Composition, biostimulant and application of biostimulant

Abstract

The invention provides a composition, a biostimulant and application of the biostimulant, and the composition comprises an acetoin aqueous solution, 2, 3-butanediol, tetramethylpyrazine and phenylalanine. According to the composition provided by the embodiment of the invention, efficient and stable targeted color change is realized through triple synergistic effects of signal transduction (acetoin and 2, 3-butanediol)-metabolic regulation (tetramethylpyrazine)-precursor supply (phenylalanine), and meanwhile, the color change effect of the composition is improved. The biostimulant containing the composition and the peptide amino acid liquid can be applied to a plurality of stages such as a post-transplanting stage, a pre-flowering stage, a fruit setting stage, a fruit swelling stage and a color changing stage, so that stable transition and synergistic promotion of crops from vegetative growth to reproductive growth are effectively realized, flower promotion, fruit retention, fruit swelling and color changing are comprehensively realized, furthermore, yield increase and advanced harvesting of the crops can be realized, and the economic benefit of the crops is increased. Meanwhile, the raw materials for improving the sweetness and the vitamin content of the fruits are easy to obtain, the preparation method is simple, industrialization is easy to achieve, and high use value is achieved.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a composition, a biostimulant, and its application. Background Technology

[0002] In modern agricultural production, consumers are increasingly demanding higher standards for fruit appearance (such as uniform coloring), nutritional value, and food safety, driving the transformation of planting models towards "high yield, high quality, and high efficiency." However, traditional management methods that rely on chemical fertilizers and pesticides can easily lead to soil degradation, decreased fruit flavor, and increased pesticide residue risks, making it difficult to meet the current market demand for high-quality agricultural products.

[0003] This contradiction is particularly prominent in the greenhouse cultivation of high-value-added cash crops such as strawberries and tomatoes. For example, low temperatures and insufficient sunlight in winter easily lead to low fruit set rates and difficulty in fruit coloring in strawberries; while continuous cropping obstacles in the soil often result in weak plant growth, poor fruit development, and uneven coloring in tomatoes. At the same time, the economic benefits of "uniform coloring" and "early market availability" of agricultural products are extremely significant, with early market prices often several times higher than those during the peak season. Therefore, the market urgently needs a safe and efficient planting technology that can comprehensively regulate crop growth and development, effectively promote fruit enlargement and coloring, and significantly advance the harvest period.

[0004] Currently, agricultural technologies used to improve fruit coloring mainly fall into two categories. The first category consists of plant growth regulators (such as ethephon), which, while capable of forcibly initiating coloring, have a single mechanism of action and easily disrupt the plant's endogenous hormone balance, leading to uneven coloring, fruit softening and rotting, flavor deterioration, and even yield reduction. The second category comprises biostimulants (such as single amino acids or simple compounds), which have higher safety profiles but generally suffer from simple components, slow onset of action, and unstable effects. Furthermore, their application is often limited to temporary intervention during the fruit coloring period, lacking strategies for systematic regulation starting from the early stages of crop growth.

[0005] Therefore, there is an urgent need in this field for a novel technical solution that can both target and activate the crop's intrinsic color-changing metabolic pathways through precisely designed signaling compositions and integrate nutritional management throughout the entire growth period to safely, stably, and efficiently achieve the comprehensive goals of promoting color change, early market entry, and increased yield. Summary of the Invention

[0006] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the object of this invention is to provide a composition, a biostimulant, and its application. The composition provided by this invention can achieve efficient and stable targeted color change, and the biostimulant containing the above composition can effectively regulate the balance of crop growth and development, promote flowering, fruit setting, fruit enlargement, and color change, achieving the effects of improved quality, increased yield, and earlier market entry, promoting high-quality crop growth and yield. At the same time, the raw materials for the biostimulant are readily available, the preparation method is simple, and it is easy to industrialize.

[0007] Therefore, in a first aspect, the present invention provides a composition for targeted color change in crops. According to an embodiment of the invention, the composition comprises an aqueous solution of acetoin, 2,3-butanediol, tetramethylpyrazine, and phenylalanine, wherein the concentration of acetoin in the aqueous solution is 80-150 g / L; and the mass ratio of acetoin, 2,3-butanediol, tetramethylpyrazine, and phenylalanine is (10-30):(1-5):(0.5-3):(1-5). The composition according to the embodiments of the present invention can effectively upregulate the expression of genes related to hormone regulation (auxin-related) and color change promotion (anthocyanin synthesis), regulate the genes related to substance transport and enhance stress resistance, and construct a triple synergistic pathway of "signal transduction (acetoin, 2,3-butanediol) - metabolic regulation (tetramethylpyrazine) - precursor supply (phenylalanine)". This composition overcomes the defects of existing technologies with single components and one-sided mechanisms. In particular, the addition of 2,3-butanediol, together with acetoin, strengthens the activation of endogenous signaling pathways such as jasmonic acid, providing a key metabolic driving force for the efficient conversion of phenylalanine into anthocyanins, thereby achieving efficient and stable targeted color change.

[0008] In a second aspect, the present invention provides a biostimulant for regulating crop color change and / or growth. According to an embodiment of the invention, the biostimulant comprises the composition described in the first aspect and a peptide amino acid solution.

[0009] In a third aspect, the present invention provides a method for preparing the biostimulant described in the second aspect. According to an embodiment of the present invention, the method comprises: (1) subjecting the composition described in the first aspect to a first mixing treatment with a peptide amino acid solution. The method according to an embodiment of the present invention can effectively prepare the aforementioned biostimulant.

