Fermented fish protein liquid fertilizer rich in dipeptides and small-molecule organic acids and preparation method thereof

Abstract

The application belongs to the biological field, and specifically discloses a fish protein fermentation liquid fertilizer rich in dipeptides and small-molecule organic acids and a preparation method. The fish protein liquid fertilizer prepared by the method is rich in active peptides necessary for plant growth and development, including leucine dipeptide, glutamic acid dipeptide and proline dipeptide, and small-molecule organic acids including lactic acid, phenyl lactic acid and gamma-aminobutyric acid. The composition and content of small-molecule metabolites are regulated by controlling the fermentation time, so as to play the regulating function of fish protein leftovers as non-nutritional elements of liquid fertilizer. The application realizes high-value utilization of fishery waste, accurately regulates the content and composition of metabolites of the prepared liquid fertilizer, and the liquid fertilizer can significantly promote the growth of apple trees, improve the soluble solid content and quality of fruits, and has multiple functions of nutrition, soil improvement and stress resistance enhancement.

Description

Technical Field

[0001] This invention belongs to the field of bio-organic fertilizer preparation, specifically relating to a fermented liquid fertilizer rich in dipeptides and small molecule organic acid fish protein and its preparation method. Background Technology

[0002] With the increasing demand for green, efficient, and environmentally friendly fertilizers in modern agriculture, amino acid liquid fertilizers, characterized by comprehensive nutrition, high absorption efficiency, and environmental friendliness, are gradually becoming an important alternative to traditional chemical fertilizers. Fish protein, as a high-quality protein resource, is rich in various amino acids and trace elements, making it an ideal raw material for preparing amino acid liquid fertilizers. Low-value marine fish (including small low-value fish and processing by-products), as a byproduct of my country's large-scale fisheries development, have a huge annual output, but their effective utilization rate is low. The current extensive treatment model, mainly based on feed production or landfilling, triggers a dual systemic contradiction: on the one hand, it causes a serious waste of nutritional resources, and on the other hand, it leads to the continuous deterioration of the ecological environment. The accumulation and decay of biomass causes the concentrations of hydrogen sulfide and ammonia nitrogen in nearshore areas to exceed safe thresholds, becoming a major contributing factor to ecological disasters such as red tides. Furthermore, traditional methods for preparing fish protein liquid fertilizers mostly rely on chemical acid hydrolysis or single enzymatic hydrolysis processes, which have problems such as high destruction rates of amino acid active peptides and significant loss of active ingredients, resulting in serious resource waste.

[0003] In recent years, microbial fermentation has gradually become a research hotspot for the resource utilization of fish protein due to its advantages such as mild reaction conditions, high product activity, and environmental friendliness. Fermenting fish protein waste with compound microbial agents can effectively improve the conversion rate of amino acids and promote the generation of small-molecule organic acids as secondary metabolites, thereby increasing the fertilizer efficiency of liquid fertilizers. Furthermore, organic acids (such as lactic acid and acetic acid), as microbial metabolites, not only promote nutrient absorption by plants but also have multiple functions such as soil improvement and pathogen inhibition.

[0004] However, current technologies lack a systematic approach to utilizing the synergistic effects of multiple functional bacteria in fish protein fermentation, and specifically enriching liquid fertilizers containing clearly defined target amino acids or peptides, as well as organic acids. Addressing these challenges, developing low-energy, high-fidelity bio-directed transformation technologies to achieve functional value-added processing of fish protein resources and synergistic environmental pollution control has become a key breakthrough for promoting sustainable fisheries development and the development of high-value agricultural formulations. Therefore, developing a liquid fertilizer based on the fermentation of fish protein using compound bacteria to prepare liquid fertilizer rich in clearly defined dipeptides and organic acids, and its preparation process, not only has significant theoretical value but also broad application prospects. Summary of the Invention

[0005] This invention provides a fermented liquid fertilizer rich in dipeptides and small molecule organic acid fish protein and its preparation method. When used as a liquid fertilizer, the fermented liquid can act as a biostimulant, promoting plant growth, increasing plant yield, and enhancing plant stress resistance. In particular, it can promote the accumulation of solids in apples.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a fish protein fermentation liquid fertilizer rich in dipeptides and small molecule organic acids, wherein the fish protein fermentation liquid fertilizer contains dipeptides derived from fish protein and small molecule organic acids produced by the microbial fermentation metabolism of fish protein; the dipeptides are at least one or more of the following: dipeptides containing glutamic acid, dipeptides containing proline, and dipeptides containing leucine; the small molecule organic acids include at least one or more of the following: lactic acid, phenyllactic acid, and γ-aminobutyric acid; the pH value of the fish protein fermentation liquid fertilizer is 4.0-4.5, the amino acid nitrogen content is ≥2.0g / L, and the total organic acid content is ≥35g / L.

