All-natural component multiple intelligent response type biological soil conditioner, preparation method and application

Abstract

The present application relates to a kind of all natural component multiple intelligent response type biological soil conditioner, preparation method and application.Bioactive core is composed of engineered microalgae, synthetic probiotic group, super stable enzyme complex and plant-derived signal molecule.The carrier system is sequentially enzyme-sensitive core, temperature-sensitive hydrogel layer, pH / ionic dual-sensitive layer and light-triggered and rhizosphere anchoring outer layer from inside to outside.Each layer cooperatively realizes the cascade sequential response of "enzyme signal→temperature signal→pH / ionic signal→light signal", and ensures that active ingredients are accurately released in rhizosphere microzone as needed.All carrier materials of the present application are food-grade natural products and can be completely biodegraded.Field tests show that under the condition of reducing chemical fertilizer by 15%-20%, the yield of tomato increases by 18.5%, the incidence of bacterial wilt decreases by 65%, and the soil microbial diversity is significantly improved.The present application has multiple synergistic effects of reducing fertilizer, increasing yield, preventing disease and improving soil, and is suitable for organic agriculture and degraded soil remediation.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to an all-natural, multi-component, intelligently responsive biological soil conditioner, its preparation method, and its application. Background Technology

[0002] Soil health is the foundation of sustainable agricultural development. Currently, mainstream biological soil conditioners (such as microbial agents and enzyme preparations) generally face three major challenges: First, uncontrollable release, with active ingredients released at non-target times or locations, resulting in low utilization rates (often below 30%); second, easy loss of biological activity, with probiotics and enzymes easily inactivated during processing, storage, transportation, and in complex soil environments; and third, material compliance risks, as many controlled-release carriers use synthetic polymers or nanomaterials, posing potential ecotoxicity risks and environmental persistence issues, making it difficult to meet increasingly stringent organic agriculture and green food production standards.

[0003] In the prior art, patent CN108157364A discloses a pH-sensitive pesticide carrier, but its material is modified mesoporous silica; CN120604776A and CN109082391B describe a pH and pectinase dual-responsive pesticide carrier and a thermosensitive hydrogel-embedded bacterial agent, respectively, but the core shell is a synthetic tannic acid-based copper polymer and a thermosensitive material, poly(N-isopropylacrylamide) (PNIPAM), whose degradation products raise environmental concerns. None of these solutions can achieve intelligent response while meeting the requirements of next-generation green agricultural inputs that are all-natural, fully degradable, and zero-toxic. Although CN121609600A also employs a multi-layered core-shell structure, its design principle is essentially 'physical barrier and layer-by-layer dissolution'. Its outermost layer dissolves rapidly upon contact with water to stimulate plant immunity, the middle layer acts as a nutrient reservoir for slow release, and the inner layer relies on the material's own water absorption and swelling to release the bacterial agent. The release behavior of such systems is mainly controlled by water diffusion and material thickness. Their 'responsiveness' is limited to passive reactions to single, non-specific environmental changes (such as the presence of abundant water), unable to distinguish the presence of rhizosphere enzymes, detect subtle changes in soil temperature (28℃ vs. 20℃), or respond to 'external light signals' for precise human intervention. More importantly, the functions of each layer are independent, lacking a synergistic amplification effect based on logical sequence. Furthermore, the hydrogel material (sodium alginate-chitosan-gelatin) used in this prior art is primarily used for encapsulating microbial agents, rather than being designed as a temperature-sensitive switch with a precisely adjustable transition temperature (28-35℃). This invention designs the outermost layer as a phototriggered layer responding to a specific wavelength of blue light, and combines this with the rhizosphere chemotactic anchoring function of chitosan oligosaccharides, achieving precise identification of rhizosphere microregions and controllable, on-demand replenishment. This design approach demonstrates the non-obviousness of this invention.

[0004] Furthermore, a long-standing technical bias in this field has been the perception that 'all-natural materials have insufficient performance': it is generally believed that achieving precise temperature response (transition temperature deviation <2℃), pH / ion dual response, and remotely controllable light triggering requires the use of structurally designable synthetic polymers (such as PNIPAM) or metal ion-containing nanomaterials. For example, the literature "Advances in the Application of Polysaccharide-Based Smart Hydrogels in Agriculture" (Chinese Agricultural Science, 2023) clearly points out that 'natural polysaccharide hydrogels typically have low response sensitivity and a narrow range of transition temperature control, making it difficult to meet the requirements of precision agriculture'. Against this technical backdrop, this invention, through innovative material blending (a synergistic gel network of gelatin-κ-carrageenan-CNC) and sophisticated sequence design, achieves response precision surpassing that of synthetic materials using all-natural materials (adjustable transition temperature 28-35℃, on / off release ratio >6 times). This breakthrough is not a simple replacement of existing materials, but rather a re-understanding and creative application of the principles of materials science.

[0005] In summary, existing technologies generally believe that achieving "multiple intelligent responses" inevitably relies on non-natural components such as synthetic polymers (e.g., PNIPAM), modified nanomaterials, or metal-organic frameworks. This approach fails to address the technical challenge of "how to construct an intelligent system capable of actively sensing and cascading responses to multiple rhizosphere signals in a logical sequence of 'biorecognition → physical adaptation → chemical initiation → artificial intervention,' based on entirely natural components." Furthermore, complex microbial community regulation is often simplified to single strains or simple combinations, lacking a systematic design for the synergistic effects of microbial communities and the ability to comprehensively sense and cascade responses to multiple environmental signals (biological, physical, and chemical). The bottleneck in this field lies in how to construct a highly efficient intelligent delivery system capable of sensing and cascading responses to multiple rhizosphere signals, while simultaneously achieving synergistic effects of active ingredients, while also considering the use of natural, green, fully degradable, and eco-friendly materials.

[0006] Therefore, this invention integrates synthetic biology, all-natural smart materials and ecological theories by designing the cascade response sequence and interface compatibility between materials. It uses food-grade safe natural materials to construct a multi-response soil conditioner with intelligent release of active ingredients, biological synergistic effect, and stable safety, providing technical support for smart agriculture and green sustainable development. Summary of the Invention

[0007] The technical solution of this invention to solve the above-mentioned technical problems is to provide an all-natural, multi-component, intelligent, responsive biological soil conditioner, comprising:

[0008] The bioactive core includes engineered microalgae, synthetic probiotics, ultrastable enzyme complexes, and plant-derived signaling molecules, including glycosylated signaling substances extracted from salicylic acid hyperaccumulating white willow.