[0010] In a fourth aspect, the present invention provides a method for promoting color change in fruit crops. According to an embodiment of the invention, the method comprises applying the composition described in the first aspect to the crop. As previously stated, the composition constructs a triple synergistic pathway of "signal transduction (acetoin, 2,3-butanediol) - metabolic regulation (tetramethylpyrazine) - precursor supply (phenylalanine)," enabling efficient and stable targeted color change.

[0011] In a fifth aspect, the present invention proposes a method for promoting color change in fruit-bearing crops, increasing the number of initial flowers, the number of initial fruits, and / or fruit enlargement. According to an embodiment of the present invention, the method includes applying the biostimulant described in the second aspect to the crop. As previously mentioned, the biostimulant constructs a triple synergistic pathway of "signal transduction (acetoin, 2,3-butanediol) - metabolic regulation (tetramethylpyrazine) - precursor supply (phenylalanine)," and, combined with peptide amino acid solutions, can meet the nutritional needs of crops at various growth stages, enhance stress resistance, and provide energy and substances for later fruit color change. Specifically, by applying the biostimulant at multiple stages, including after transplanting, before flowering, during fruit setting, during fruit enlargement, and during color change, a smooth transition and synergistic promotion from vegetative growth to reproductive growth are achieved, comprehensively promoting flowering, fruit retention, fruit enlargement, and color change.

[0012] In a sixth aspect, the present invention provides a method for increasing early yield, sweetness, vitamin content, and / or enhancing stress resistance in fruit crops. According to an embodiment of the invention, the method includes applying the biostimulant described in the second aspect to the crop. By applying the aforementioned biostimulant at multiple stages, such as after transplanting, before flowering, during fruit setting, during fruit enlargement, and during color change, a smooth transition and synergistic promotion of the crop from vegetative to reproductive growth can be effectively achieved, comprehensively promoting flowering, fruit retention, fruit enlargement, and color change. Furthermore, it can increase crop yield and achieve earlier harvest, while simultaneously improving the sweetness and vitamin content of the fruit.

[0013] Compared with the prior art, the beneficial effects of the present invention are at least as follows: (1) The composition provided by the present invention can effectively upregulate the expression of hormone regulation (auxin-related) and color change promotion (anthocyanin synthesis) related genes, regulate the related genes of substance transport and enhance stress resistance, and construct a triple synergistic pathway of "signal transduction (acetoin, 2,3-butanediol) - metabolic regulation (tetramethylpyrazine) - precursor supply (phenylalanine)". This composition overcomes the defects of the existing technology of single components and one-sided mechanism. In particular, the addition of 2,3-butanediol, together with acetoin, strengthens the activation of endogenous signaling pathways such as jasmonic acid, providing a key metabolic driving force for the efficient conversion of phenylalanine into anthocyanins, thereby achieving efficient and stable targeted color change.

[0014] (2) The biostimulants containing the above composition can promote the targeted color change of the fruit and can also be applied at multiple growth stages from the time the crop is transplanted and the seedlings have recovered to the time the fruit is about to turn color, so as to achieve synergistic nutritional stress management, promote the flowering, fruit setting, fruit expansion and color change of the crop, and achieve increased yield, improved quality and earlier market launch.

[0015] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The diagram shows the biostimulant of Example 11 of the present invention and the transcriptome sequencing results of each group of dwarf tomatoes after treatment. Detailed Implementation

[0017] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0018] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "multiple" or "a plurality of" means at least two, two types, such as two, two, three, three, etc., unless otherwise explicitly specified.

[0019] In this document, the terms “comprising,” “having,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0020] In this document, the term “optionally” generally means that an event or condition described below may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] In this article, the term "early yield" refers to the yield obtained from the first 3 to 5 concentrated harvests after the crop has entered the harvest period. For example, for continuously harvested strawberries, it can be "the cumulative yield of the first 5 harvests"; for tomatoes, it can be "the cumulative yield of the first 4 harvests".

[0023] In this article, "fruit crops" refers to plants whose main edible part is fruit, including fruits, vegetables, nuts, etc. For example, fruits include, but are not limited to, berries (e.g., strawberries, grapes, kiwis), pome fruits (e.g., apples, pears), drupes (e.g., peaches, plums, cherries), citrus fruits (oranges, tangerines, lemons), and melons (watermelons, cucumbers); vegetables include, but are not limited to, tomatoes and eggplants.

[0024] In this paper, the term "initial flowering" refers to the total number of open flowers observed on a fixed sampling branch (plant) during the initial flowering period.

[0025] In this article, the term "early fruit" refers to the number of young fruits that have set fruit (ovary enlarged, fruit length ≥1cm or reaching the size specified by the variety) on a fixed sampling branch (plant) during the fruit-setting period.

[0026] In this article, the term "color change" refers to the process by which the pigment composition of the fruit peel changes towards ripening characteristics.

[0027] Therefore, in some embodiments of the present invention, the present invention provides a composition for targeted color change of crops, the composition comprising an aqueous solution of acetoin, 2,3-butanediol tetramethylpyrazine and an amino acid, wherein the concentration of acetoin in the aqueous solution of acetoin is 80-150 g / L; and the mass ratio of acetoin, 2,3-butanediol, tetramethylpyrazine and phenylalanine is (10-30):(1-5):(0.5-3):(1-5). The composition according to the embodiments of the present invention can effectively upregulate the expression of genes related to hormone regulation (auxin-related) and color change promotion (anthocyanin synthesis), regulate the genes related to substance transport and enhance stress resistance, and construct a triple synergistic pathway of "signal transduction (acetoin, 2,3-butanediol) - metabolic regulation (tetramethylpyrazine) - precursor supply (phenylalanine)". This composition overcomes the defects of existing technologies with single components and one-sided mechanisms. In particular, the addition of 2,3-butanediol, together with acetoin, strengthens the activation of endogenous signaling pathways such as jasmonic acid, providing a key metabolic driving force for the efficient conversion of phenylalanine into anthocyanins, thereby achieving efficient and stable targeted color change.