[0007] Preferably, the dipeptide containing glutamic acid is at least one or more selected from the following: valine-glutamic acid, glutamic acid-glutamic acid, lysine-glutamic acid, alanine-glutamic acid, glutamic acid-glycine, glutamic acid-leucine, glutamic acid-lysine, glutamic acid-arginine, glutamic acid-threonine, and glutamic acid-asparagine; the dipeptide containing proline is at least one or more selected from the following: isoleucine-proline, valine-proline, proline-arginine, proline-glutamic acid, proline-lysine, proline-serine, proline-phenylalanine, proline-glutamine, proline-methionine, glutamic acid-proline, proline-threonine, and proline-proline; the dipeptide containing leucine is at least one or more selected from the following: leucine-valine, isoleucine-leucine, leucine-isoleucine, leucine-glutamic acid, threonine-leucine, His-leucine, leucine-leucine, and leucine-tyrosine.

[0008] Preferably, the fish protein fermentation liquid fertilizer also includes at least one or more of the following organic acids: isoleucine, leucine, valine, alanine, proline, aspartic acid, and glycine; the other organic acids also include at least one or more of the following non-protein amino acids: ornithine, glycine, methionine, naphthylcarbamate, DL-m-tyrosine, sarcosine, and norleucine.

[0009] This invention also provides a method for preparing a fish protein fermentation liquid fertilizer rich in dipeptides and small molecule organic acids, the method comprising the following steps: S1. Preparation of compound microbial agents: Compound microbial agents are prepared by combining microbial strains with complementary functions. S2. Raw material pretreatment: Crush and homogenize low-value marine fish and / or their processing by-products, and mix them with water to obtain fermentation substrate; S3. Fermentation condition control: The compound microbial agent is introduced into the fermentation substrate, and fermentation is carried out under controlled conditions of 30-42℃ for a controlled time. S4. Endpoint determination: When the pH value of the fermentation broth drops to 4.0-4.5, the amino acid nitrogen content is ≥2.0g / L, and the total organic acid content is ≥35g / L, the fermentation ends and the fish protein fermentation liquid fertilizer is obtained.

[0010] Preferably, the compound microbial agent in step S1 contains at least two or more of the following: *Lactobacillus reuteri*, lactic acid bacteria, *Planctomyces*, *Lactobacillus symbiosis*, lactobacillus, lactic acid bacteria, and *Acinetobacter*.

[0011] Preferably, the mass ratio of *Lactobacillus reuteri*: *Lactobacillus*: *Planctomyces*: *Lactobacillus*: *Lactobacillus*: *Lactobacillus*: *Acinetobacter* is 55:35:2:2:2:2:2.

[0012] Preferably, the inoculation amount of the compound microbial agent in step S3 is 2%-8% of the total weight of the fermentation substrate.

[0013] Preferably, in step S3, the fermentation time is controlled to be 80-100 days; during the fermentation process, intermittent stirring is used, stirring once every 5-8 days, and stirring for 15-30 minutes each time. The fermentation temperature is 30-42℃.

[0014] Preferably, the fermentation temperature is 37±1℃.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. It achieves precise and targeted regulation of metabolite composition, giving liquid fertilizer diversified and targeted functions.

[0016] This invention, for the first time, achieves precise guidance of microbial community succession and metabolic pathways through a core control method centered on "fermentation time," thereby producing a peak enrichment of functional small-molecule metabolites rich in dipeptides and small-molecule organic acids. The product is rich in dipeptides such as leucine, glutamic acid, and proline, as well as small-molecule organic acids such as lactic acid, phenyllactic acid, and γ-aminobutyric acid (GABA), whose core function is to enhance crop physiological functions and stress resistance. The active peptides are easily absorbed by plants and act as signaling molecules, directly participating in protein synthesis and metabolic regulation, promoting cell division and elongation, resulting in thicker leaves and stronger stems. The small-molecule organic acid group exerts multiple non-nutritive regulatory functions: lactic acid activates rhizosphere nutrients and stimulates the root system; phenyllactic acid effectively inhibits soil-borne pathogens and prevents root diseases; and GABA, as a key stress-resistance substance, significantly enhances the crop's tolerance to drought, salinity, and other adverse conditions, ensuring continuous crop growth in harsh environments.

