A fermented shell liquid calcium fertilizer composition, a preparation method and application thereof

Abstract

The present application relates to shell liquid calcium fertilizer technical field, more specifically, it is a kind of fermented shell liquid calcium fertilizer composition and its preparation method and application.Fermented shell liquid calcium fertilizer composition includes: shell powder 100~300 parts, chitin 5~30 parts, compound microbial inoculant 5~20 parts, molasses 30~80 parts, old brine 10~50 parts, potassium dihydrogen phosphate 3~10 parts, magnesium sulfate 1~5 parts, water 800~1200 parts;The compound microbial inoculant is compounded by bacillus amyloliquefaciens, lactobacillus plantarum and aspergillus niger according to (1~3):(2~4):(1~2) volume ratio, and total viable count is not less than 1×10 8 CFU / g.

Description

Technical Field

[0001] This invention relates to the field of liquid calcium fertilizer technology based on seashells, and more specifically, to a fermented liquid calcium fertilizer composition based on seashells, its preparation method, and its application. Background Technology

[0002] my country is a major producer and consumer of shellfish, generating a massive amount of shell waste annually. Statistics show that my country's total shellfish production exceeds 12 million tons, with large quantities of shells being dumped as waste, causing not only resource waste but also serious environmental pollution. Shells contain up to 95% calcium carbonate, making them a high-quality natural calcium source. Utilizing them for fertilizer production has significant economic and environmental implications.

[0003] Currently, common methods for preparing fertilizer from seashells include mechanical crushing, acid hydrolysis, and microbial degradation. Mechanical crushing suffers from low seashell utilization and a long degradation cycle of seashell powder in soil; acid hydrolysis is costly and prone to environmental pollution. Microbial decomposition has become a research hotspot due to its low cost and good decomposition effect. However, current microbial decomposition of seashells mainly uses lactic acid bacteria and Xanthomonas, which show significant differences in decomposition effects on different types of seashells, resulting in poor universality. Furthermore, existing seashell fermentation fertilizers are mostly in solid form or only focus on the release of calcium, lacking systematic design and functional enhancement of the active ingredients in the fermentation products.

[0004] Therefore, developing a fermented shell liquid calcium fertilizer composition with high decomposition efficiency, good versatility, and multiple nutritional functions is of great practical significance. Summary of the Invention

[0005] In view of this, the purpose of this invention is to at least partially solve one of the technical problems in the above-mentioned related technologies. To this end, this invention provides a fermented shell liquid calcium fertilizer composition, its preparation method, and its application.

[0006] The first aspect of this invention discloses a fermented shell liquid calcium fertilizer composition, comprising the following components by weight: 100-300 parts shell powder, 5-30 parts chitin, 5-20 parts compound microbial agent, 30-80 parts molasses, 10-50 parts old brine, 3-10 parts potassium dihydrogen phosphate, 1-5 parts magnesium sulfate, and 800-1200 parts water; the compound microbial agent is composed of Bacillus amyloliquefaciens, Lactobacillus plantarum, and Aspergillus niger in a volume ratio of (1-3):(2-4):(1-2), with a total viable count of not less than 1×10⁻⁶. 8 CFU / g.

[0007] Furthermore, the shell powder includes at least one of oyster shell powder, scallop shell powder, and clam shell powder, with a particle size of 100-300 mesh.

[0008] Furthermore, the chitin is crude chitin extracted from shrimp and crab shells, with a purity of ≥85%.

[0009] Furthermore, the old brine is concentrated brine produced during the salt production process in salt fields, with a density of 1.20~1.30 g / mL.

[0010] Furthermore, the molasses includes cane molasses or beet molasses.

[0011] A second aspect of this invention provides a method for preparing a fermented shell liquid calcium fertilizer composition, comprising the following steps: (1) After washing and drying the seashells, pulverize them to 100-300 mesh to obtain seashell powder; (2) Bacillus amyloliquefaciens, Lactobacillus plantarum and Aspergillus niger were activated and cultured separately, and then mixed in proportion to obtain a compound microbial agent; (3) Mix shell powder, chitin, molasses, potassium dihydrogen phosphate, magnesium sulfate and water evenly, inoculate with compound microbial agent, and carry out staged variable temperature fermentation at 28~37℃. The total fermentation time is 7~14 days. (4) After fermentation, add old brine, stir well, and continue the chelation reaction at 28~32℃ for 2~3 days; (5) After the reaction is complete, filter to remove solid residue, and the fermented shell liquid fertilizer composition is obtained.