[0009] The four-layer core-shell structured intelligent carrier system encapsulating the bioactive core consists of, from the inside out: an enzyme-sensitive core, a temperature-sensitive hydrogel layer, a pH / ion dual-sensitive layer, and a light-triggered and rhizosphere-anchored outer layer.

[0010] Furthermore, the temperature-sensitive hydrogel layer is composed of gelatin, κ-carrageenan, and cellulose nanocrystals, and its gel-sol transition temperature is controlled between 28-35℃ by adjusting the mass ratio of gelatin to κ-carrageenan.

[0011] Furthermore, the mass ratio of gelatin to κ-carrageenan is 2.5:1 to 3.5:1, and the amount of cellulose nanocrystals added is 2%-5% of the total mass of gelatin and κ-carrageenan.

[0012] Furthermore, the pH / ion dual-sensing layer is formed by cross-linking chitosan, pectin, and calcium alginate with the natural cross-linking agent genipin, wherein the mass ratio of chitosan to pectin is 1.5:1 to 2.5:1.

[0013] Furthermore, the light-triggered and rhizosphere anchoring outer layer is composed of zein, gum arabic, photosensitizer, and root chemotactic agent; the photosensitizer is riboflavin or curcumin; and the root chemotactic agent is chitosan oligosaccharide.

[0014] Furthermore, the engineered microalgae is the gene-edited Synechocystis XL-1 strain, whose genome integrates a nitrogen-fixing gene cluster and the phytase gene phyA, and overexpresses a key gene for endogenous strigolactone synthesis.

[0015] Furthermore, the synthetic probiotic group is composed of Bacillus subtilis EA-1, Acinetobacter AB-3 and Bacillus amyloliquefaciens BA-5 in a live bacteria ratio of 5:3:2.

[0016] Furthermore, the ultrastable enzyme complex includes the thermostable xylanase Xyn10B-DM4, the broad-spectrum phosphatase PhoA-M2, and the highly efficient laccase LacS-EC1, all obtained through directed evolution.

[0017] A method for preparing the above-mentioned all-natural multi-component intelligent responsive bio-soil conditioner includes the following steps:

[0018] S1. Preparation of bioactive core freeze-dried powder: The engineered microalgae and the synthetic probiotic group are cultured and harvested separately, mixed with stable enzyme complex and plant-derived signaling molecules, and then freeze-dried.

[0019] S2. Construction of enzyme-sensitive core: Using microfluidic technology, the lyophilized active core powder obtained in S1 was dispersed in an aqueous solution of sodium alginate and chondroitin sulfate, and then added dropwise to a calcium chloride solution to solidify and form primary microspheres;

[0020] S3. Layer-by-layer self-assembly intelligent response layer:

[0021] Thermosensitive coating: The microspheres obtained from S2 are immersed in a composite aqueous solution of gelatin, κ-carrageenan and cellulose nanocrystals at 50-60℃, and cooled to room temperature to form a gel coating layer;

[0022] pH / ion-sensitive layer coating: Through layer-by-layer self-assembly technology, chitosan solution and pectin-calcium alginate mixture are alternately deposited on the surface of the temperature-sensitive layer, and finally immersed in genipin solution for cross-linking.

[0023] Phototriggered and anchored outer coating: Microspheres were immersed in an ethanol-water solution containing zein, gum arabic, riboflavin and chitosan oligosaccharide, and then dried to form a film;

[0024] S4. Post-processing of the product: Freeze-dry the coated microspheres to obtain the final powder product;

[0025] Furthermore, in step S3, the mass ratio of gelatin to κ-carrageenan is 2.5:1 to 3.5:1, and the amount of cellulose nanocrystals added in step S3 is 2%-5% of the total mass of gelatin and κ-carrageenan; the mass ratio of chitosan to pectin in step S3 is 1.5:1 to 2.5:1; and the photosensitizer in step S3 is riboflavin with a concentration of 0.01%-0.1% w / v.

[0026] Application of a multi-component, intelligent, responsive bio-soil conditioner with all-natural components as described above in improving crop yield and stress resistance, restoring degraded soil, or in organic agricultural production.

[0027] Compared with the prior art, the technical solution of the present invention has the following technical effects:

[0028] 1. A "signal-guided cascading release" intelligent response system was constructed to achieve precise on-demand replenishment;

[0029] This invention breaks through the limitations of traditional single-layer or double-layer responses, pioneering a cascade response mechanism that unlocks in a four-stage sequence: enzyme → temperature → pH / ion → light. This design strictly follows the succession logic of the rhizosphere microecology: release is initiated by root-secreted enzymes (biorecognition), release intensity is regulated by soil temperature (environmental adaptation), precise rhizosphere targeting is achieved through pH / ion changes (chemical gating), and finally, active human intervention (on-demand replenishment) is achieved through external blue light. In vitro experiments show that the product of this invention exhibits significant "on-off" release characteristics in the crop's optimal temperature range of 28-35℃ (24-hour release rate reaches 82%-88%), with a 12-hour release rate of 78% in an enzyme-containing rhizosphere environment, compared to only 12% in an enzyme-free environment. This achieves a high degree of synchronization between release kinetics and crop physiological needs, increasing nutrient utilization by more than 50%.

[0030] 2. It achieves a five-fold synergistic effect of "reducing fertilizer use and increasing yield, improving quality and preventing disease, and improving soil and promoting growth," with a comprehensive effect far exceeding the simple sum of the components;

[0031] Field trials have confirmed that, even under the stringent condition of reducing chemical fertilizer application by 15%-20%, this invention can still increase tomato yield by 18.5% (5747 kg / mu) and strawberry yield by 15.3% (2832 kg / mu), significantly outperforming commercially available microbial agents under conventional fertilization (tomato +12.7%, strawberry +8.6%). More importantly, the increased yield does not come at the expense of quality and health: the incidence of bacterial wilt in tomatoes decreased from 12.5% ​​to 4.4% (65% control efficacy), and the incidence of powdery mildew in strawberries decreased from 18.5% to 6.2% (66.5% control efficacy); the soluble solids content of strawberries increased to 12.8%, fruit firmness increased by 15%, and shelf life was extended by 3 days; the Shannon diversity index of rhizosphere soil bacterial communities (tomato 9.25, strawberry 9.18) and the relative abundance of actinomycetes (tomato 22.5%, strawberry 24.3%) were both significantly improved. This invention is one of the few agricultural inputs that can simultaneously achieve the four goals of "increasing yield, improving quality, preventing disease, and improving soil" under the condition of reducing fertilizer use.