[0017] 2. An innovative composite microbial synergistic fermentation system achieves targeted enrichment of product functions: This invention does not simply use a single or a few microbial species, but scientifically combines multiple functional microorganisms such as *Lactobacillus reuteri*, lactic acid bacteria, and *Aeromonas vaginalis*, utilizing their synergistic effects. This composite microbial system can not only efficiently enzymatically hydrolyze fish protein to generate a large amount of easily absorbed amino acids and small molecule peptides, but also directionally metabolize and produce abundant small molecule organic acids such as lactic acid and acetic acid, significantly enhancing the functionality and added value of the liquid fertilizer—something difficult to achieve with a single microbial species or chemical methods.

[0018] 3. Significantly improved product quality and fertilizer efficacy of liquid fertilizer: The fermentation products prepared using compound microbial agents are rich in amino acids, small molecule peptides, organic acids, vitamins, nucleic acids, and other active substances, providing comprehensive nutrition and making them easier for plants to directly absorb and utilize, with a utilization rate far exceeding that of traditional chemical fertilizers. Furthermore, the high concentration of small molecule organic acids in the product not only activates fixed nutrients such as phosphorus and potassium in the soil, improves soil compaction, inhibits the reproduction of soil-borne pathogens, and creates a healthy rhizosphere microecological environment, but also stimulates crop root development, enhancing the crop's resistance to stress, cold, drought, and disease. In addition, its effects are more precise, especially for cash crops such as apples. This invention effectively promotes fruit enlargement and coloring, significantly increases the content of soluble solids (sugar and acidity) in the fruit, and improves taste and flavor, demonstrating outstanding effects in enhancing fruit quality and commercial value.

[0019] 4. Broad application prospects and significant economic benefits: This invention's product possesses multiple functions, including nutrient supply, soil improvement, stress resistance induction, and quality enhancement. It is a highly efficient, multifunctional bio-organic liquid fertilizer that can be widely applied to drip irrigation, fertigation, and foliar spraying of cash crops such as apples, citrus, vegetables, and flowers, with broad market application prospects. The promotion of this technology will not only find a high-value outlet for fishery by-products and improve industrial efficiency, but also provide high-quality production materials for green agriculture, reduce reliance on chemical fertilizers, and generate significant economic, ecological, and social benefits.

[0020] Other features and advantages of this disclosure will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments or related technologies of this disclosure, the accompanying drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the accompanying drawings described below are only embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1This is a diagram showing the composition of the microbial community in the fish protein liquid fertilizer compound of the present invention; Figure 2 This is a diagram showing the aroma and flavor index of fish protein liquid fertilizer according to the present invention; Figure 3 This is a Simpson index diagram of the biological community of the fish protein liquid fertilizer of the present invention; Figure 4 This is a diagram showing the Chao1 richness index of the fish protein liquid fertilizer biological community of the present invention. Figure 5 This is a graph showing the relative content of organic matter in the fish protein liquid fertilizer of the present invention. Figure 6 This is a graph showing the relative content of fats in the fish protein liquid of this invention; Figure 7 This is a graph showing the relative sugar content of the fish protein liquid fertilizer of the present invention; Figure 8 This is a graph showing the content of organic acid compounds in the fish protein liquid fertilizer of the present invention; Figure 9 This is a graph showing the content of amino acid compounds in the fish protein liquid fertilizer of the present invention; Figure 10 This is a diagram showing the nitrogenous organic matter content of the fish protein liquid fertilizer of the present invention; Figure 11 This is a stacked diagram showing the peptide content of various amino acids in the fish protein liquid fertilizer of the present invention. Figure 12 This is a stacked diagram showing the percentage of peptide content of each amino acid in the fish protein liquid fertilizer of the present invention. Figure 13 This is a diagram showing the content of the main amino acids in the fish protein liquid fertilizer of the present invention; Figure 14 This is a graph showing the lactic acid content analysis of the fish protein liquid fertilizer of the present invention; Figure 15 This is a chromatogram showing the phenyllactic acid content analysis of the fish protein liquid fertilizer of the present invention; Figure 16 This is a graph showing the γ-aminobutyric acid (GABA) content analysis of the fish protein liquid fertilizer of this invention. Figure 17 This is a graph showing the content analysis of small molecule organic acids in the fish protein liquid fertilizer of the present invention. Figure 18 This is a metabolometric thermogram of the long-term fermented organic fertilizer product made from fish protein liquid fertilizer of the present invention; Figure 19 The Venn diagram shows the number of small molecule differential metabolites in the fish protein liquid fertilizer of this invention. Figure 20 This is a graph showing the content analysis of proline dipeptide in the fish protein liquid fertilizer of the present invention. Figure 21 This is a graph showing the content analysis of leucine dipeptide in the fish protein liquid fertilizer of the present invention. Figure 22This is a graph showing the content analysis of glutamic acid dipeptide in the fish protein liquid fertilizer of the present invention; Figure 23 This is a diagram showing the signaling pathways for the enrichment of major differential metabolites in the fish protein liquid fertilizer of this invention. Figure 24 This is an interaction diagram of the core differential metabolites of the fish protein liquid fertilizer of the present invention; Figure 25 This is a diagram showing the appearance of apple fruit after applying the fish protein liquid fertilizer of the present invention in an apple orchard. Figure 26 This is a graph showing the effect of the fish protein liquid fertilizer of the present invention on the titratable acid content of apples; Figure 27 This is a graph showing the effect of the fish protein liquid fertilizer of the present invention on the soluble solids content of apples. Figure 28 This is a graph showing the effect of the fish protein liquid fertilizer of the present invention on the total phenol content of apples; Figure 29 This is a graph showing the effect of the fish protein liquid fertilizer of the present invention on the soluble sugar content of apples. Figure 30 This is a graph showing the effect of the fish protein liquid fertilizer of the present invention on the reducing sugar content of apples. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0025] Please see Figure 1-30The present invention provides a technical solution: 1. Construction and optimization of a compound microbial agent: Highly efficient and complementary functional strains were screened and obtained, including but not limited to: *Lactobacillus reuteri*, *Lactobacillus*, *Pediococcus*, *Companilactobacillus*, *Lactobacillus*, *Latilactobacillus*, and *Acinetobacter*. The optimal ratio and inoculation proportion of these strains were investigated: *Lactobacillus reuteri*: *Lactobacillus*: *Pediococcus*: *Companilactobacillus*: *Lactobacillus*: *Lactobacillus*: *Acinetobacter* = 55:35:2:2:2:2:2, with an inoculation amount of approximately 2%-8%. This ensures synergistic effects and avoids antagonism during the fermentation process for liquid fertilizer production. Lactobacillus primarily produces acid to lower pH and inhibit contaminating bacteria, while other strains assist in protein degradation and provide precursor substances, forming a stable symbiotic fermentation system. To construct a stable symbiotic and functionally complementary microbial community system during fermentation, so as to ensure efficient degradation of fish protein and targeted accumulation of small molecule organic acid secondary metabolites.