[0012] Further, the staged variable-temperature fermentation in step (3) includes the following stages performed sequentially: an aerobic enzyme production stage, an anaerobic acid production stage, and an aerobic acid production stage; the conditions for the aerobic enzyme production stage are: temperature 35~37℃, pH 6.0~7.0, rotation speed 150~200rpm, aeration rate 0.5~1.5vvm, and fermentation for 2~4 days; the conditions for the anaerobic acid production stage are: temperature 28~30℃, pH naturally decreasing, rotation speed 0~50rpm, and fermentation for 3~5 days; the conditions for the aerobic acid production stage are: temperature 30~32℃, pH 4.0~5.0, rotation speed 150~200rpm, aeration rate 0.5~1.0vvm, and fermentation for 2~5 days.

[0013] Furthermore, at the end of the aerobic enzyme production stage described in step (3), the protease activity in the fermentation broth is ≥500U / mL, and the chitosanase activity is ≥50U / mL; At the end of the aerobic acid production stage, the pH value of the fermentation broth drops to 4.0~5.0, and the water-soluble calcium content is ≥15g / L.

[0014] A third aspect of the present invention provides the application of the fermented shell liquid calcium fertilizer composition in the preparation of liquid calcium fertilizer.

[0015] A fourth aspect of the present invention provides a fermented shell liquid calcium fertilizer, wherein the liquid calcium fertilizer comprises the fermented shell liquid fertilizer composition.

[0016] First, the three strains of *Bacillus amyloliquefaciens*, *Lactobacillus plantarum*, and *Aspergillus niger* in the compound microbial agent each perform their respective functions and complement each other, forming a three-level synergistic chain of enzymatic hydrolysis, rapid acid production, and deep chelation. No single strain can be replaced by the others. As the vanguard of the fermentation process, the irreplaceable nature of *Bacillus amyloliquefaciens* is reflected in its unique enzyme production spectrum. Studies have shown that *Bacillus amyloliquefaciens* can produce a variety of important extracellular enzyme systems, including polysaccharide-degrading enzymes such as α-amylase, cellulase, β-1,3-1,4-glucanase, lignocellulose degrading enzyme, and chitosanase, as well as protein-degrading enzymes such as proteases, thrombolytic enzymes, and rennet. These enzyme systems can effectively decompose residual organic matter and chitin in shells, disrupting the organic matter-calcium carbonate complex structure of the shells and creating favorable conditions for subsequent acid hydrolysis. In contrast, while other common bacterial strains, such as *Bacillus subtilis*, can also produce proteases, their ability to produce chitosanase is far less than that of *Bacillus amyloliquefaciens*. Yeasts and lactic acid bacteria, on the other hand, essentially lack the ability to produce chitosanase, thus failing to complete the crucial enzymatic hydrolysis and cell wall breaking step. As the primary acid-producing bacterium in the fermentation process, *Lactobacillus plantarum*'s irreplaceable role lies in its high efficiency in lactic acid production and its compatibility with calcium ion dissolution. Studies have shown that fermenting calcium-containing substrates using *Lactobacillus plantarum* can achieve efficient calcium dissolution through lactic acid production. In the fermentation system of razor clam shells, lactic acid bacteria fermentation of shell powder can effectively convert free calcium; in the fermentation system of chicken bone paste, *Lactobacillus plantarum* also demonstrates a significant effect on calcium release. *Lactobacillus plantarum* metabolizes molasses to produce a large amount of lactic acid, rapidly lowering the system pH and promoting the dissolution of calcium carbonate into soluble calcium ions. Other acid-producing bacteria, such as acetic acid bacteria, can also produce acid, but their acid production is mainly acetic acid, and their ability to chelate calcium ions is far less than that of lactic acid. While *Aspergillus niger* produces citric acid, its acid production rate is slow and the process is lengthy, making it difficult to quickly establish an acidic environment in the early stages of fermentation and provide suitable pH conditions for subsequent reactions in a short time. As a deep-conversion bacterium in the fermentation process, *Aspergillus niger*'s irreplaceable role lies in its ability to produce multiple organic acids. During fermentation, *Aspergillus niger* can secrete various organic acids such as citric acid, gluconic acid, and oxalic acid. These multiple organic acids not only further dissolve calcium carbonate, but more importantly, they can form organic acid-calcium chelates with calcium ions, significantly improving the bioavailability of calcium. While *Bacillus amyloliquefaciens* and *Lactobacillus plantarum* can also produce acid, their acid production is limited; the former mainly produces acetic acid, and the latter mainly produces lactic acid. They cannot produce a complex system of multiple organic acids such as citric acid, gluconic acid, and oxalic acid like *Aspergillus niger*, thus showing significant shortcomings in deep calcium chelation and conversion. It is evident that the enzymatic hydrolysis of Bacillus amyloliquefaciens removes obstacles to the acid hydrolysis of Lactobacillus plantarum. The rapid acid production of Lactobacillus plantarum establishes an acidic environment, thereby creating favorable pH conditions for the growth of Aspergillus niger. The deep chelation of Aspergillus niger further converts dissolved calcium ions into highly bioactive organic acid calcium. These three processes are interconnected and indispensable. The absence of any one strain would mean the breakage of the entire synergistic chain.