[0028] According to embodiments of the present invention, the composition may further include at least one of the following additional technical features: According to an embodiment of the present invention, the mass ratio of acetoin, 2,3-butanediol, tetramethylpyrazine and phenylalanine is (10-20):(2-4):(0.5-2):(2-4).

[0029] According to an embodiment of the present invention, the mass ratio of acetoin, 2,3-butanediol, tetramethylpyrazine and phenylalanine is (10-20):(2-3):(0.5-1.5):(2-3).

[0030] According to an embodiment of the present invention, the mass ratio of acetoin, 2,3-butanediol, tetramethylpyrazine and phenylalanine is (10-20):(2-3):(0.5-1):(2-3).

[0031] According to an embodiment of the present invention, the mass ratio of acetoin, 2,3-butanediol, tetramethylpyrazine and phenylalanine is (10-20):(2-3):(0.5-1):(1-2).

[0032] According to an embodiment of the present invention, the concentration of acetoin in the acetoin aqueous solution is 80-150 g / L. Exemplarily, the concentration of acetoin in the acetoin aqueous solution is 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 g / L, or a range between any two of the above values.

[0033] According to an embodiment of the present invention, the acetoin is 3-hydroxy-2-butanone.

[0034] It should be noted that the above-mentioned 2,3-butanediol, tetramethylpyrazine, and phenylalanine are all industrial raw materials with a content and / or purity of not less than 95%, and their content and / or purity can be 95%, 96%, 97%, 98%, 99%, or 100%.

[0035] According to a specific embodiment of the present invention, the 2,3-butanediol is preferably (2R,3R)-(-)-2,3-butanediol, with a content and / or purity ≥95%.

[0036] According to a specific embodiment of the present invention, the tetramethylpyrazine is 2,3,5,6-tetramethylpyrazine with a content and / or purity ≥95%.

[0037] According to a specific embodiment of the present invention, the phenylalanine is L-phenylalanine with a content and / or purity ≥95%.

[0038] According to an embodiment of the present invention, the composition is prepared by the following method: (1) Add 2,3-butanediol to the aqueous solution of acetoin and stir to dissolve; (2) After heating the product from step (1) to 50°C to 80°C, add tetramethylpyrazine and phenylalanine in sequence and stir until completely dissolved to obtain the composition.

[0039] In some embodiments of the present invention, a biostimulant is provided, comprising the aforementioned composition and a peptide amino acid solution. By mixing the composition targeting color change with a nutritionally complete peptide amino acid solution rich in small peptides and functional amino acids, the biostimulant prepared according to the embodiments of the present invention can not only meet the nutritional needs of crops at various growth stages and enhance their stress resistance, but also store energy and substances in advance for later fruit color change. By applying it at multiple stages such as after transplanting, before flowering, during fruit setting, during fruit expansion, and during color change, a smooth transition and synergistic promotion from vegetative growth to reproductive growth is achieved, ultimately comprehensively promoting flowering, fruit retention, fruit expansion, color change, and yield increase, and can also improve early harvest yield.

[0040] According to embodiments of the present invention, the above-mentioned biostimulant may further include at least one of the following additional technical features: According to an embodiment of the present invention, the mass ratio of the composition to the peptide amino acid solution is (10~30):(40~80). Exemplarily, the mass ratio of the composition to the peptide amino acid solution is 1:8, 1:7, 1:6, 1:5, 1:4, 2:7, 1:3, 2:5, 1:2, 3:8, 3:7, 3:5, or 3:4, or a range between any two of the above values.

[0041] According to an embodiment of the present invention, the biostimulant further comprises elemental fertilizer and functional adjuvants; the functional adjuvants include wetting synergists and / or film-forming agents.

[0042] According to an embodiment of the present invention, based on the total weight of the biostimulant, the content of each component is as follows: The composition: 10%~30%; The peptide amino acid solution contains 40% to 80% of the total content. The elemental fertilizer content is 5%~10%; The functional additives: 5%~10%; The remainder is water.

[0043] According to an embodiment of the present invention, the total amino acid content in the peptide amino acid solution is 30%~60%, the free amino acid content is 5%~15%, and the peptide content is 25%~50%. Free amino acids are more easily absorbed by crops and are an important indicator for evaluating the quality of peptide raw materials.

[0044] According to an embodiment of the present invention, the content of peptides with a molecular weight ≤5000 Da in the peptide is not less than 70%. More specifically, the content of peptides with a molecular weight ≤5000 Da in the peptide (total peptide) is not less than 70%. Exemplarily, the content of peptides with a molecular weight ≤5000 Da in the peptide is 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any value between any two of the above values.

[0045] According to an embodiment of the present invention, the elemental fertilizer includes potassium fertilizer.

[0046] According to an embodiment of the present invention, the peptide amino acid solution includes at least one of plant-derived peptides and animal-derived peptides. The peptide requirements of crops are met by adding peptides other than those derived from microbial fermentation.

[0047] According to an embodiment of the present invention, the peptide amino acid solution includes at least one of glycine, alanine, glutamic acid and proline.

[0048] According to an embodiment of the present invention, the peptide amino acid solution is prepared by the following method: (1) Animal-derived protein raw materials and plant-derived protein raw materials are mixed with water at a mass ratio of (1~2):(8~10) and homogenized to obtain animal-derived protein slurry and plant-derived protein slurry; (2) The animal-derived protein slurry and the plant-derived protein slurry were respectively subjected to enzymatic hydrolysis with proteases to obtain animal-derived protease hydrolysate and plant-derived protease hydrolysate; (3) The animal-derived protease hydrolysate and the plant-derived protease hydrolysate are subjected to a second mixing treatment and fermentation treatment in a mass ratio of (1~2):(2~3) to obtain the peptide amino acid solution.