[0026] 2. Preparation process of fish protein fermentation liquid fertilizer: Raw material pretreatment: Low-value marine fish or processing by-products (such as fish heads, bones, viscera, and skin) are pulverized to a particle size passing through a 20-mesh sieve, homogenized until visually uniform and fine, and the optimal material-to-water ratio of 1:2-3 is optimized for fermentation conditions: precisely controlling the fermentation process temperature (30-42℃), pH (3.0-4.5), and fermentation time (20-180 days). The optimal combination of process parameters is determined using single-factor experiments to maximize the content of small-molecule organic acids (lactic acid, acetic acid, etc.) and amino acids.

[0027] 3. Analysis of the composition of active substances in fish protein liquid fertilizer: By comparing the content of carbon and nitrogen sources beneficial to plant growth in fish protein liquid fertilizers produced at different fermentation times, and using 16S microbiome and metabolomics techniques, the types and concentrations of small-molecule organic acids, the composition and content of free amino acids, and other core indicators in the liquid fertilizer products obtained at different fermentation times were quantitatively analyzed to clarify their material basis differences. Furthermore, carbohydrates and their analogues, as well as lipids and their analogues, can serve as carbon sources required for plant growth, providing nutrition for plants at different growth stages. Moreover, small-molecule organic acids can directly or indirectly participate in the synthesis of key signaling substances and functional compounds in plant nutrition, development, and stress resistance processes.

[0028] 4. Analysis of fermentation products, their application in apple trees, and functional evaluation: Liquid chromatography-mass spectrometry (LC-MS) and other techniques were used to qualitatively and quantitatively analyze the types and contents of small molecule organic acids (such as lactic acid, acetic acid, and propionic acid), free amino acids, and small molecule active peptides in the fermentation broth. The liquid fertilizer was then applied to apple trees in a field trial to study its effects on soluble solids, sugar content, acidity, sugar-acid ratio, vitamin C content, and coloring of apple fruits, thus verifying its core efficacy in improving fruit quality.

[0029] The research content of this invention revolves around the main line of "microbial agent construction - product development - efficacy verification", systematically solving the key technical problems in the process of preparing high-value-added, functional liquid fertilizer by fermenting low-value fish protein with compound bacteria, and finally forming a complete, mature and industrializable production technology solution.

[0030] More specifically, the present invention provides a fermented liquid fertilizer rich in dipeptides and small molecule organic acid fish protein. The composition of the secondary metabolites of the liquid fertilizer was analyzed, and the analysis results are shown in Tables 1-3 below.