[0017] Secondly, the co-fermentation of shell powder and chitin achieves the in-situ generation of chitosan oligosaccharide-calcium complex, which is another core innovation of this disclosure. This invention is the first to place two marine biomass resources, shell powder (calcium carbonate source) and chitin (aminopolysaccharide source), in the same fermentation system for co-fermentation, allowing them to react in situ under the action of microorganisms. Specifically, under the action of chitosanase secreted by Bacillus amyloliquefaciens, chitin is partially degraded into chitosan oligosaccharides; simultaneously, organic acids produced by Lactobacillus plantarum and Aspergillus niger dissolve the calcium carbonate in the shell into calcium ions. The chitosan oligosaccharide molecular chain is rich in amino and hydroxyl groups, and these active groups can undergo complexation reactions with calcium ions to generate chitosan oligosaccharide-calcium complex in situ. Scientific research shows that calcium can be rapidly absorbed into crops by chitosan oligosaccharides, significantly improving calcium absorption and utilization. Chitosan oligosaccharide-calcium complexes possess three advantages: calcium exists in an organic complex form, resulting in a much higher absorption rate than inorganic calcium ions; chitosan oligosaccharides themselves have multiple functions, including inducing plant disease resistance, promoting root growth, and enhancing crop stress resistance; and chitosan oligosaccharide-calcium complexes combine calcium supplementation and biostimulation, achieving a synergistic effect greater than the sum of its parts. More importantly, while existing technologies have reported the use of shells and chitin separately in fertilizers, a technique for co-fermenting both in the same fermentation system and utilizing in-situ enzymatic hydrolysis by microorganisms to generate chitosan oligosaccharide-calcium complexes has not been reported in existing patents or literature. This invention, through ingenious strain combination and fermentation process design, enables this in-situ generation reaction. The resulting chitosan oligosaccharide-calcium complex exhibited biological effects far exceeding those of conventional calcium fertilizers in subsequent pot experiments.

[0018] Finally, at the fermentation process level, the staged variable-temperature fermentation process adopted in this invention further ensures the full realization of the aforementioned synergistic effects. This process, designed for the different metabolic characteristics of the three strains, involves a three-stage variable-temperature fermentation: aerobic enzyme production → anaerobic acid production → aerobic acid production. The temperature, pH, and dissolved oxygen conditions of each stage have been specifically optimized: The first stage, high-temperature aerobic (35~37℃), promotes the growth and extracellular enzyme secretion of *Bacillus amyloliquefaciens*. At the end of the aerobic enzyme production stage, the protease activity in the fermentation broth is no less than 500 U / mL, and the chitosanase activity is no less than 50 U / mL. The second stage, mesophilic anaerobic (28~30℃), promotes lactic acid fermentation by *Lactobacillus plantarum*. The third stage, mesophilic aerobic (30~32℃), promotes organic acid fermentation by *Aspergillus niger*. At the end of the aerobic acid production stage, the pH value of the fermentation broth drops to 4.0~5.0, and the water-soluble calcium content is no less than 15 g / L. The final product contains no less than 18 g / L of water-soluble calcium, no less than 2 g / L of chitosan oligosaccharide, and no less than 5 × 10⁻⁶ effective viable bacteria. 7CFU / mL. This staged and precisely controlled fermentation process allows all three strains to fully exert their metabolic functions under their respective optimal conditions, maximizing fermentation efficiency. When the fermented shell liquid fertilizer composition prepared according to this invention is diluted 300-500 times and applied as rhizosphere irrigation during the tomato seedling and flowering / fruiting stages, the soil improvement effect is also significant: the soil pH value increases from 5.7 to 6.6, and the soil exchangeable calcium content increases by 48.3%, indicating that the product of this invention has the dual functions of crop nutrient supply and soil improvement. Detailed Implementation

[0019] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0020] 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 or 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.

[0021] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0022] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0023] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0024] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0025] All raw materials used in the examples are commercially available.

[0026] All strains used in the examples were purchased from Beina Biotechnology; among them, Bacillus amyloliquefaciens was numbered BNCC336388; Lactobacillus plantarum was numbered BNCC364479; and Aspergillus niger was numbered BNCC392698.