[0049] According to an embodiment of the present invention, the fermentation process is carried out using Bacillus subtilis.

[0050] According to an embodiment of the present invention, the fermentation conditions are 37°C, aerobic fermentation, and the fermentation time is 40-70 hours.

[0051] According to an embodiment of the present invention, the elemental fertilizer includes potassium fertilizer.

[0052] According to an embodiment of the present invention, the elemental fertilizer includes at least one of potassium formate, potassium acetate, and potassium citrate.

[0053] According to an embodiment of the present invention, the additive includes at least one of fatty alcohol polyoxyethylene ether phosphate and modified polyethylene wax emulsion. The fatty alcohol polyoxyethylene ether phosphate is used as a wetting synergist, and the modified polyethylene wax emulsion is used as a film-forming agent to prevent the volatilization of the active ingredient and to promote rapid drying.

[0054] In some embodiments of the present invention, the present invention provides a method for preparing the aforementioned biostimulant, the method comprising: (1) subjecting the aforementioned composition to a first mixing treatment with a peptide amino acid solution.

[0055] According to embodiments of the present invention, the method for preparing the aforementioned biostimulant may further include at least one of the following additional technical features: According to an embodiment of the present invention, the method further includes: (2) subjecting the first mixed treatment product to a second mixed treatment with elemental fertilizer.

[0056] According to an embodiment of the present invention, the method further includes: (3) subjecting the second mixed treatment product to a third mixed treatment with an adjuvant, wherein the third mixed treatment is carried out until the adjuvant is completely dissolved to obtain the biostimulant.

[0057] According to an embodiment of the present invention, the first, second and third mixing processes are carried out at 20°C to 30°C.

[0058] In some embodiments of the present invention, a method for promoting color change in fruit crops is proposed. According to an embodiment of the present invention, the method includes applying the aforementioned composition to the crop. As previously described, the composition constructs a triple synergistic pathway of "signal transduction (acetoin, 2,3-butanediol) - metabolic regulation (tetramethylpyrazine) - precursor supply (phenylalanine)," enabling efficient and stable targeted color change.

[0059] According to a specific embodiment of the present invention, the application includes fertigation and / or spraying.

[0060] According to a specific embodiment of the present invention, the application stage includes at least one of the late fruit expansion stage and the color change stage.

[0061] In some embodiments of the present invention, a method is proposed to promote the color change of fruit-bearing crops, increase the number of initial flowers, the number of initial fruits, and / or fruit enlargement. According to an embodiment of the present invention, the method includes applying the aforementioned biostimulant to the crop. As previously mentioned, the biostimulant constructs a triple synergistic pathway of "signal transduction (acetoin, 2,3-butanediol) - metabolic regulation (tetramethylpyrazine) - precursor supply (phenylalanine)," and, combined with peptide amino acid solutions, can meet the nutritional needs of crops at various growth stages, enhance stress resistance, and reserve energy and substances in advance for later fruit color change. Specifically, by applying the biostimulant at multiple stages, including after transplanting, before flowering, during fruit setting, during fruit enlargement, and during color change, a smooth transition and synergistic promotion from vegetative growth to reproductive growth are achieved, comprehensively promoting flowering, fruit retention, fruit enlargement, and color change.

[0062] It should be noted that the types of fruit crops mentioned are not particularly limited. As a preferred option, the crops mentioned are tomatoes and / or strawberries.

[0063] According to a specific embodiment of the present invention, the application includes fertigation and / or spraying.

[0064] According to a specific embodiment of the present invention, the application stage includes, but is not limited to, at least one of the following: after transplanting and seedling establishment, before flowering, during flowering and fruit setting, and during fruit enlargement.

[0065] According to a specific embodiment of the present invention, the application is performed by applying 500-1000 ml / mu to the roots at at least during at least one of the following stages: after transplanting and seedling establishment, before flowering, during flowering and fruit setting, and during fruit enlargement, and by foliar spraying 50-100 ml / mu (with a dilution ratio of 600-1000 times).

[0066] In some embodiments of the present invention, a method is proposed to improve the early yield, sweetness, vitamin content, and / or stress resistance of fruit crops. According to an embodiment of the present invention, the method includes applying the aforementioned biostimulants to the crop. By applying the above-mentioned biostimulants at multiple stages, such as after transplanting, before flowering, during fruit setting, during fruit expansion, and during color change, a smooth transition and synergistic promotion of the crop from vegetative growth to reproductive growth can be effectively achieved, comprehensively promoting flowering, fruit retention, fruit expansion, and color change. Furthermore, it can increase crop yield and achieve earlier harvest, while simultaneously improving the sweetness and vitamin content of the fruit.

[0067] It should be noted that the types of fruit crops mentioned are not particularly limited. As a preferred option, the crops mentioned are tomatoes and / or strawberries.

[0068] According to a specific embodiment of the present invention, the application includes fertigation and / or spraying.

[0069] According to a specific embodiment of the present invention, the application stage includes, but is not limited to, at least one of the following: after transplanting and seedling establishment, before flowering, during flowering and fruit setting, and during fruit enlargement.

[0070] According to a specific embodiment of the present invention, the application is performed by applying 500-1000 ml / mu to the roots at at least during at least one of the following stages: after transplanting and seedling establishment, before flowering, during flowering and fruit setting, and during fruit enlargement, and by foliar spraying 50-100 ml / mu (with a dilution ratio of 600-1000 times).

