, the plants are infected with a spore suspension of Botrytis cinerea.

[0114] Monitoring: The development of disease symptoms is monitored for two weeks.

[0115] Disease Assessment: The severity of the infection is assessed based on leaf surface coverage by the fungus.

[0116] > Treated plants will show significantly reduced disease progression compared to untreated controls.

[0117] Example 3: Phytotoxicity Tests

[0118] Evaluating whether the hydrolysate has harmful effects on plant growth and development.

[0119] Plant Material: Seedlings of lettuce and spinach are chosen for this test.

[0120] - Treatment: Plants are sprayed with hydrolysate concentrations of 1%, 5%, and 10%.

[0121] Growth Monitoring: Growth and development of plants are observed over a period of 30 days.

[0122] - Toxicity Assessment: Signs of phytotoxicity, such as chlorosis, leaf necrosis, and growth inhibition, are noted.

[0123] Results No visible signs of phytotoxicity will be observed at concentrations up to 10%.

[0124] Example 4: Field Trials Establishing the effectiveness and practical applicability of the hydrolysate in real agricultural conditions.

[0125] Location: The trial is conducted on a commercial farm with tomatoes and cucumbers. - Treatment: Fields are treated with a 5% hydrolysate solution and a commercial fungicide as a control.

[0126] Monitoring: The incidence and severity of fungal diseases are tracked throughout the growing season.

[0127] Data Analysis: The results are statistically analyzed to compare effectiveness.

[0128] Results The hydrolysate will show comparable or better effectiveness in reducing fungal diseases compared to the commercial fungicide.

[0129] Example 5

[0130] It was demonstrated that the uptake of feather hydrolysates by plants results in an upregulation of the glutathione metabolism after 24 hours. This increased glutathione metabolism modulates the activity of genes involved in defense, resulting in the production of antimicrobial and antifungal components that are toxic to fungi. Additionally, glutathione plays a role in the synthesis of lignin and other cell wallstrengthening components, making it more difficult for fungi to penetrate the cell wall and hindering further infection.

[0131] Example 6

[0132] It was demonstrated that the uptake of feather hydrolysates by plants results in an upregulation of sucrose synthase (SUSY) after 24 hours. This enzyme plays a role in cell wall reinforcement, contributing to a better physical barrier against pathogens. Additionally, sucrose synthase functions as a metabolic signal involved in the activation of defense mechanisms in plants, thereby enhancing resistance to infections.

[0133] Example 7

[0134] It was demonstrated that the uptake of feather hydrolysates by plants leads to an upregulation of defensins after just 2 hours. Defensins are capable of forming pores in fungal cell membranes and inducing reactive oxygen species (ROS), which can ultimately lead to fungal cell death.

[0135] Example 8

[0136] It has been found that cationic keratin peptides with a molecular weight of more than 2500 Daltons, although not absorbed by the plant, have a direct effect on fungi. These peptides disrupt the structure of cell membranes by interacting with lipids or membrane proteins, resulting in a delay in fungal growth.

Claims

CLAIMS1. A composition for use as a fungicide comprising a hydrolysate of keratin- containing material.

2. Composition according to claim 1, wherein the composition further does not comprise biocides.

3. Composition according to any one of the preceding claims, wherein the keratin- containing material substantially consists of poultry slaughterhouse waste.

4. Composition according to any one of the preceding claims, wherein the composition comprises the hydrolysate of keratin-containing material in an amount of at least 50 wt%.

5. Composition according to any one of the preceding claims, wherein the composition comprises the hydrolysate of keratin-containing material in an amount of at least 95 wt%.

6. Composition according to any one of the preceding claims, wherein the composition comprises more than 80 wt% keratin and / or derivatives thereof on the total amount of dry matter of the composition.

7. Composition according to any one of the preceding claims, wherein the hydrolysate comprises sulfoxide derivatives of sulfide and cysteine.

8. Use of a composition according to any one of the preceding claims for use as a fungicide on plants.

9. Use of a composition according to claim 8, wherein the plants are agricultural crops.

10. Use of a composition according to claim 8 or 9, wherein the agricultural crops are chosen from the list of: carrots, beans, onions, lettuce, celery, spinach, kale, herbs, cucumber, melons, squash, zucchini, parsnip, beetroot, potato, bitter melon, tomato, pepper, Brussels sprouts, cabbage, grapes, soy, grains, corn, and rutabagas.

11. Use of a composition according to any one of the preceding claims 8-10 for use as a fungicide on plant foliage.

12. Use of a composition according to any one of the preceding claims 8-11, as a fungicide against fungal diseases chosen from the list of: downy mildew, powdery mildew, white rust, clubroot, leaf spot disease, Fusarium head blight, Sclerotinia rot, southern wilt, yellow rust, and gray mold.

13. Use of a composition according to any one of the preceding claims 8-12, as a fungicide against fungal diseases caused by fungi chosen from the list of: Phytophthora species, Aphanomyces species, Erysiphales, Albugo species,Plasmodiophora brassicae, Alternaria species, Fusarium species, Sclerotinia sclerotiorum, Sclerotium rolfsii, Puccinia species, and Botrytis cinerea.

14. Use of a composition according to any one of the preceding claims 8-13, wherein the composition is applied in a total amount between 20 and 40 L / hectare per year.

15. Use of a composition according to any one of the preceding claims 8-14, wherein the composition is applied in a total amount between 10 and 15 kg dry matter / hectare per year.

16. A method for producing a composition according to any one of the claims 1-7, comprising the steps of: a. providing keratin-containing material; b. thermally hydrolyzing the keratin-containing material, whereby a hydrolysate of keratin-containing material is obtained.

17. Method according to claim 16, wherein in step b, an enzymatic hydrolysis, a fermentation, a filtration, or a combination hereof is also performed on the keratin-containing material.

18. Method for regulating the glutathione metabolism in a plant, wherein a composition according to any one of the claims 1-7 is applied to the plant.

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