[0027] Example 1

[0028] This embodiment provides a fermented shell liquid calcium fertilizer, comprising the following components by weight: 200 parts shell powder, 20 parts chitin, 15 parts compound microbial agent, 50 parts molasses, 30 parts old brine, 5 parts potassium dihydrogen phosphate, 3 parts magnesium sulfate, and 1000 parts water; the compound microbial agent is composed of Bacillus amyloliquefaciens, Lactobacillus plantarum, and Aspergillus niger in a volume ratio of 2:3:1.5, with a total viable count of not less than 1×10⁻⁶. 8 CFU / g.

[0029] The shell powder includes oyster shell powder with a particle size of 100-300 mesh; the chitin is crude chitin extracted from shrimp and crab shells with a purity of ≥85%; the old brine is concentrated brine produced during the salt production process in salt fields with a density of 1.25 g / mL; and the molasses is beet molasses.

[0030] The preparation method of the above-mentioned fermented shell liquid calcium fertilizer includes the following steps: (1) After washing and drying the seashells, pulverize them to 100-200 mesh to obtain seashell powder; (2) Bacillus amyloliquefaciens, Lactobacillus plantarum and Aspergillus niger were activated and cultured separately, and then mixed in proportion to obtain a compound microbial agent; (3) Mix shell powder, chitin, molasses, potassium dihydrogen phosphate, magnesium sulfate and water evenly, inoculate with compound microbial agent, and carry out staged variable temperature fermentation at 32℃. The total fermentation time is 8 days. The staged variable-temperature fermentation includes the following sequentially occurring stages: an aerobic enzyme production stage, an anaerobic acid production stage, and an aerobic acid production stage. The conditions for the aerobic enzyme production stage are: temperature 36℃, pH 6.5, rotation speed 160 rpm, aeration rate 0.8 vvm, and fermentation for 3 days. The conditions for the anaerobic acid production stage are: temperature 29℃, pH naturally decreasing, rotation speed 30 rpm, and fermentation for 4 days. The conditions for the aerobic acid production stage are: temperature 31℃, pH 4.5, rotation speed 180 rpm, aeration rate 0.75 vvm, and fermentation for 3 days. At the end of the aerobic enzyme production stage, the protease activity in the fermentation broth is ≥500 U / mL, and the chitosanase activity is ≥50 U / mL. At the end of the aerobic acid production stage, the pH of the fermentation broth drops to 4.5, and the water-soluble calcium content is ≥15 g / L. (4) After fermentation, add old brine, stir well, and continue the chelation reaction at 30℃ for 2.5 days; (5) After the reaction is complete, filter to remove solid residue, and the fermented shell liquid fertilizer composition is obtained.

[0031] Example 2

[0032] This embodiment provides a fermented shell liquid calcium fertilizer, comprising the following components by weight: 100 parts shell powder, 5 parts chitin, 5 parts compound microbial agent, 30 parts molasses, 10 parts old brine, 3 parts potassium dihydrogen phosphate, 1 part magnesium sulfate, and 800 parts water; the compound microbial agent is composed of Bacillus amyloliquefaciens, Lactobacillus plantarum, and Aspergillus niger in a volume ratio of 1:2:1, with a total viable count of not less than 1×10⁻⁶. 8 CFU / g.

[0033] The shell powder is clam shell powder with a particle size of 200-300 mesh; the chitin is crude chitin extracted from shrimp and crab shells with a purity of ≥85%; the old brine is concentrated brine produced during the salt production process in salt fields with a density of 1.20 g / mL; and the molasses is sugarcane molasses.

[0034] The preparation method of the above-mentioned fermented shell liquid calcium fertilizer includes the following steps: (1) After washing and drying the seashells, pulverize them to 100-300 mesh to obtain seashell powder; (2) Bacillus amyloliquefaciens, Lactobacillus plantarum and Aspergillus niger were activated and cultured separately, and then mixed in proportion to obtain a compound microbial agent; (3) Mix shell powder, chitin, molasses, potassium dihydrogen phosphate, magnesium sulfate and water evenly, inoculate with compound microbial agent, and carry out staged variable temperature fermentation at 28℃. The total fermentation time is 7~14 days. The staged variable-temperature fermentation includes the following sequentially performed stages: an aerobic enzyme production stage, an anaerobic acid production stage, and an aerobic acid production stage. The conditions for the aerobic enzyme production stage are: temperature 35℃, pH 6.0, rotation speed 150 rpm, aeration rate 0.5 vvm, and fermentation time 2 days. The conditions for the anaerobic acid production stage are: temperature 28℃, pH naturally decreasing, rotation speed 50 rpm, and fermentation time 3 days. The conditions for the aerobic acid production stage are: temperature 30℃, pH 4.0, rotation speed 150 rpm, aeration rate 0.5 vvm, and fermentation time 2 days. At the end of the aerobic enzyme production stage, the protease activity in the fermentation broth is ≥500 U / mL, and the chitosanase activity is ≥50 U / mL. At the end of the aerobic acid production stage, the pH of the fermentation broth drops to 4.0, and the water-soluble calcium content is ≥15 g / L. (4) After fermentation, add old brine, stir well, and continue the chelation reaction at 28℃ for 2 days; (5) After the reaction is complete, filter to remove solid residue, and the fermented shell liquid fertilizer composition is obtained.