[0032] 3. The specific order of the four-layer structure is a core technical feature; misalignment of the layers will lead to a significant reduction in functionality.

[0033] This invention demonstrates through a systematic sequential substitution comparative experiment that the four-layer response sequence (enzyme → temperature → pH / ion → light) is itself a non-obvious inventive point. When the temperature-sensitive layer is placed on the outermost layer (Comparative Example 4), the activity loss during storage and transportation (35℃ / 7 days) reaches as high as 42%, and the yield increase rate drops to only about 4980 kg / mu. When the photo-triggered layer is placed on the inner layer (Comparative Example 5), blue light cannot penetrate the outer layer, causing the photoresponse to fail, and the yield increase rate is only 5120 kg / mu. However, after the present invention is assembled in a specific sequence, the storage and transportation loss is only 9%, the blue light response multiplier reaches 4.8 times, and the yield increase rate reaches 5747 kg / mu. This sequence causes a non-linear synergistic amplification of the functions of each layer, with a synergistic enhancement index (SEI) of 0.44 (yield increase) and 1.23 (disease prevention), proving that its effect is not a simple addition of the layers, but rather produces a significant "1+1>2" qualitative change effect.

[0034] 4. The use of all-natural materials to construct complex intelligent systems breaks the technological prejudice that "intelligent responses must rely on synthetic materials";

[0035] All carrier materials used in this invention (gelatin, κ-carrageenan, cellulose nanocrystals, chitosan, pectin, calcium alginate, genipin, zein, gum arabic, riboflavin, and chitosan oligosaccharides) are food-grade or organically licensed natural products, requiring no complex toxicological or environmental safety assessments. A head-to-head comparison with existing technologies (CN121609600A) and commercially available products shows that, under the same fertilizer reduction conditions, this invention significantly increases yield (+18.5%), far exceeding the control sample's +6.2% to +7.3%. In contrast, commercially available pH-responsive microbial agents, photosensitive bio-fertilizers, and all-natural enzyme fertilizers only increase yield by +6.8%, +4.5%, and +2.1% respectively under the same conditions. This invention achieves a more complex and logically integrated multi-functional intelligent response than synthetic polymers using all-natural materials, opening a new path for the development of green agricultural inputs.

[0036] 5. Excellent long-term stability, meeting the requirements of commercial storage and transportation;

[0037] Thanks to the tiered protection of four layers of all-natural carriers, the probiotic survival rate of the freeze-dried product remained at 84.6% (4.4 × 10⁻⁶) after 24 months of storage at 4°C. 8 The product exhibits a CFU / g concentration, a xylanase activity retention rate of 91%, and good appearance and flowability. Even after 6 months of storage at 25°C, the survival rate remains at 78.8%, with 88% enzyme activity retained and only slight clumping. These stability indicators meet the commercial requirements of the entire agricultural product chain, from production and logistics to end-use, and provide the basic conditions for industrial-scale promotion.

[0038] 6. Fully biodegradable, environmentally friendly, and non-ecotoxic;

[0039] All carrier materials exhibited a 97% weight loss rate in soil within 180 days. GPC testing showed a significant shift in molecular weight distribution towards lower molecular weight regions, indicating that they have been essentially and completely degraded into small molecules, posing no risk of microplastics or persistent residues. Ecotoxicity testing showed that at the maximum test concentration of 1000 mg / kg dry soil, earthworms maintained a 100% survival rate after 14 days. At 10 times the recommended dose, the germination rate and root length of radish and Shanghai bok choy seeds showed no significant difference compared to the control group (inhibition rate <5%). The product complies with organic agricultural production standards (GB / T 19630 and EC 834 / 2007) and is highly safe for crops and the environment.

[0040] 7. The light-triggered layer is practical and operable, addressing fundamental technical concerns in practical applications;

[0041] Soil penetration experiments confirmed that 470 nm blue light irradiation can still effectively trigger release under a shallow soil cover of 0.5-1.0 cm (acceleration ratios of 4.5 times and 3.9 times, respectively). This penetration depth is entirely feasible in precision agronomic operations such as root dipping before transplanting, irradiation before covering with soil after seed furrow application, and shallow burial of drip irrigation tape. This design provides a practical technical solution for a "manual intervention switch" in smart agriculture scenarios, distinguishing this invention from all existing technologies that rely solely on natural conditions for release, and achieving true "on-demand replenishment."

[0042] 8. Production costs are controllable, and it has the market potential to replace chemical inputs on a large scale;

[0043] The gelatin, κ-carrageenan, chitosan, and zein used in this invention are all commercially available food industry additives with low costs. Engineered microalgae and probiotics can be produced on a large scale through conventional fermentation processes. The overall production cost of the product is controllable, and its field effects are significant. It possesses the market potential to replace a portion of chemical fertilizers and pesticides on a large scale in food crops, cash crops, fruits and vegetables, and in the remediation of degraded soils, thus having significant economic and social value in promoting the green transformation of agriculture. Detailed Implementation

[0044] This invention proposes an all-natural, multi-component, intelligent, responsive biological soil conditioner, its preparation method, and its application. The aim is to design a soil conditioner that combines intelligent and ecological effects, suitable for precision agriculture, organic agriculture, and the remediation of degraded soils.

[0045] The following specific embodiments will illustrate the all-natural, multi-component, intelligently responsive bio-soil conditioner proposed in this invention:

[0046] Example 1:

[0047] A multi-component, intelligently responsive bio-soil conditioner consisting of all-natural components, including:

[0048] The bioactive core includes engineered microalgae, synthetic probiotics, ultrastable enzyme complexes, and plant-derived signaling molecules, including glycosylated signaling substances extracted from salicylic acid hyperaccumulating white willow.

[0049] The four-layer core-shell structured intelligent carrier system encapsulating the bioactive core consists of, from the inside out: an enzyme-sensitive core, a temperature-sensitive hydrogel layer, a pH / ion dual-sensitive layer, and a light-triggered and rhizosphere-anchored outer layer.

[0050] Furthermore, the temperature-sensitive hydrogel layer is composed of gelatin, κ-carrageenan, and cellulose nanocrystals, and its gel-sol transition temperature is controlled between 28-35℃ by adjusting the mass ratio of gelatin to κ-carrageenan.

[0051] Furthermore, the mass ratio of gelatin to κ-carrageenan is 2.5:1 to 3.5:1, and the amount of cellulose nanocrystals added is 2%-5% of the total mass of gelatin and κ-carrageenan.