[0071] The embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, unless otherwise specified, all reagents used in the following embodiments are commercially available or can be synthesized according to the methods described herein or known to others. For reaction conditions not listed, they are also readily available to those skilled in the art.

[0072] Example 1 This embodiment provides a composition containing a small molecule signaling substance, comprising: 10 parts by weight of 150 g / L aqueous solution of acetoin; 2 parts by weight of (2R,3R)-(-)-2,3-butanediol (hereinafter referred to as 2,3-butanediol) with a purity of 95%; 95% 2,3,5,6-Tetramethylpyrazine, 1 part by weight; 95% L-phenylalanine, 2 parts by weight; The preparation method is as follows: According to the above proportions, weigh out an aqueous solution of acetoin, add 2,3-butanediol and stir to dissolve, then heat the solution to 80°C, and then add tetramethylpyrazine and phenylalanine in sequence, stirring until completely dissolved to obtain the composition.

[0073] Example 2 This embodiment provides a composition containing a small molecule signaling substance, comprising: 15 parts by weight of 100 g / L aqueous solution of acetoin; 95% (2R,3R)-(-)-2,3-butanediol, 2 parts by weight; 95% 2,3,5,6-Tetramethylpyrazine, 1 part by weight; 2 parts by weight of L-phenylalanine with 95% purity; The preparation method is as follows: Weigh out the above acetoin aqueous solution according to the above ratio, add 2,3-butanediol and stir to dissolve, then heat the solution to 80°C, and then add tetramethylpyrazine and phenylalanine in sequence, stirring until completely dissolved.

[0074] Example 3 This embodiment provides a composition containing a small molecule signaling substance, comprising: 20 parts by weight of an 80 g / L aqueous solution of acetoin; 3 parts by weight of (2R,3R)-(-)-2,3-butanediol with a purity of 95%; 95% 2,3,5,6-Tetramethylpyrazine, 1 part by weight; 2 parts by weight of L-phenylalanine with 95% purity; The preparation method is as follows: According to the above proportions, weigh out the acetoin aqueous solution, add 2,3-butanediol and stir to dissolve, then heat the solution to 80°C, and then add tetramethylpyrazine and phenylalanine in sequence, stirring until completely dissolved.

[0075] Example 4 This embodiment provides a comparative composition, comprising: 20 parts by weight of an 80 g / L aqueous solution of acetoin; 95% 2,3,5,6-Tetramethylpyrazine, 1 part by weight; 2 parts by weight of L-phenylalanine with 95% purity; The preparation method is as follows: Weigh out the acetoin aqueous solution according to the above ratio, heat the solution to 80°C, and then add tetramethylpyrazine and phenylalanine in sequence, stirring until completely dissolved.

[0076] Example 5 This embodiment prepares a peptide amino acid solution, and the preparation method is as follows: Raw material pretreatment: Equal amounts of pigskin and cowhide were selected as animal-derived materials, and soybean meal was selected as plant-derived material. Both raw materials were separately crushed to a particle size of less than 5 mm and then washed with clean water. Then, the two raw materials were separately mixed with water at a mass ratio of 1:10 and homogenized.

[0077] Enzymatic hydrolysis: 0.5% (dry matter mass) of compound protease (Novozymes Protex 6L) was added to both animal-derived and plant-derived homogenates. The pH of the system was adjusted to 7.0 with sodium hydroxide solution. The homogenates were stirred and hydrolyzed at a constant temperature of 50°C for 14 hours to obtain animal-derived hydrolysate and plant-derived hydrolysate.

[0078] Mixing and Fermentation: The above-mentioned animal-derived and plant-derived enzymatic hydrolysates were mixed at a mass ratio of 1:2 and inoculated with Bacillus subtilis CICC 10085 at an inoculation volume of 3% (v / v) of the mixed liquid. Aerobic fermentation was carried out at 37°C with aeration and stirring (1.0 vvm) for 60 hours. Samples were taken periodically during fermentation, and the content and molecular weight distribution of free amino acids were determined by high-performance liquid chromatography (HPLC).

[0079] Post-processing: After fermentation, the fermentation broth was heated to 90℃ and maintained for 30 minutes to inactivate the cells. Subsequently, the inactivated fermentation broth was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected. Finally, the supernatant was concentrated under reduced pressure at 60℃ and -0.09 MPa until the target amino acid content was reached, thus obtaining the peptide amino acid solution. Analysis showed that the total amino acid content in the peptide amino acid solution was 30%, of which the free amino acid content was 5%, the total peptide content was 25%, and the content of peptides with a molecular weight less than 5000 Da was 85% of the total peptide content.

[0080] Example 6 This embodiment prepares a peptide amino acid solution, and the preparation method is as follows: Raw material pretreatment: Equal amounts of fish skin and fish meat were selected as animal-based materials, and soybean meal was selected as plant-based material. Both raw materials were separately crushed to a particle size of less than 5 mm and then washed with clean water. Then, the two raw materials were separately mixed with water at a mass ratio of 1:10 and homogenized.

[0081] Enzymatic hydrolysis: 0.5% (dry matter mass) of compound protease (Novozymes Protex 6L) was added to both animal-derived and plant-derived homogenates. The pH of the system was adjusted to 7.0 with sodium hydroxide solution. The homogenates were stirred and hydrolyzed at a constant temperature of 50°C for 14 hours to obtain animal-derived hydrolysate and plant-derived hydrolysate.

[0082] Mixing and Fermentation: The above-mentioned animal-derived and plant-derived enzymatic hydrolysates were mixed at a mass ratio of 1:1 and inoculated with Bacillus subtilis CICC 10085 at an inoculation volume of 3% (v / v) of the mixed liquid. Aerobic fermentation was carried out at 37°C with aeration and stirring (1.0 vvm) for 60 hours. Samples were taken periodically during fermentation, and the free amino acid content and molecular weight distribution were determined using high-performance liquid chromatography (HPLC).