[0035] Example 3

[0036] This embodiment provides a fermented shell liquid calcium fertilizer, comprising the following components by weight: 300 parts shell powder, 30 parts chitin, 20 parts compound microbial agent, 80 parts molasses, 50 parts old brine, 10 parts potassium dihydrogen phosphate, 5 parts magnesium sulfate, and 1200 parts water; the compound microbial agent is composed of Bacillus amyloliquefaciens, Lactobacillus plantarum, and Aspergillus niger in a volume ratio of 3:4:2, with a total viable count of not less than 1×10⁻⁶. 8 CFU / g.

[0037] The shell powder is scallop shell powder with a particle size of 100-300 mesh; the chitin is crude chitin extracted from shrimp and crab shells with a purity of ≥85%; the old brine is concentrated brine produced during the salt production process in salt fields with a density of 1.30 g / mL; and the molasses is sugarcane molasses.

[0038] The preparation method of the above-mentioned fermented shell liquid calcium fertilizer includes the following steps: (1) After washing and drying the seashells, pulverize them to 150-220 mesh to obtain seashell powder; (2) Bacillus amyloliquefaciens, Lactobacillus plantarum and Aspergillus niger were activated and cultured separately, and then mixed in proportion to obtain a compound microbial agent; (3) Mix shell powder, chitin, molasses, potassium dihydrogen phosphate, magnesium sulfate and water evenly, inoculate with compound microbial agent, and carry out staged variable temperature fermentation at 37℃. The total fermentation time is 14 days. The staged variable-temperature fermentation includes the following sequentially performed stages: aerobic enzyme production, anaerobic acid production, and aerobic acid production. The conditions for the aerobic enzyme production stage are: temperature 37℃, pH 7.0, rotation speed 200 rpm, aeration rate 1.5 vvm, and fermentation time 4 days. The conditions for the anaerobic acid production stage are: temperature 30℃, pH naturally decreasing, rotation speed 30 rpm, and fermentation time 5 days. The conditions for the aerobic acid production stage are: temperature 32℃, pH 5.0, rotation speed 200 rpm, aeration rate 1.0 vvm, and fermentation time 5 days. At the end of the aerobic enzyme production stage, the protease activity in the fermentation broth is ≥500 U / mL, and the chitosanase activity is ≥50 U / mL. At the end of the aerobic acid production stage, the pH of the fermentation broth drops to 5.0, and the water-soluble calcium content is ≥15 g / L. (4) After fermentation, add old brine, stir well, and continue the chelation reaction at 32℃ for 3 days; (5) After the reaction is complete, filter to remove solid residue, and you will get fermented shell liquid calcium fertilizer.

[0039] Comparative Example 1 Compared with Example 1, Comparative Example 1 differs in that Bacillus amyloliquefaciens BNCC336388 is replaced with an equal amount of Lactobacillus plantarum BNCC364479.

[0040] Comparative Example 2 Compared with Example 1, Comparative Example 2 differs in that Lactobacillus plantarum BNCC364479 is replaced with Aspergillus niger BNCC392698 in equal amounts.

[0041] Comparative Example 3 Compared with Example 1, Comparative Example 3 differs in that Bacillus amyloliquefaciens BNCC336388 is replaced with an equal amount of Aspergillus niger BNCC392698.

[0042] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that chitin was replaced with shell powder in equal amounts.

[0043] Comparative Example 5 Compared with Example 1, Comparative Example 5 differs in that chitosan is replaced in an equal amount with a compound microbial agent.

[0044] Comparative Example 6 Compared with Example 1, Comparative Example 6 differs in that the compound microbial agent is replaced with chitin in equal amounts.

[0045] Pot experiments were conducted using the fermented shell liquid fertilizers prepared in Examples 1 to 3 and Comparative Examples 1 to 6.