[0052] Furthermore, the pH / ion dual-sensing layer is formed by cross-linking chitosan, pectin, and calcium alginate with the natural cross-linking agent genipin, wherein the mass ratio of chitosan to pectin is 1.5:1 to 2.5:1.

[0053] Furthermore, the light-triggered and rhizosphere anchoring outer layer is composed of zein, gum arabic, photosensitizer, and root chemotactic agent; the photosensitizer is riboflavin or curcumin; and the root chemotactic agent is chitosan oligosaccharide.

[0054] Furthermore, the engineered microalgae is the gene-edited Synechocystis XL-1 strain, whose genome integrates the nitrogen fixation gene cluster nifH and the phytase gene phyA, and overexpresses the endogenous strigolactone synthesis key gene MAX3.

[0055] Furthermore, the synthetic probiotic group is composed of Bacillus subtilis EA-1, Acinetobacter AB-3 and Bacillus amyloliquefaciens BA-5 in a live bacteria ratio of 5:3:2.

[0056] Furthermore, the ultrastable enzyme complex includes the thermostable xylanase Xyn10B-DM4, the broad-spectrum phosphatase PhoA-M2, and the highly efficient laccase LacS-EC1, all obtained through directed evolution.

[0057] Specifically, the glycosylation signaling substance is salicin, an active ingredient extracted from the bark of white willow (Salix alba L.). Salicin is a phenolic β-glycoside, composed of one molecule of D-glucose and one molecule of salicylol linked by a glycosidic bond. On a dried basis, the aqueous extract of white willow bark contains 15 wt% to 98 wt% salicin, and its total glycoside content is not less than 80 wt%.

[0058] The parent of “Syntrophus XL-1” is Syntrophus PCC 6803, a single-celled, freshwater cyanobacterium.

[0059] The engineered algal strain's integrated gene consists of three parts:

[0060] 1. Nitrogen fixation module: The complete nif gene cluster from Cyanothece sp. ATCC 51142 is introduced, which includes nifB, nifS, nifU, nifE, nifN, nifV, nifP, nifZ, nifT, nifX, nifW and auxiliary genes such as hesA and hesB.

[0061] 2. Phosphorus fixation module: A highly efficient signal peptide (Slr0161 protein signal peptide of Synechocystis itself) is fused to the N-terminus of the phyA gene to guide the secretion of the enzyme protein into the extracellular space.

[0062] 3. Strigolactone module: Key genes for strigolactone synthesis, MAX3 (encoding carotenoid cleavage dioxygenase CCD7), MAX4 (encoding CCD8), and MAX1 (encoding cytochrome P450 monooxygenase), are overexpressed. A polycistronic operon is constructed from MAX3, MAX4, and MAX1 to achieve co-expression.

[0063] The suffixes "EA-1", "AB-3", and "BA-5" in Bacillus subtilis EA-1, Acinetobacter AB-3, and Bacillus amyloliquefaciens BA-5 are internal designations used to distinguish between various strains that have been constructed or screened. They can be replaced by commercially available Bacillus subtilis, Acinetobacter, and Bacillus amyloliquefaciens strains.

[0064] Example 2:

[0065] Preparation of an all-natural intelligent biological soil conditioner; comprising the following steps:

[0066] S1. Preparation of bioactive core:

[0067] Engineered microalgae XL-1 and synthetic probiotics (EA-1:AB-3:BA-5=5:3:2) were cultured at high density by fermentation, and algal cells and bacterial cells were collected by centrifugation.

[0068] The harvested algal cells, bacterial cells, ultrastable enzyme complex (Xyn10B-DM4, PhoA-M2, LacS-EC1 in a ratio of 2:2:1), and freeze-dried powder of white willow extract were mixed evenly in a dry weight ratio of 3:4:1:2.

[0069] The mixture was pre-frozen at -50°C and then dried in a freeze dryer for 48 hours to obtain the active core freeze-dried powder.

[0070] S2. Enzyme-sensitive kernel construction:

[0071] Prepare a mixed aqueous solution of 2.0% (w / v) sodium alginate and 0.5% (w / v) chondroitin sulfate, add the lyophilized active core powder obtained from S1, and ultrasonically disperse it evenly as the internal phase.

[0072] Using a coaxial microfluidic device, the internal phase flow rate was 0.5 mL / h, and the external phase (soybean oil containing 2% Span80) flow rate was 5 mL / h, to generate uniform microdroplets.

[0073] The microdroplets were collected in a 0.1 M CaCl2 solution and gently stirred to solidify for 30 minutes. The mixture was washed three times with deionized water to obtain primary microspheres with a particle size of 150 ± 23 μm.

[0074] S3. Layer-by-layer self-assembly intelligent response layer:

[0075] Thermosensitive layer coating: An aqueous solution containing 3% (w / v) gelatin, 1% (w / v) κ-carrageenan, and 0.12% (w / v) cellulose nanocrystals (CNC) was prepared and dissolved uniformly in a water bath at 55°C. The microspheres obtained in S2 were immersed in this hot solution for 5 minutes, and gently stirred to ensure uniform coating. The entire system was then cooled to 4°C and allowed to stand for 1 hour to allow complete gel formation. Filtration yielded microspheres coated with a thermosensitive layer.

[0076] pH / ion-sensitive layer coating: A solution of 0.5% (w / v) chitosan (dissolved in 1% acetic acid) and 0.5% (w / v) pectin (containing 1% sodium alginate) was prepared. A layer-by-layer self-assembly method was used: first, the temperature-sensitive microspheres were immersed in the chitosan solution for 5 minutes and washed with water; then immersed in the pectin-sodium alginate solution for 5 minutes and washed with water; this process was repeated 3 times. Finally, the microspheres were immersed in a 0.2% (w / v) genipin aqueous solution and crosslinked at 37°C for 2 hours. After washing with water, microspheres coated with the pH / ion-sensitive layer were obtained.

[0077] Phototriggered and anchored outer coating: A 70% ethanol-water solution containing 2% (w / v) zein, 1% (w / v) gum arabic, 0.05% (w / v) riboflavin, and 0.1% (w / v) chitosan oligosaccharide was prepared. The pH / ion-sensitive layer microspheres were immersed in this solution for 10 seconds, quickly removed, and dried at 30°C under ventilation for 20 minutes to form a dense coating film.

[0078] S4. Product post-processing:

[0079] The microspheres with the final coating were placed in a freeze dryer and dried for 36 hours to obtain a light yellow powder with good flowability, which was labeled as Sample A.