[0083] Post-processing: After fermentation, the fermentation broth was heated to 90℃ and maintained for 30 minutes to inactivate the cells. Subsequently, the inactivated fermentation broth was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected. Finally, the supernatant was concentrated under reduced pressure at 60℃ and -0.09 MPa until the target amino acid content was reached, thus obtaining the peptide amino acid solution. Analysis showed that the peptide amino acid solution contained 60% total amino acids, of which 10% were free amino acids, and 50% total peptides. 90% of the peptides had a molecular weight less than 5000 Da.

[0084] Example 7 This embodiment prepares a peptide amino acid solution, and the preparation method is as follows: Raw material pretreatment: Equal amounts of pig skin, fish skin, and fish meat were selected as animal-based materials, and soybean meal was selected as plant-based material. Both raw materials were separately crushed to a particle size of less than 5 mm and then washed with clean water. The two raw materials were then mixed separately with water at a mass ratio of 1:10 and homogenized.

[0085] Enzymatic hydrolysis: 0.5% (dry matter mass) of compound protease (Novozymes Protex 6L) was added to both animal-derived and plant-derived homogenates. The pH of the system was adjusted to 7.0 with sodium hydroxide solution. The homogenates were stirred and hydrolyzed at a constant temperature of 50°C for 14 hours to obtain animal-derived hydrolysate and plant-derived hydrolysate.

[0086] Mixing and Fermentation: The above-mentioned animal-derived and plant-derived enzymatic hydrolysates were mixed at a mass ratio of 2:1 and inoculated with Bacillus subtilis CICC 10085 at an inoculation volume of 3% (v / v) of the mixed liquid. Aerobic fermentation was carried out at 37°C with aeration and stirring (1.0 vvm) for 60 hours. Samples were taken periodically during fermentation, and the free amino acid content and molecular weight distribution were determined using high-performance liquid chromatography (HPLC).

[0087] Post-processing: After fermentation, the fermentation broth was heated to 90℃ and maintained for 30 minutes to inactivate the cells. Then, the inactivated fermentation broth was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected. Finally, the supernatant was concentrated under reduced pressure at 60℃ and -0.09 MPa until the target amino acid content was reached, thus obtaining the peptide amino acid solution. Analysis showed that... The peptide amino acid solution contains 50% total amino acids, of which 8% are free amino acids, 42% total peptides, and 88% of the total peptides are peptides with a molecular weight less than 5000 Da.

[0088] Example 8 This embodiment provides a biostimulant, comprising: The composition prepared in Example 3: 30%; The peptide amino acid solution prepared in Example 6: 40%; 75% potassium formate solution: 5%; Fatty alcohol polyoxyethylene ether phosphate: 5%.

[0089] Replenish with the remaining water.

[0090] The preparation method is as follows: After mixing the composition and peptide amino acid solution evenly according to the above mass ratio, add the above potassium fertilizer and functional additives in sequence, and stir evenly to obtain the final product.

[0091] Example 9 This embodiment provides a biostimulant, comprising: The composition prepared in Example 3: 10%; The peptide amino acid solution prepared in Example 6: 80%; 75% potassium formate solution: 5%; Modified polyethylene wax emulsion: 5%.

[0092] The preparation method is as follows: After mixing the composition and peptide amino acid solution evenly according to the above mass ratio, add the above potassium fertilizer and functional additives in sequence, and stir evenly to obtain the final product.

[0093] Example 10 This embodiment provides a biostimulant, comprising: The composition prepared in Example 3: 20%; The peptide amino acid solution prepared in Example 6: 60%; 75% potassium formate solution: 10%; Fatty alcohol polyoxyethylene ether phosphate: 5%; Modified polyethylene wax emulsion: 5%.

[0094] The preparation method is as follows: After mixing the composition and peptide amino acid solution evenly according to the above mass ratio, add the above potassium fertilizer and functional additives in sequence, and stir evenly to obtain the final product.

[0095] Example 11 This embodiment is an application embodiment. Transcriptome sequencing was used to verify the mechanism of action of the composition of the present invention and the synergistic effect of 2,3-butanediol.

[0096] This embodiment was conducted in Hedong District, Linyi City, where a hydroponic experiment of dwarf tomatoes was carried out in an artificial climate chamber, with 6 treatments set up: CK: Water control.

[0097] T1: Acetonitrile 80g / L aqueous solution.

[0098] T2: 80 g / L aqueous solution of acetoin + 2,3-butanediol (the ratio and concentration of the two are the same as in Example 3).

[0099] T3: The ternary comparative composition without 2,3-butanediol prepared in Example 4.

[0100] T4: The quaternary composition prepared in Example 3.

[0101] T5: Biostimulant prepared in Example 9.

[0102] The specific implementation method is as follows: Dwarf tomato seedlings with uniform growth were selected, and each treatment group had at least 12 biological replicates. Ten days after germination, the seedlings were carefully transplanted into transparent hydroponic bottles containing 200 mL of the corresponding treatment solution (800-fold dilution) and secured with planting cotton. All hydroponic bottles were randomly placed on a shaker in an artificial climate chamber for hydroponics. The culture conditions were set as follows: a photoperiod of 14 hours light / 10 hours dark, and a light intensity of 250 μmol·m⁻². - ²·s - ¹ Day / night temperature was 25℃ / 20℃, and relative humidity was 60%–70%. The shaker speed was 80 rpm to ensure aeration of the solution. Ten days after transplanting, healthy lateral roots were randomly selected from each plant, and root tips of about 1 cm were cut. All samples from the same treatment group were mixed and quickly frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80℃ for subsequent non-targeted transcriptome detection.