[0046] Tomato pot experiment Test materials The tomato variety used in the experiment was "Xinzhongshu No. 4" (Solanum lycopersicum L.). Plump, uniformly sized seeds were selected, soaked in 55℃ warm water for 15 minutes, and then germinated at 28℃ before being sown in seedling trays. When the seedlings had 3-4 true leaves, healthy, uniformly growing seedlings were transplanted into plastic flowerpots (25 cm in diameter and 20 cm in height). The cultivation substrate was peat moss:vermiculite:perlite = 3:1:1 (volume ratio), with 3.5 kg of substrate per pot and one plant per pot. The experiment was conducted in a greenhouse under natural light, with daytime temperatures controlled at 25-30℃ and nighttime temperatures at 18-22℃, and relative humidity at 60%-70%.

[0047] Experimental Design The experiment consisted of 6 treatments: CK: Watered with an equal amount of clean water (blank control); Liquid fertilizer 2: Apply the liquid fertilizer prepared in Comparative Example 1 by watering; Liquid fertilizer 3: Apply the liquid fertilizer prepared in Comparative Example 2 by watering; Liquid fertilizer 4: Apply the liquid fertilizer prepared in Comparative Example 3 by watering; Liquid fertilizer 5: Apply the liquid fertilizer prepared in Comparative Example 4 by watering; Liquid fertilizer 6: Apply the liquid fertilizer prepared in Comparative Example 5 by watering; Liquid fertilizer 7: Apply the liquid fertilizer prepared in Comparative Example 6 by watering; Liquid fertilizer 1: Apply the liquid fertilizer prepared in Example 1 (the present invention) by watering.

[0048] Each treatment was repeated 3 times (3 pots per replicate, 1 plant per pot), for a total of 63 pots, arranged in a randomized block. All pots were placed randomly in the greenhouse and their positions were rotated periodically to reduce edge effects.

[0049] All liquid fertilizers were diluted 100 times (by volume) with water before use, shaken thoroughly, and applied immediately. The first fertilization was given on the 7th day after transplanting, with 100 mL of diluted solution applied to each pot. Subsequent fertilizations were given every 7 days for a total of 4 applications (on days 7, 14, 21, and 28 after transplanting). The control (CK) treatment received the same amount of water. Except for the type of fertilizer, all treatments maintained the same water management (daily quantitative watering to maintain substrate moisture content at approximately 70% field capacity) and pest and disease control measures.

[0050] Seven days after the last fertilization (35 days after transplanting), plants with uniform growth were selected from each treatment for growth and physiological index measurements. Fruits from each treatment were harvested at the fruit ripening stage (red ripening stage) for yield and quality index measurements. The methods for measuring each index are as follows: (1) Plant height: The height from the substrate surface to the highest growth point of the plant is measured with a ruler, in cm.

[0051] (2) Stem diameter: The stem diameter at the second internode above the base of the plant is measured using an electronic vernier caliper, in mm.

[0052] (3) Relative chlorophyll content (SPAD value): The SPAD value of the third fully unfolded leaf from the top was measured using a SPAD-502 chlorophyll meter (Konica Minolta, Japan). The average value was taken from three different points on each leaf.

[0053] (4) Fresh weight of aboveground parts and fresh weight of underground parts: The plant was cut from the base of the stem, and the aboveground parts were weighed directly; the roots were rinsed with clean water and the surface moisture was absorbed before weighing. The unit is g. Root-to-shoot ratio = Fresh weight of underground parts / Fresh weight of aboveground parts.

[0054] (5) Root activity: Triphenyltetrazolium chloride (TTC) method was used. A cleaned root tip (approximately 0.5 g) was placed in a 10 mL test tube, and 5 mL each of 0.4% TTC solution and 0.1 mol / L phosphate buffer (pH 7.0) were added. The mixture was incubated at 37°C in the dark for 1 h. After removal, 2 mL of 1 mol / L sulfuric acid was added to terminate the reaction. The root segment was removed, blotted dry with filter paper, and extracted by grinding with ethyl acetate. The volume was adjusted to 10 mL, and the absorbance was measured at 485 nm. The amount of TTF generated was calculated based on the standard curve. Root activity is expressed in μgTTF·g. -1 Fresh weight·h -1 express.

[0055] (6) Number of fruits per plant: The total number of commercial fruits harvested from each plant during its entire growth period.

[0056] (7) Average weight of a single fruit: 10 commercial fruits were randomly selected from each treatment, weighed using an electronic balance, and the average weight was calculated in g.

[0057] (8) Yield per plant: Yield per plant (g) = number of fruits per plant × average weight of a single fruit.