[0080] Comparative Example 1: Microspheres were made by encapsulating the same active core with sodium alginate and labeled as Sample B.

[0081] Experiment 1: In vitro intelligent response performance test:

[0082] Thermosensitive release characteristics: 100 mg of sample A and sample B were placed in PBS buffer at pH 7.0 and incubated at 20°C, 25°C, 30°C, and 35°C with constant shaking (100 rpm). Samples were taken at predetermined time points, centrifuged, and the concentration of vitamin B12 (model compound) in the supernatant was measured to calculate the cumulative release rate. The results showed that sample B exhibited slow diffusion release at 20-35°C, with a 24-hour release rate between 35-50%, and no significant thermosensitivity. Sample A showed strong inhibition at 20°C and 25°C, with 24-hour release rates of only 10% and 15%, respectively. When the temperature rose to 30°C and 35°C, the release curves increased sharply within 4-12 hours, with 24-hour release rates reaching 82% and 88%, respectively, exhibiting significant "on / off" thermosensitive characteristics. Its critical response temperature was approximately 28-30°C.

[0083] Enzyme response degradation and release: Sample A was placed in simulated rhizosphere solution (containing 5 U / mL β-glucanase, pH 5.8) and shaken at 30°C. An identical buffer solution without the enzyme was used as a control. Samples were taken periodically to observe morphology and determine the release rate.

[0084] SEM observation: The microspheres remained structurally intact at 0 hours; after 12 hours in the enzyme-containing solution, numerous pores and cracks appeared on the surface of the microspheres, indicating significant structural disintegration. The control group maintained structural integrity within 72 hours.

[0085] Release curve: The enzyme-treated group achieved a release rate of 78% within 12 hours, while the control group only achieved a release rate of 12% during the same period. This demonstrates that the enzyme-sensitive design of the core can efficiently achieve targeted release based on biometrics.

[0086] Phototriggered response test: A thin film coated only with the outer layer was prepared, and its water contact angle and permeability to NaCl solution were measured before and after irradiation with 470 nm blue light (50 mW / cm²). The results showed that after 5 minutes of irradiation, the water contact angle decreased significantly from 102° to 48°; the permeability of NaCl solution increased by 4.8 times. After 60 minutes of irradiation, the contact angle recovered to 95°, indicating that the phototriggered response is partially reversible and can achieve instantaneous regulation of permeability.

[0087] Soil penetration verification of the photo-triggered layer: To demonstrate the feasibility of the photo-triggered layer of this invention in practical rhizosphere applications, a soil penetration experiment was conducted. Empty microspheres coated with the photo-triggered layer were buried in standard loam (15% moisture content, 1.2 g / cm³ bulk density) at different depths. The soil surface was irradiated with a 470 nm blue light source (50 mW / cm²) for 10 minutes. The microspheres were then removed and their release rate was measured. The direct irradiation group without soil cover served as a control.

[0088] result:

[0089] 0cm (direct irradiation): the release acceleration ratio (irradiated / unirradiated) is 4.8 times (same as Example 2).

[0090] 0.5cm soil cover: release acceleration ratio is 4.5 times, and the decrease is not significant.

[0091] With a 1.0cm soil cover: the release acceleration ratio is 3.9 times, and it still maintains a significant light-triggered response.

[0092] 2.0cm soil cover: release acceleration ratio is 2.5 times, response is weakened but still detectable.

[0093] With a 3.0cm soil layer covering: the release acceleration ratio is 1.2 times, and the response basically disappears.

[0094] Conclusion: Under a shallow soil cover of 1.0 cm, 470 nm blue light can still effectively penetrate and trigger a response. In practical agricultural applications, regulator particles are usually buried in the topsoil layer 5-15 cm deep along with seeds or seedling roots. However, the photo-triggered design of this invention does not require direct blue light irradiation of all particles. Instead, it utilizes the light scattering channels formed by soil pore water in the rhizosphere and the window period during which the surface cover of the crop is not yet fully formed to achieve effective triggering in local root zones (such as near-surface roots and the surface layer after irrigation). More importantly, the photo-triggered layer of this invention acts as an "artificial intervention switch," allowing irradiation during the short window period before root dipping before transplanting, after seed furrow application and before covering with soil, or under conditions of shallow burial of drip irrigation tape. Therefore, an effective penetration depth of 0.5-1.0 cm is entirely feasible in precision agriculture operations. This experiment demonstrates the practicality and operability of the photo-triggered layer of this invention, addressing concerns in existing technologies that photo-triggered technology is not suitable for the rhizosphere.

[0095] Comparative Example 2: Preparation of a modulator (three-layer structure) lacking the outer light triggering / anchoring layer;

[0096] Preparation method:

[0097] The preparation method of Example 2 is followed, but only (a) thermosensitive layer coating and (b) pH / ion-sensitive layer coating are performed in step S3, omitting (c) phototriggered and anchored outer layer coating. All other steps and parameters are exactly the same as in Example 2, as follows:

[0098] S1 (Preparation of bioactive core): Same as Example 2.

[0099] S2 (Enzyme-sensitive core construction): Same as in Example 2, primary microspheres with a particle size of 150±23μm were obtained.

[0100] S3(a) (Thermosensitive layer coating): Same as in Example 2, microspheres were impregnated with a 55°C composite aqueous solution of 3% gelatin, 1% κ-carrageenan and 0.12% cellulose nanocrystals (CNC), and then cooled to 4°C for 1 hour to cure.

[0101] S3(b) (pH / ion-sensitive layer coating): Same as in Example 2, chitosan solution and pectin-calcium alginate mixture were deposited alternately through layer-by-layer self-assembly (3 cycles), and finally crosslinked with 0.2% genipin at 37°C for 2 hours.

[0102] S4 (Product Post-processing): The microspheres coated with the pH / ion-sensitive layer were directly freeze-dried for 36 hours to obtain a powder product with a three-layer core-shell structure, which was labeled as Sample C.

[0103] Sample characteristics:

[0104] Structure: From the inside out, it consists of an enzyme-sensitive core, a temperature-sensitive hydrogel layer, and a pH / ion dual-sensitive layer (without an outermost light-triggered and rhizosphere anchoring layer).

[0105] Appearance: Light yellow powder, with slightly lower flowability than sample A (due to the lack of protection from the outermost zein membrane).

[0106] Particle size: The particle size of the dried microspheres is 120±18μm (slightly smaller than 140±20μm of sample A due to the absence of an outer membrane).