[0031] Table 1. Compositional analysis of liquid fertilizer metabolites

[0032] A total of 70,279 metabolites were detected in the entire metabolome under both positive and negative ion modes, including 44,110 positive ions and 26,169 negative ions. After filtering for deviations and missing values, imputing missing values, and standardizing the raw data, 45,452 metabolites were retained, resulting in a final annotation of 3,389 metabolites, including 2,073 positive ions and 1,316 negative ions. The most frequently detected metabolites in this analysis were organic heterocyclic compounds, followed by organic acids and their derivatives, benzene rings, lipids, and lipid molecules.

[0033] Table 2 Main components of substances in each category under positive ion mode

[0034] Table 3 Main components of each category of substances under negative ion mode

[0035] Tables 2 and 3 show the three substances with the highest content in various metabolites under positive and negative ion modes. Among the organic acids, lactic acid, 3-amino-4-methylvaleric acid, and 2-hydroxybutyric acid have the highest content under negative ion mode.

[0036] Table 4. Composition and characteristics of dipeptides rich in leucine, glutamic acid, and proline in fish protein liquid fertilizer.

[0037] The dipeptide composed of leucine, glutamic acid and proline in the liquid fertilizer of this invention serves as the fertilizer matrix. Its core biological function is not to provide a large number of elements like traditional nitrogen, phosphorus and potassium, but to act as a highly efficient biostimulant. By regulating the physiological metabolic processes of plants, it enhances the crop's resistance to adversity, promotes growth and ultimately improves yield and quality.

[0038] This invention analyzes the microbial community composition and content distribution characteristics of fermented liquid fertilizer. Analysis of the microbial community structure of the fermented liquid fertilizer shows that: At the phylum level, Firmicutes was the dominant bacterial group in the 20-day fermentation broth sample, with a relative abundance of 98.37%. In the 90-day fermentation broth sample, the dominant bacterial groups shifted to Firmicutes and Proteobacteria, with Actinobacteriota also appearing, with relative abundances of approximately 82.05%, 14.82%, and 2.40%, respectively. In the 180-day fermentation broth sample, Firmicutes again became the absolutely dominant group, accounting for 96.15%. Firmicutes microorganisms are typically Gram-positive bacteria with thick cell walls. Representative groups such as Bacillus and Clostridium can form spores and have strong tolerance to adversity (see...). Figure 1 ).

[0039] The Aroma Index and Simpson Index were used to primarily reflect community diversity. The Aroma Index, derived from information entropy, indicates greater uncertainty. Higher uncertainty means more unknown factors within the community, thus indicating higher diversity. The 180-day fermentation broth showed the highest Simpson Index value, indicating the richest microbial diversity in the 180-day group. The 90-day fermentation broth showed the highest Aroma Index value, indicating the highest microbial diversity in the 90-day group (see...). Figure 2 and Figure 3 ).

[0040] Biodiversity indices primarily reflect community richness and are used to estimate the number of species within a community; higher values ​​indicate a greater number of species. The results showed that the 90-day fermentation broth had the highest biodiversity value, indicating the richest microbial diversity in the 90-day group, with the most microorganisms participating during the 90-day fermentation process, suggesting an active fermentation state. Detailed mass spectrometry analysis results can be found in [link to analysis]. Figure 4 .

[0041] Depend on Figure 5 Analysis revealed that at 20 days, the most abundant substances in the fermentation broth were lipids and lipid molecules, while at 90 and 180 days, the most abundant substances were benzene ring compounds. Except for organic oxygen-containing compounds, the content of all substances increased with fermentation time, with the most significant changes occurring between 20 and 90 days.

[0042] Depend on Figure 6-10 Analysis revealed that, except for carbohydrates and nitrogenous organic matter, which showed a trend of first increasing and then decreasing, the contents of all other substances increased with increasing fermentation time, with lipids showing the most significant differences at different fermentation times. Among all the small molecules in the fermentation broth, nitrogenous organic matter had the highest content, accounting for nearly half of all small molecules. It can be decomposed by soil microorganisms into nitrogen sources needed by plants or directly absorbed by plants.

[0043] Depend on Figure 11-12 As can be seen, a total of 103 dipeptides or polypeptides were detected. In the 20-day fermentation broth, peptides containing valine were the most abundant, followed by isoleucine, leucine, and proline. With increasing fermentation time, the amount of peptides in the fermentation broth showed a very significant decreasing trend. As fermentation progresses, microorganisms consume some peptides as an energy source or to synthesize other metabolites. Furthermore, peptides may be broken down into smaller molecules, such as amino acids, under the action of microorganisms.

[0044] Depend on Figure 13 It is known that the majority of amino acids in the fermentation broth that can synthesize proteins are isoleucine, leucine, valine, alanine, phenylalanine, methionine, and valine. Isoleucine and leucine constitute a significant portion of this composition. Among these, leucine and isoleucine show a trend of first decreasing and then increasing with increasing fermentation time, while the others all increase with increasing fermentation time.