[0058] (9) Vitamin C content: determined by 2,6-dichlorophenol titration method, referring to GB 5009.86-2016, unit mg / 100g fresh weight.

[0059] (10) Soluble sugar content: determined by anthrone-sulfuric acid colorimetric method, with glucose as standard, unit:

[0060] (11) Soluble protein content: determined by Coomassie Brilliant Blue G-250 staining method, with bovine serum albumin as standard, unit mg / g fresh weight.

[0061] All indicators were measured in triplicate. Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 27.0 software, and multiple comparisons were performed using Duncan's method. The significance level was set at p < 0.05. The results are shown in Table 1.

[0062]

[0063] Note: Data in the table are "mean ± standard deviation" (n=3). Different lowercase letters in the same row indicate significant differences between treatments (*p* <0.05, Duncan's method). FW = fresh weight; root-to-shoot ratio is the direct calculation of the mean (no standard deviation).

[0064] The results above show that, in terms of strain synergy, the experimental data of Comparative Examples 1-3 exhibit a regular pattern. Comparative Example 1, by replacing *Bacillus amyloliquefaciens* with *Lactobacillus plantarum*, lost its ability to produce chitosanase and protease, resulting in the inability to effectively enzymatically break down the shell organic matter-calcium carbonate complex, thus hindering subsequent calcium dissolution. Its single-strain yield (691.2 g) and root activity (108.5 μg TTF·g) were significantly lower. -1 ·h -1The yield of the first comparative example was the lowest among all the comparative examples. Comparative example 2, which replaced *Lactobacillus plantarum* with *Aspergillus niger*, lost its ability to rapidly produce lactic acid and establish an acidic environment. The pH decreased slowly in the early stages, calcium dissolution efficiency decreased, and the yield per plant was 787.4 g. Comparative example 3, which replaced both *Bacillus amyloliquefaciens* and *Lactobacillus plantarum* with *Aspergillus niger*, lacked both enzymatic cell-wall breaking ability and rapid acid production, relying solely on the slow acid production of *Aspergillus niger*. The yield per plant was 876.1 g, higher than the previous two but still far lower than Example 1 (1736.1 g). The data gradient of the three comparative examples clearly shows that *Bacillus amyloliquefaciens* is responsible for enzymatic cell-wall breaking, *Lactobacillus plantarum* is responsible for rapid acid production, and *Aspergillus niger* is responsible for deep chelation and transformation. All three are indispensable; the absence of any one strain will lead to the breakage of the entire synergistic chain, resulting in a significant decrease in the yield and efficiency of the final product. Regarding the synergy between chitin and the microbial agent, the experimental data of comparative examples 4-6 further verified the synergistic relationship between shell powder, chitin, and the compound microbial agent. Comparative Example 4 replaced chitin with shell powder. The system lacked an amino polysaccharide source, preventing the in-situ formation of chitosan oligosaccharide-calcium complexes during fermentation. Although the basic calcium fertilizer effect remained, fruit quality indicators significantly decreased. Its vitamin C content (22.4 mg / 100g FW), soluble sugar content (3.32%), and soluble protein content (3.05 mg / g FW) were all significantly lower than in Example 1 (28.5 mg / 100g FW, 4.38%, and 4.52 mg / g, respectively). (FW); Comparative Example 5 replaced chitin with a compound microbial agent. While microbial metabolites increased, the lack of chitin as an enzymatic substrate prevented the formation of chitosan oligosaccharide-calcium complexes. Although the quality indicators were slightly better than Comparative Example 4, they still showed a significant difference from Example 1. Comparative Example 6 replaced chitin with a compound microbial agent. Insufficient total microbial volume led to low fermentation efficiency, limited organic acid production and enzyme activity, and insufficient calcium dissolution from shell powder and formation of chitosan oligosaccharides. All indicators were generally lower than those of Comparative Examples 4 and 5. The comparison between the above four comparative examples and Example 1 fully demonstrates that: shell powder provides a calcium source, chitin provides an amino polysaccharide source, and the compound microbial agent provides enzymes and organic acids. Only through the synergistic effect of these three components in the same fermentation system can chitosan oligosaccharide-calcium complexes be generated in situ, achieving an organic unity of calcium supplementation and biostimulation functions, ultimately resulting in a dual improvement in yield and quality. Based on the comparison results of all comparative examples and Example 1, it can be seen that the present invention forms an inseparable overall technical solution through the synergistic fermentation of three functional bacteria, combined with the joint fermentation design of shell powder and chitin, and the scientific formulation of auxiliary materials such as old brine and molasses. The absence or replacement of any element will lead to a significant reduction in the overall performance of the final product.