[0107] Application effect comparison:

[0108] In the tomato greenhouse experiment, the same dose of sample C (2 kg / ha) was applied, and the experiment was conducted under the same condition of reducing fertilizer application by 20%, but without 470 nm blue light irradiation (because the photo-triggering layer was missing, irradiation was meaningless). Sample A from Example 2 (complete four-layer structure, combined with blue light irradiation) was used as a control.

[0109] Yield: The tomato yield of treatment group C was 5250±156 kg / mu, which was significantly lower than that of treatment group A (5747±175 kg / mu) (p<0.05), with a yield reduction of about 8.6%.

[0110] Incidence of bacterial wilt: The incidence rate of bacterial wilt in sample C treatment group was 7.8%, which was higher than that of sample A group (4.4%), and the control efficacy decreased by about 43%.

[0111] Soil microbial Shannon index: The index of treatment group C was 8.92±0.11, which was lower than that of treatment group A (9.25), but still better than that of the conventional fertilization control group (T1).

[0112] This comparative analysis proves that:

[0113] After the outer light-triggered / anchored layer was missing, the yield-increasing and disease-preventing effects of the regulator both decreased significantly, indicating that the outer light-triggered function and blue light management measures are key components of the present invention to achieve "superior efficiency under reduced application conditions".

[0114] Sample C still retained a certain improvement effect (better than conventional fertilization), indicating that the inner three-layer structure still has basic functions. However, the existence of the outermost layer and its light-triggered response are necessary conditions for achieving precise on-demand replenishment and amplifying synergistic effects, and are not irrelevant additional features.

[0115] Experiment 2: Application Effect Test

[0116] Tomato greenhouse experiment, location and crop: Changchun, Jilin, tomato greenhouse.

[0117] Experimental design: Three treatments were set up, with three replicates for each treatment. T1: Conventional fertilization (CK); T2: Conventional fertilization + commercially available compound microbial agent (10 kg / ha); T3: Conventional fertilizer reduction of 20% + sample A of this invention (2 kg / ha).

[0118] Management: During the initial flowering and fruit enlargement stages of tomatoes, the root zone was irradiated twice using a portable 470nm LED lamp, for 10 minutes each time.

[0119] Yield: The yield of tomatoes in the T3 treatment was 5747 kg per mu, which was significantly higher than that of T1 (4850 kg) and T2 (5100 kg), with yield increases of 18.5% and 12.7% respectively (p<0.01).

[0120] Disease control: At the end of the growing season, the incidence of bacterial wilt in the T3 treatment was 4.4%, which was significantly lower than that in T1 (12.5%) and T2 (8.2%), with control efficacy of 65% and 46%, respectively.

[0121] Soil microorganisms: High-throughput sequencing showed that the Shannon diversity index of the bacterial community in the rhizosphere soil treated with T3 was 9.25, significantly higher than that of T1 (8.56) and T2 (8.78). The relative abundance of beneficial bacteria such as actinomycetes reached 22.5% in T3, higher than that of T1 (15.2%) and T2 (16.8%).

[0122] Strawberry greenhouse experiment; Location and crop: Shuangyang, Jilin Province, strawberry greenhouse.

[0123] Experimental design: Three treatments were set up, with three replicates for each treatment. T1: Conventional fertilization (CK); T2: Conventional fertilization + commercially available compound microbial agent (12 kg / ha); T3: Conventional fertilizer reduction of 15% + sample A of this invention (3 kg / ha).

[0124] Management: During the strawberry budding and fruit coloring stages, the root zone was irradiated twice using a portable 470nm LED lamp, for 15 minutes each time.

[0125] Yield: The strawberry yield of the T3 treatment was 2832 kg / mu, which was significantly higher than that of T1 (2457 kg) and T2 (2608 kg), with yield increases of 15.3% and 8.6% respectively (p<0.01).

[0126] Fruit quality: The soluble solids content of T3-treated fruit reached 12.8%, which was significantly higher than that of T1 (10.5%) and T2 (11.2%); fruit firmness was increased by 15%, and shelf life was extended by 3 days.

[0127] Disease control: The incidence of powdery mildew in T3 treatment was 6.2%, which was significantly lower than that in T1 (18.5%) and T2 (11.4%), with control efficacy of 66.5% and 45.6%, respectively.

[0128] Soil microorganisms: The Shannon index of rhizosphere soil bacteria community in treatment T3 was 9.18, which was significantly higher than that in T1 (8.42) and T2 (8.71); the relative abundance of Actinobacteria increased to 24.3%.

[0129] Experiment 3: Product stability and material environmental safety testing;

[0130] 1. Storage stability: Sample A was sealed and stored at 4°C and 25°C respectively. Periodic testing was performed.

[0131] Table 1. Product stability evaluation of the product of the present invention:

[0132] Conclusion: The active ingredients remained stable after 24 months of storage at 4°C, meeting commercial requirements.

[0133] 2. Material biodegradability (ISO 17556:2019):

[0134] The pure carrier material (without active ingredients) was buried in standard soil. After 90 days, the weight loss rate reached 72%, and after 180 days, the weight loss rate reached 97%. GPC analysis showed that the molecular weight distribution shifted significantly to the low molecular weight region, indicating that it had been almost completely degraded into small molecules.

[0135] 3. Ecotoxicity:

[0136] Acute toxicity of earthworms (OECD207): At the maximum test concentration of 1000 mg / kg dry soil, the earthworm survival rate was 100% after 14 days.

[0137] Seed germination test: At 10 times the recommended dose of regulator, the germination rate and root length of small radish and Shanghai bok choy seeds were not significantly different from the control group (inhibition rate <5%).

[0138] Conclusion: The product is safe for environmental organisms and crops, and has no ecotoxicity.

[0139] Experiment 4. Effect of cellulose nanocrystal (CNC) addition amount on the mechanical strength and release performance of the temperature-sensitive layer;

[0140] To determine the optimal range of cellulose nanocrystals (CNCs) added to the thermosensitive hydrogel layer, this embodiment sets up a series of comparative experiments with different CNC addition amounts to comprehensively evaluate their effects on the mechanical strength of the gel layer, gel-sol transition temperature, and response sensitivity.

[0141] Experimental Design: Following the preparation method of the temperature-sensitive layer in Example 2, the mass ratio of gelatin to κ-carrageenan was fixed at 3:1 (total concentration 4% w / v), and cellulose nanocrystals (CNCs) were added at proportions of 1%, 2%, 5%, 8%, and 12% relative to the total mass of gelatin and κ-carrageenan, respectively. The specific formulations are as follows:

[0142] Table 2. Formulations of temperature-sensitive layers with different CNC additive amounts;

[0143] Each formulation was prepared into a gel sheet with a thickness of 2 mm for mechanical strength testing; at the same time, microspheres coated with temperature-sensitive layers of different CNC content were prepared according to the temperature-sensitive layer coating process of Example 2 for release performance testing.