[0045] In agricultural products and agriculture, lactic acid can be used as a green feed preservative and forage ripening agent; it can also be used as a feed preservative and a microbial enhancer and stabilizer for by-products of feed, grain, and meat processing; therefore, lactic acid is a green and pollution-free chelating agent, and its use in combination with fertilizers may enhance fertilizer effects and achieve better results. The lactic acid content in the liquid fertilizer of this invention also changes over time (see...). Figure 14 ).

[0046] Phenyl lactic acid (PLA) is a small-molecule organic acid produced by lactic acid bacteria through the metabolism of phenylalanine. It is a novel substance that regulates plant growth, significantly promoting root growth in rice and lettuce. Further research using existing technologies has confirmed that LPA treatment, while promoting root growth in rice seedlings, also promotes the accumulation of dry matter and vegetative growth in the aboveground parts of rice seedlings. The phenyl lactic acid content in the liquid fertilizer of this invention also changes over time (see...). Figure 15 ).

[0047] Research on alleviating environmental stress in plants through the application of exogenous substances has become a hot topic. Related studies have shown that γ-aminobutyric acid (GABA) plays an important role in regulating the germination period of plant seeds. The main method of applying GABA is pre-germination seed soaking. Numerous research experiments have shown that soaking seeds in GABA can improve seed germination rate, accelerate germination speed, increase the conversion rate of stored substances, promote the elongation of the radicle and plumule, and increase the fresh and dry weight of early seedlings. The GABA content in the liquid fertilizer of this invention also changes over time (see...). Figure 16 ).

[0048] Metabolomics results showed that the fish protein liquid fertilizer prepared by long-term fermentation of this invention contains abundant small-molecule organic nitrogen-containing compounds, including plant growth hormone analogs (such as 1-methyl-3-indoleacetic acid), micronutrient chelators (such as ethacrylic acid), and various amides that can serve as slow-release nitrogen sources. These metabolites collectively constitute a complex functional system integrating nutrition, regulation, and stress resistance. Through multiple pathways, including stimulating root development, improving micronutrient availability, providing organic nitrogen sources, and regulating endogenous plant hormone levels, they work synergistically to ultimately achieve a comprehensive effect of significantly promoting crop growth, improving fruit quality, and enhancing stress resistance. The content of small-molecule organic acids in this invention changes with fermentation time (see...). Figure 17 ).

[0049] Heatmap analysis of metabolite profiles from different fermentation cycles (see...) Figure 18 The fermentation time significantly affected the composition and content of its metabolites, indicating that as the number of fermentation days increased, macromolecules in the fish protein fermentation broth underwent continuous degradation under the catalysis of the compound microbial agent, forming small-molecule protein peptides, amino acids, and organic acids. This provides direct evidence for the growth characteristics of plants at different stages.

[0050] The number of differentially metabolites identified by LC-MS / MS in the fish protein fermentation broth is as follows: Figure 19 As shown, 747 metabolites were identified in all three fermentation cycles (short-term 20 days, medium-term 90 days, and long-term 180 days). 83 metabolites were identified in both the long-term and short-term cycles, 623 metabolites in both the medium-term and short-term cycles, and 85 metabolites in both the long-term and short-term cycles. This result indicates that the composition of the fish protein fermentation broth undergoes drastic changes in the early stages of fermentation, while the product gradually stabilizes after fermentation (see...). Figure 19 Given that there are little difference in the quantity and composition of metabolites between long-term fermentation products and mid-term fermentation products, and taking into account both production costs and product quality, the content and characteristics of mid-term fermentation products will be the focus of attention.

[0051] Metabolomics also revealed that fish protein liquid fertilizer contains small peptide molecules rich in proline (see...). Figure 20These small peptides play an important role in plant osmotic regulation and free radical scavenging, and are also involved in plant flowering and embryonic development, as well as in signal transduction pathways leading to stress tolerance as second messengers. Furthermore, because proline is closely related to plant stress resistance, it can promote plant stress resistance to a certain extent.

[0052] Metabolomics analysis of the amino acid, peptide, and analogue composition in fish protein liquid fertilizer revealed small peptides containing leucine in the long-term fermentation broth (see...). Figure 21 These small peptides can promote cell division, thereby promoting plant growth. They can also affect plant growth and development by regulating plant hormone signal transduction. In addition, leucine participates in the plant's disease resistance response during the plant growth process.