[0103] A classification analysis was performed on all genes enriched in popular KEGG pathways, and they were categorized into different biological function categories through functional annotation. The results are shown in Table 1 (list of upregulated genes). Figure 1As shown in the gene count statistics for each category, compared to the CK group, T1 (acetoin) mainly upregulated the expression of genes related to hormone regulation (auxin-related) and promoting color change (anthocyanin synthesis). T2 (acetoin + 2,3-butanediol) further enhanced the expression of key genes related to hormone regulation (jasmonic acid signaling pathway) based on T1. T3 (comparative composition without 2,3-butanediol) and T4 (quaternary composition) also significantly regulated genes related to substance transport and enhanced stress resistance. In particular, it can be found that T4 is significantly better than T3 in regulating core genes of hormone and anthocyanin synthesis pathways, demonstrating a synergistic effect of 1+1+1+1>4. T5 (biostimulant) showed a balanced and prominent function in multiple aspects such as hormone regulation, substance transport, reproductive growth, promoting color change, and enhancing stress resistance. It is predicted that it may have better resistance to abiotic stress, regulate the balance of crop growth and development, promote crop flowering, fruit setting, fruit enlargement and color change, and achieve the effects of improving quality, increasing yield, and early market availability.

[0104] Table 1

[0105] Example 12 This embodiment is an application embodiment. Based on embodiment 11, a pot experiment with dwarf tomatoes is conducted to further verify the actual efficacy of the present invention in fruit coloring.

[0106] This embodiment was conducted in Hedong District, Linyi City, where a soil-cultivation experiment of dwarf tomatoes was carried out in a glass greenhouse, with 6 treatments set up: CK: Water control.

[0107] T1: Acetonitrile 80g / L aqueous solution.

[0108] T2: 80 g / L aqueous solution of acetoin + 2,3-butanediol (the ratio and concentration of the two are the same as in Example 3).

[0109] T3: The ternary comparative composition without 2,3-butanediol prepared in Example 4.

[0110] T4: The quaternary composition prepared in Example 3.

[0111] T5: Biostimulant prepared in Example 9. The specific implementation method was as follows: Healthy, uniformly growing dwarf tomato seedlings were selected and transplanted at the four-leaf-one-heart stage. Each treatment group had at least 8 biological replicates, using a completely randomized block design. Each seedling was planted in an independent, uniformly sized pot filled with the same mass of substrate. The substrate was a uniformly sterilized mixture of vermiculite and perlite (volume ratio 1:1). All treatment groups were cultivated in the same glass greenhouse under natural light, with a daytime temperature of 25±2℃, a nighttime temperature of 18±2℃, and a relative humidity of 50%-70%. After transplanting, routine water and basic nutrient supply management was uniformly implemented. The first treatment was administered 10 days after transplanting, with 50 mL of the corresponding treatment solution (500 times diluted) applied to the roots of each plant, repeated every 7 days for a total of 4 applications. Fruit color-changing rate and anthocyanin content were investigated during the color-changing period, and yield and fruit sugar content were investigated at harvest. The experimental results are shown in Table 2.

[0112] Table 2

[0113] As shown in the table above, T4 (quaternary composition) outperformed T3 (ternary composition) in all color-changing, quality, and yield indicators, indicating that 2,3-butanediol has a significant synergistic effect with other components in the composition. T5 (biostimulant) showed the best overall crop effect, indicating that by integrating targeted functional technology (quaternary composition), it regulates nutrient absorption and enhances stress resistance, further improving various crop effects and demonstrating significant potential for improved quality, increased yield, and earlier market entry.

[0114] Example 13 This embodiment is an application example. The composition containing small molecule signaling substances, peptide amino acid solution, and biostimulant prepared in Examples 3, 6, and 9 were tested on strawberries. A comparison was made with commercially available amino acid products (total amino acid content 50%, free amino acid 8%, peptide content 42%, and the ratio of peptides with a molecular weight ≤5000 Da to the total peptide content ≥70%).

[0115] The experiment was conducted in Hedong District, Linyi. Four replicates were designed for each treatment group, with each replicate ≥10m. 2 The corresponding treatments are as follows: Treatment 1, water control; Treatment 2, the above-mentioned competitor control; Treatment 3, the composition of Example 3; Treatment 4, peptide amino acid solution of Example 6; Treatment 5, biostimulant of Example 9.

[0116] The specific application method was as follows: Strawberry seedlings (variety: Akihime) with 3-4 true leaves, propagated from runners, were transplanted. The first treatment was initiated 15 days after transplanting. Each treatment group received 500 ml / mu (1000 times dilution) via irrigation and 50 ml / mu (600 times dilution) via foliar spray, for a total of 3 applications, with an interval of 10 days between each application. The number of initial flowers was assessed at the initial flowering stage, the number of initial fruits before fruit expansion, the color-changing rate at the color-changing stage, and the sugar content and harvest quantity at harvest. The yield from the first 5 harvests was achieved within 25 days after the first batch of strawberries matured. The experimental results are shown in Table 3.

[0117] Table 3

[0118] As can be seen from the table above, each component of this invention exhibits a positive regulatory effect on strawberry growth and development. Specifically, the small molecule signaling composition (Example 3) alone is significantly effective in promoting color change and increasing sugar content; the peptide amino acid solution (Example 6) shows advantages in promoting flower bud differentiation and fruit setting. The biostimulant (Example 9), a synergistic combination of the two, integrates the advantages of both, exhibiting the most comprehensive promoting effect: it not only significantly increases the number of initial flowers and fruit setting rate, laying the foundation for high yield, but more importantly, it greatly improves the uniformity and speed of fruit color change, while simultaneously improving sugar content. This results in a significant increase in the marketable fruit rate of strawberries, with high concentration during early harvesting. The yield of the first five harvests is significantly higher than the water control by 41.2%, effectively achieving the synergistic goals of earlier market entry, improved quality, and increased profits.