[0144] Mechanical strength test:

[0145] The gel sheets were immersed in PBS buffer (pH 7.0) at 30°C and shaken for 12 hours (simulating a swollen state). The breaking strength of the gel was then measured using a texture analyzer (probe diameter 5 mm, compression speed 1 mm / s). Each group was repeated 5 times.

[0146] result:

[0147] C1 (CNC1%): After swelling, the gel texture is soft and mushy, with a breaking strength of only 12.3±2.1kPa. It breaks into fragments under slight vibration and cannot maintain an intact encapsulation structure.

[0148] C2 (CNC2%): The structure remains intact after swelling, and the breaking strength is increased to 28.7±3.4kPa, with no visible cracks.

[0149] C3 (CNC5%): The fracture strength further increases to 35.2±2.8kPa, with a dense structure and good elasticity.

[0150] C4 (CNC8%): The breaking strength is as high as 41.5±3.1kPa, but the gel hardens and becomes more brittle.

[0151] C5 (CNC12%): The breaking strength reached 44.8±2.9kPa, but the gel surface was rough and microcracks appeared in some areas.

[0152] Transition temperature and response sensitivity testing:

[0153] Microspheres coated with temperature-sensitive layers of varying CNC content (without an active core, only empty) were placed in PBS buffer and heated from 20°C to 45°C at a rate of 1°C / min. Changes in microsphere diameter and the cumulative release curve of the model compound (vitamin B12) were recorded. The gel-sol transition temperature was defined as the temperature at which the microsphere diameter begins to increase significantly (>5%) and the release rate shows a sharp inflection point.

[0154] Table 3. Effects of different CNC addition amounts on conversion temperature and release performance;

[0155] Overall conclusion: When the amount of CNC added is less than 2% (e.g., 1%), the mechanical strength of the gel layer is insufficient, it is easy to break after swelling, and the transition temperature is too low (26℃), which leads to premature leakage of active ingredients in non-target low temperature environments.

[0156] When the CNC addition is higher than 5% (e.g., 8% or 12%), although the mechanical strength is further improved, the gel-sol transition temperature is significantly increased (≥36℃), which exceeds the optimal growth temperature range of crops (28-35℃). At the same time, the response is delayed, and the "on / off" rapid release characteristic is lost.

[0157] When the CNC addition is 2-5%, the temperature-sensitive layer has both good mechanical strength (bursting strength ≥28kPa) and precise temperature response (transition temperature 29-31℃). It can achieve 80% release within 4-5 hours under 35℃ conditions, exhibiting ideal "on / off" controlled release behavior.

[0158] Therefore, the present invention preferably determines the amount of cellulose nanocrystals added to be 2-5% of the total mass of gelatin and κ-carrageenan.

[0159] Experiment 5: Criticality verification of the mass ratio of thermosensitive gelatin to κ-carrageenan:

[0160] To demonstrate that the gelatin:κ-carrageenan ratio range (2.5:1 to 3.5:1) selected in this invention is not a conventionally preferred design but produces unexpected technical effects, comparative experiments with different ratios were conducted. The total concentration was fixed at 4% w / v, and the CNC addition amount was 3% (relative to the total mass).

[0161] Table 4. Experimental results on the mass ratio of gelatin to κ-carrageenan;

[0162] Conclusion: The ratio of gelatin to κ-carrageenan is crucial in determining the function of the thermosensitive layer. A ratio that is too low (≤2:1) results in an excessively high transition temperature, failing to trigger within the crop's optimal temperature range; a ratio that is too high (≥4:1) results in an excessively low transition temperature, losing its gating function. The 2.5:1 to 3.5:1 range defined in this invention is the only critical interval that can simultaneously achieve the two contradictory goals of 'stable storage and transportation (leakage <15% at 25°C)' and 'rapid release within the optimal temperature range (release >75% at 30-35°C)'. The determination of this critical range is non-obvious.

[0163] Experiment 6: The impact of cascaded response sequence on product performance:

[0164] To demonstrate the inventiveness of the specific sequence (enzyme → temperature → pH / ion → light) of the four-layer structure of this invention, two control products with disordered sequences were prepared and compared with the product of this invention (sample A).

[0165] Preparation of control product: Comparative Example 4 (thermosensitive layer placed on the outermost layer): Refer to Example 2, but place the thermosensitive layer coating (S3a) in the last step, and move the phototriggered layer (S3c) to the inner layer. Keep the materials of each layer and the total thickness unchanged. Marked as sample D.

[0166] Comparative Example 5 (Phototriggered layer placed in the inner layer): Refer to Example 2, but the order of the phototriggered layer coating (S3c) and the pH / ion-sensitive layer (S3b) is interchanged, i.e., the outermost layer is the pH / ion-sensitive layer. Labeled as Sample E.

[0167] Table 5. Storage and transportation stability and release tests;

[0168] Table 6. Field application effects (tomato, under the same greenhouse test conditions as tomato).

[0169] In summary, altering the four-layer response sequence severely impairs the product's storage and transportation stability, release accuracy, or light control capabilities, leading to a significant decrease in yield increase and disease prevention effects. This demonstrates that the "enzyme → temperature → pH / ion → light" cascade sequence determined in this invention is not obvious and is one of the core technical features of this invention.

[0170] Comparative experiment with existing multilayer core-shell technology (CN121609600A):

[0171] Preparation of control product (named sample F): Organic fertilizer granules with a four-layer structure were prepared strictly according to the core scheme of Example 1 in the comparative document CN121609600A: the innermost layer is a humic acid-trace element core, the second layer is a composite antagonistic bacteria (Bacillus subtilis, Trichoderma harzianum, and gelatin-encapsulated microcapsules) encapsulated in sodium alginate-chitosan-gelatin, the third layer is an organic fertilizer matrix layer, and the outermost layer is a biodegradable coating layer containing polyglutamic acid and strigolactone GR24.

[0172] Comparative test: The effects of the product sample A and the control sample F of this invention were compared under the same conditions (tomato greenhouse, fertilizer application reduced by 20%). At the same time, a 'sample F + blue light irradiation' group was added (to verify whether it responds to non-design signals).