[0053] Metabolomics analysis of the amino acid, peptide, and analogue composition in fish protein liquid fertilizer revealed small peptides containing glutamic acid in the long-term fermentation broth (see...). Figure 22 In terms of plant growth and development, glutamate promotes the growth of plant roots and leaves. In plant metabolic pathways, glutamate can be directly converted into glycolic acid as a sugar, and it can also combine with other amino acids in protein synthesis to form new proteins. Furthermore, glutamate can inhibit the damage of some stress factors to plants and influence plant growth, development, and metabolic pathways through its interaction with the synthesis of some plant hormones.

[0054] from Figure 23 A comparison of the number of metabolites between the two groups revealed 83 differentially expressed metabolites between the long-fermentation-prepared fish protein liquid fertilizer and the short-fermentation product. Further analysis of the signaling pathways regulated by these differentially expressed metabolites showed that they primarily affect energy metabolism, especially amino acid synthesis and the transduction and transport of substance signals. This suggests that metabolites in fish protein directly participate in plant growth and development. Finally, analysis of the interactions between the differentially expressed metabolites revealed that the main differentially expressed metabolites are amino acids, particularly lysine, glutamic acid, proline, and alanine (see...). Figure 24 The results also confirm that fish protein liquid fertilizer regulates plant growth and development through small molecules of organic acids and amino acids. The aforementioned substances mainly accumulate in the mid-term and long-term fermented products. Considering the quality of the products and the finished product, the mid-term fermented product was finally selected as the core product and applied to apple cultivation.

[0055] In an orchard, the mid-stage fermented fish protein liquid fertilizer was compared with that applied to apple orchards using inorganic fertilizer. In apple orchards with flat terrain, uniform fertility, ample sunlight, and convenient irrigation, the fish protein organic fertilizer was evenly sprayed onto the leaves of the apple trees using a sprayer, ensuring the leaf surface was fully moistened. The yield and quality of the apples were analyzed. Visually, the average weight of the apples increased, their color became more vibrant red, and their shape became rounder and fuller. Furthermore, the fish protein organic fertilizer significantly improved the titratable acid, reducing sugar, total sugar and total phenols, and soluble solids content of the apples compared to apples without organic fertilizer. This indicates that the sensory flavor characteristics of apples treated with organic fertilizer are superior to those treated with inorganic fertilizer (see...). Figure 25-30 ). Example:

[0056] The fish protein fermentation liquid fertilizer of the present invention is prepared by the following steps: S1. Preparation of compound microbial agent: Complementary microbial strains are combined to obtain compound microbial agent. The compound microbial agent includes Lactobacillus reuteri: Lactobacillus: Planar cocci: Associated Lactobacillus: Lactobacillus: Lactobacillus: Lactobacillus: Acinetobacter in a mass ratio of 55:35:2:2:2:2:2:2. S2. Raw material pretreatment: Low-value marine fish and their processing by-products are crushed and homogenized. The crushed particle size is reduced to pass through a 20-mesh sieve, and the homogenized material is homogenized until it is uniform and fine to the naked eye. It is then mixed with water at a ratio of 1:2 to obtain the fermentation substrate. S3. Fermentation conditions control: Add more than 5% compound microbial agent to the above fermentation substrate, and carry out fermentation at 37℃ for 90 days. Stir once every 6 days during the fermentation process, and stir for 25 minutes each time. S4. Endpoint determination: When the pH value of the fermentation broth drops to 4.2, the amino acid nitrogen content is ≥2.0g / L, and the total organic acid content is ≥35g / L, the fermentation ends and the fish protein fermentation liquid fertilizer is obtained.

[0057] Analysis of the fish protein fermentation liquid fertilizer obtained above revealed that it contains dipeptides derived from fish protein and small-molecule organic acids produced by the microbial fermentation metabolism of fish protein. The dipeptides include: glutamic acid dipeptides, including valine-glutamic acid, glutamic acid-glutamic acid, lysine-glutamic acid, alanine-glutamic acid, glutamic acid-glycine, glutamic acid-leucine, glutamic acid-lysine, glutamic acid-arginine, glutamic acid-threonine, and glutamic acid-asparagine; and proline dipeptides, rich in isoleucine-proline, valine-proline, proline-arginine, proline-glutamic acid, proline-lysine, proline-serine, and proline- It contains phenylalanine, proline-glutamine, proline-methionine, glutamic acid-proline, proline-threonine, and proline-proline; leucine dipeptide; and is rich in leucine-valine, isoleucine-leucine, leucine-isoleucine, leucine-glutamic acid, threonine-leucine, histidine-leucine, leucine-leucine, and leucine-tyrosine. Small molecule organic acids include isoleucine, leucine, valine, alanine, proline, aspartic acid, and glycine. It also contains other organic acids such as ornithine, glycineamide, methionineamide, naphthylcarbamate, DL-m-tyrosine, sarcosine, and norleucine.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A fermented liquid fertilizer rich in dipeptides and small-molecule organic acid fish protein, characterized in that: The fish protein fermentation liquid fertilizer contains dipeptides derived from fish protein and small-molecule organic acids produced by the microbial fermentation and metabolism of fish protein; the dipeptides are at least one or more of the following: dipeptides containing glutamic acid, dipeptides containing proline, and dipeptides containing leucine; the small-molecule organic acids include at least one or more of the following: lactic acid, phenyllactic acid, and γ-aminobutyric acid; the pH value of the fish protein fermentation liquid fertilizer is 4.0-4.5, the amino acid nitrogen content is ≥2.0g / L, and the total organic acid content is ≥35g / L.