[0119] Example 14 This embodiment is an application example, in which the compositions, peptide amino acid solutions, and biostimulants obtained in Examples 3, 6, and 9 above are tested on tomatoes. A comparison is made with a well-known commercial amino acid product (total amino acid content 50%, free amino acid 8%, peptide content 42%, and the ratio of peptides with a molecular weight ≤5000 Da to the total peptide content ≥70%).

[0120] The experiment was conducted in Tangwang Street, Licheng District, Jinan City. Four replicates were designed for each treatment, with each replicate lasting ≥10 minutes. 2 The corresponding treatments are as follows: Treatment 1, water control; Treatment 2, competitor control; Treatment 3, Example 3; Treatment 4, Example 6; Treatment 5, Example 9.

[0121] The specific application method was as follows: Tomatoes (variety: Tianzheng 1567) with 4-5 true leaves were transplanted. The first treatment was initiated 14 days after transplanting, with 500 ml / mu (diluted 1000 times) applied via irrigation and simultaneously 50 ml / mu (diluted 600 times) as a foliar spray. This was repeated three times, with a 10-day interval between each application. The number of initial flowers was measured at the initial flowering stage, the number and diameter of initial fruits were measured at the fruit expansion stage, the color change rate was measured at the color change stage, and the vitamin C content and harvest quantity were measured at harvest. The first four harvests were completed within 20 days after the first batch of tomatoes matured. The experimental results are shown in Table 4.

[0122] Table 4

[0123] Experimental results show that the present invention has a comprehensive promoting effect on tomatoes. Specifically, the small molecule signaling composition (Example 3) has a clear effect in targeting and promoting color change and increasing the vitamin C content of the fruit; the peptide amino acid solution (Example 6) is more conducive to fruit enlargement (increased fruit diameter); and the complete biostimulant (Example 9) exhibits the strongest synergistic effect and comprehensive effect, significantly increasing the number of early flowers, promoting uniform fruit enlargement and rapid color change, and simultaneously improving the nutritional quality of the fruit (vitamin C content). This lays the foundation for higher early yields, with the yield of the first four harvests increasing by 36.6% compared to the water control, demonstrating the great potential of the biostimulant of the present invention in promoting increased tomato yield and quality and earlier market availability.

[0124] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0125] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A composition for targeted color change in crops, characterized in that, It contains an aqueous solution of acetoin, 2,3-butanediol, tetramethylpyrazine, and phenylalanine, among which... The concentration of acetoin in the aqueous solution is 80~150 g / L; The mass ratio of acetoin, 2,3-butanediol, tetramethylpyrazine and phenylalanine is (10-30):(1-5):(0.5-3):(1-5).

2. A biostimulant for regulating crop color change and / or growth, characterized in that, Includes the composition of claim 1 and the peptide amino acid solution.

3. The biostimulant according to claim 2, characterized in that, The mass ratio of the composition to the peptide amino acid solution is (10~30):(40~80).

4. The biostimulant according to claim 2 or 3, characterized in that, It also contains elemental fertilizers and functional additives; the functional additives include wetting synergists and / or film-forming agents.

5. The biostimulant according to claim 4, characterized in that, Based on the total weight of the biostimulants, the content of each component is as follows: The composition: 10%~30%; The peptide amino acid solution contains 40% to 80% of the total content. The elemental fertilizer content is 5%~10%; The functional additives: 5%~10%; The remainder is water.

6. The biostimulant according to claim 2 or 5, characterized in that, The total amino acid content in the peptide amino acid solution is 30%~60%, the free amino acid content is 5%~15%, and the peptide content is 25%~50%. Optionally, the content of peptides with a molecular weight ≤5000 Da in the peptide is not less than 70%; Optionally, the peptide amino acid solution contains at least one of plant-derived peptides and animal-derived peptides; Optionally, the peptide amino acid solution contains at least one of glycine, alanine, glutamic acid, and proline; Optionally, the elemental fertilizer includes potassium fertilizer; Optionally, the elemental fertilizer includes at least one of potassium formate, potassium acetate, and potassium citrate. Optionally, the functional additives include at least one of the following: fatty alcohol polyoxyethylene ether phosphate and modified polyethylene wax emulsion.

7. A method for preparing the biostimulant according to any one of claims 2-6, characterized in that, include: (1) The composition of claim 1 is subjected to a first mixing treatment with peptide amino acid solution.

8. The method according to claim 7, characterized in that, The method further includes: (2) performing a second mixing treatment on the first mixed treatment product and elemental fertilizer; Optionally, the method further includes: (3) subjecting the second mixed treatment product to a third mixed treatment with the functional additive, wherein the third mixed treatment is carried out until the functional additive is completely dissolved to obtain the biostimulant; Optionally, the first, second and third mixing treatments are carried out at 20°C to 30°C.

9. A method for promoting color change in fruit-bearing crops, characterized in that, include: The composition of claim 1 is applied to the crop.

10. A method for promoting color change in fruit-bearing crops, increasing the number of initial flowers, the number of initial fruits, and / or fruit enlargement in fruit-bearing crops, characterized in that, include: The biostimulant according to any one of claims 2 to 6 is applied to the crop.

11. A method for improving early yield, sweetness, vitamin content, and / or stress resistance of fruit crops, characterized in that, include: The biostimulant according to any one of claims 2 to 6 is applied to the crop.

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