[0065] The parts of this invention not described in detail are techniques known to those skilled in the art.

[0066] The basic principles of the present invention have been described above with reference to specific embodiments. However, it should be noted that the advantages, benefits, and effects mentioned in the present invention are merely examples and not limitations, and should not be considered as essential features of each embodiment of the present invention. Furthermore, the specific details disclosed above are for illustrative and facilitative purposes only, and are not limitations. These details do not limit the present invention to the necessity of employing the aforementioned specific details.

[0067] In the foregoing description of this specification, references to terms such as "one embodiment," "another embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of this application. 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, 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, without contradiction. Additionally, it should be noted that in this specification, 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.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermented seashell liquid calcium fertilizer composition, characterized in that, The product comprises the following components by weight: 100-300 parts shell powder, 5-30 parts chitin, 5-20 parts compound microbial agent, 30-80 parts molasses, 10-50 parts old brine, 3-10 parts potassium dihydrogen phosphate, 1-5 parts magnesium sulfate, and 800-1200 parts water; the compound microbial agent is composed of Bacillus amyloliquefaciens, Lactobacillus plantarum, and Aspergillus niger in a volume ratio of (1-3):(2-4):(1-2), with a total viable count of not less than 1×10⁻⁶. 8 CFU / g.

2. The fermented seashell liquid calcium fertilizer composition according to claim 1, characterized in that, The shell powder includes at least one of oyster shell powder, scallop shell powder, and clam shell powder, with a particle size of 100-300 mesh.

3. The fermented seashell liquid calcium fertilizer composition according to claim 1, characterized in that, The chitin is crude chitin extracted from shrimp and crab shells, with a purity of ≥85%.

4. The fermented seashell liquid calcium fertilizer composition according to claim 1, characterized in that, The old brine is concentrated brine produced during the salt production process in salt fields, with a density of 1.20~1.30 g / mL.

5. The fermented seashell liquid calcium fertilizer composition according to claim 1, characterized in that, The molasses includes sugarcane molasses or beet molasses.

6. A method for preparing the fermented seashell liquid calcium fertilizer composition according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) After washing and drying the seashells, pulverize them to 100-300 mesh to obtain seashell powder; (2) Bacillus amyloliquefaciens, Lactobacillus plantarum and Aspergillus niger were activated and cultured separately, and then mixed in proportion to obtain a compound microbial agent; (3) Mix shell powder, chitin, molasses, potassium dihydrogen phosphate, magnesium sulfate and water evenly, inoculate with compound microbial agent, and carry out staged variable temperature fermentation at 28~37℃. The total fermentation time is 7~14 days. (4) After fermentation, add old brine, stir well, and continue the chelation reaction at 28~32℃ for 2~3 days; (5) After the reaction is complete, filter to remove solid residue, and the fermented shell liquid fertilizer composition is obtained.

7. The preparation method according to claim 6, characterized in that, The staged variable-temperature fermentation described in step (3) includes the following stages performed sequentially: aerobic enzyme production stage, anaerobic acid production stage, and aerobic acid production stage. The conditions for the aerobic enzyme production stage are: temperature 35~37℃, pH 6.0~7.0, rotation speed 150~200rpm, aeration rate 0.5~1.5vvm, and fermentation time 2~4 days. The conditions for the anaerobic acid production stage are: temperature 28~30℃, pH naturally decreasing, rotation speed 0~50rpm, and fermentation time 3~5 days. The conditions for the aerobic acid production stage are: temperature 30~32℃, pH 4.0~5.0, rotation speed 150~200rpm, aeration rate 0.5~1.0vvm, and fermentation time 2~5 days.

8. The preparation method according to claim 7, characterized in that, At the end of the aerobic enzyme production stage described in step (3), the protease activity in the fermentation broth is ≥500U / mL and the chitosanase activity is ≥50U / mL; At the end of the aerobic acid production stage, the pH value of the fermentation broth drops to 4.0~5.0, and the water-soluble calcium content is ≥15g / L.

9. The use of the fermented shell liquid calcium fertilizer composition according to any one of claims 1 to 5 or the fermented shell liquid calcium fertilizer composition prepared by the preparation method according to any one of claims 6 to 8 in the preparation of liquid fertilizer.

10. A fermented seashell liquid calcium fertilizer, characterized in that, The liquid calcium fertilizer includes the fermented shell liquid calcium fertilizer composition according to any one of claims 1 to 5 or the fermented shell liquid calcium fertilizer composition prepared by the preparation method according to any one of claims 6 to 8.

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