2. The fermented liquid fertilizer rich in dipeptides and small molecule organic acid fish protein according to claim 1, characterized in that: The dipeptide containing glutamic acid is at least one or more of the following: valine-glutamic acid, glutamic acid-glutamic acid, lysine-glutamic acid, alanine-glutamic acid, glutamic acid-glycine, glutamic acid-leucine, glutamic acid-lysine, glutamic acid-arginine, glutamic acid-threonine, and glutamic acid-asparagine; the dipeptide containing proline is at least one or more of the following: isoleucine-proline, valine-proline, proline-arginine, proline-glutamic acid, proline-lysine, proline-serine, proline-phenylalanine, proline-glutamine, proline-methionine, glutamic acid-proline, proline-threonine, and proline-proline; the dipeptide containing leucine is at least one or more of the following: leucine-valine, isoleucine-leucine, leucine-isoleucine, leucine-glutamic acid, threonine-leucine, histidine-leucine, leucine-leucine, and leucine-tyrosine.

3. The fermented liquid fertilizer rich in dipeptides and small molecule organic acid fish protein according to claim 1, characterized in that: The fish protein fermentation liquid fertilizer also includes at least one or more of the following organic acids: isoleucine, leucine, valine, alanine, proline, aspartic acid, and glycine; the other organic acids also include at least one or more of the following non-protein amino acids: ornithine, glycine, methionine, naphthylcarbamate, DL-m-tyrosine, sarcosine, and norleucine.

4. A method for preparing fish protein fermentation liquid fertilizer rich in dipeptides and small molecule organic acids as described in any one of claims 1-3, characterized in that: The preparation method of the fish protein fermentation liquid fertilizer includes the following steps: S1. Preparation of compound microbial agents: Compound microbial agents are prepared by combining microbial strains with complementary functions. S2. Raw material pretreatment: Crush and homogenize low-value marine fish and / or their processing by-products, and mix them with water to obtain fermentation substrate; S3. Fermentation condition control: The compound microbial agent is introduced into the fermentation substrate, and fermentation is carried out under controlled conditions of 30-42℃ for a controlled time. S4. Endpoint determination: When the pH value of the fermentation broth drops to 4.0-4.5, the amino acid nitrogen content is ≥2.0g / L, and the total organic acid content is ≥35g / L, the fermentation ends and the fish protein fermentation liquid fertilizer is obtained.

5. The method for preparing a fish protein fermentation liquid fertilizer rich in dipeptides and small molecule organic acids according to claim 4, characterized in that: The compound microbial agent in step S1 contains at least two or more of the following: *Lactobacillus reuteri*, lactic acid bacteria, *Planctomyces*, *Lactobacillus symbioticus*, *Lactobacillus*, *Lactobacillus*, and *Acinetobacter*.

6. The method for preparing a fish protein fermentation liquid fertilizer rich in dipeptides and small molecule organic acids according to claim 5, characterized in that: The mass ratio of *Lactobacillus reuteri*: *Lactobacillus*: *Planctomyces*: *Lactobacillus*: *Lactobacillus*: *Lactobacillus*: *Acinetobacter* is 55:35:2:2:2:2:

2.

7. The method for preparing a fish protein fermentation liquid fertilizer rich in dipeptides and small molecule organic acids according to claim 4, characterized in that: In step S3, the inoculation amount of the compound microbial agent is 2%-8% of the total weight of the fermentation substrate.

8. The method for preparing a fish protein fermentation liquid fertilizer rich in dipeptides and small molecule organic acids according to claim 4, characterized in that: In step S3, the fermentation time is controlled to be 80-100 days; during the fermentation process, intermittent stirring is used, stirring once every 5-8 days, and stirring for 15-30 minutes each time; the fermentation temperature is 30-42℃.

9. The method for preparing a fish protein fermentation liquid fertilizer rich in dipeptides and small molecule organic acids according to claim 8, characterized in that: The fermentation temperature is 37±1℃.

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