[0173] Table 7. Comparison Results;

[0174] In summary, although the control sample F has a similar four-layer physical structure, its release behavior is still mainly passive diffusion due to the lack of the 'cascaded response sequence' and 'specific functional material layers' (such as the temperature-sensitive layer, Ca²⁺ response layer, and phototriggered layer) of this invention. It cannot sense temperature changes to initiate a 'switch' release, nor can it respond to external blue light for artificial intervention. Its yield-increasing and colonization-promoting effects are far inferior to those of this invention, proving that this invention is not a simple material replacement of the multi-layered structure in the prior art, but rather a novel intelligent delivery system based on a completely new principle. Particularly noteworthy is the yield increase (18.5%) achieved by this invention under a 20% reduction in fertilizer use, far exceeding the yield increase (17.2%) reported in the prior art under conventional fertilization conditions, further highlighting the technological advancement and significant energy-saving and efficiency-enhancing advantages of this invention.

[0175] Experiment 7: Comparative test of material substitution while maintaining the existing technological order;

[0176] Preparation of the control product (named Sample G): Following the four-layer sequence of comparative document CN121609600A (from inside to outside: trace element core → microbial agent embedding layer → organic fertilizer matrix layer → biodegradable coating layer), the material of its second layer (microbial agent embedding layer) was replaced with the thermosensitive layer material of this invention, 'gelatin-κ-carrageenan-CNC' (same ratio as in Example 2), instead of 'sodium alginate-chitosan-gelatin'. The outermost layer remained a water-soluble coating layer (polyglutamic acid + starch). The active core remained the same as the bioactive core of this invention (engineered microalgae + probiotics + enzymes + signaling molecules). The remaining preparation steps followed the method of CN121609600A.

[0177] Comparative test: Sample G and sample A of the present invention were compared under the same conditions (tomato greenhouse, fertilizer application reduced by 20%). At the same time, a 'sample G + blue light irradiation' group was set up (to verify whether a light response was unexpectedly obtained).

[0178] Table 8. Test Results:

[0179] In summary, simply replacing the materials in the prior art with the thermosensitive material of this invention, without changing the layer order (i.e., the phototriggered layer is not placed on the outermost layer, and the thermosensitive layer is not placed on the second layer), not only fails to achieve photoresponse capability, but also leads to decreased storage and transportation stability because the thermosensitive material is directly exposed under the coating layer, resulting in a far less efficient production increase than this invention. This demonstrates that the technical effect of this invention does not depend on the replacement of individual materials, but rather on the systematic combination of a 'specific order + specific responsive material'. This combination, as a whole, produces an unpredictable synergistic effect that cannot be replicated by any unilateral imitation. This result further proves the non-obviousness of this invention.

[0180] The core of this invention lies in constructing a system that deeply integrates a "synthetic bioactive core" with an "all-natural intelligent response carrier".

[0181] 1. Synergistic design of bioactive cores:

[0182] Engineered microalgae (Synechocystis sp. XL-1): As a "photosynthetic factory", it simultaneously achieves biological nitrogen fixation (introducing nifH cluster), organic phosphorus activation (introducing phyA) and endogenous growth-promoting signal (overexpressing MAX3) through gene editing, providing initial nutrients and signals for the system.

[0183] Synthetic probiotic flora: including Bacillus subtilis EA-1 (water retention and growth promotion) which produces polyglutamic acid, Acinetobacter AB-3 (nutrition and antagonism) which has dual functions of nitrogen fixation and siderophore production, and Bacillus amyloliquefaciens BA-5 (biocontrol) which expresses quorum sensing quenching enzyme. The three have evolved through co-culture to form a stable functionally complementary symbiotic organism.

[0184] The ultra-stable enzyme complex contains the thermostable xylanase Xyn10B-DM4 (stable at 85°C), the broad-spectrum phosphatase PhoA-M2 (stable at pH 4-10), and the highly efficient laccase LacS-EC1, which together are responsible for the biochemical improvement of the soil matrix.

[0185] Plant-derived signaling molecules: Glycosylated signaling substances extracted from salicylic acid hyperaccumulating white willow serve as "starting keys" for pre-activating plant systemic resistance.

[0186] The all-natural intelligent response carrier features a quadruple-gated design: employing a biomimetic multi-layer structure, with each layer composed of natural materials, to achieve cascading response.

[0187] First layer (core): Enzyme-sensitive gating. The sodium alginate-chondroitin sulfate gel network can be efficiently degraded by rhizosphere-specific secreted β-glucanase, ensuring that release begins in healthy root microzones.

[0188] The second layer (thermosensitive layer): temperature gating. Gelatin-κ-carrageenan-cellulose nanocrystal composite hydrogel. By adjusting the ratio of gelatin (gelation temperature below 25℃, melting point ~35℃) to carrageenan (gelation temperature ~40℃), the gel-sol transition temperature of the system is precisely controlled within the optimal growth range of 28-35℃ for crops. Upon increasing temperature, the hydrogen bond network breaks down, the gel swells, and the pores open. Cellulose nanocrystals (CNC) act as a green reinforcing agent, preventing gel collapse.

[0189] The third layer (pH / ion-sensitive layer): chemically gated. A chitosan / pectin / calcium alginate composite membrane, cross-linked with genipin. Chitosan protonates and swells in an acidic (pH < 6.5) rhizosphere environment; the calcium alginate network is sensitive to Ca²⁺ concentration. Genipin, as a natural cross-linking agent, replaces toxic chemical cross-linking agents, ensuring safe cross-linking.

[0190] The fourth layer (outer layer): phototriggered and bio-anchored gating. A zein-gum arabic composite membrane, loaded with riboflavin and chitosan oligosaccharides. Riboflavin (vitamin B2) undergoes a photosensitizing reaction under 470nm blue light irradiation, generating trace amounts of reactive oxygen species, causing localized oxidation of the protein membrane and a transient increase in permeability. Chitosan oligosaccharides, as natural root chemotactic agents, enhance particle adhesion and recognition on the root surface.

[0191] 2. Key constraints and core identification features of the technical solution of this invention:

[0192] The specific sequence described in this invention—'from inside to outside: enzyme-sensitive core, temperature-sensitive hydrogel layer, pH / ion dual-sensitive layer, and phototriggered outer layer'—is the key technology for achieving 'signal-guided cascade release' and is non-obvious. This sequence strictly follows the natural succession logic of the rhizosphere microecology:

[0193] (1) Logical starting point (enzyme): Ensure that the release only begins around healthy, metabolically active roots to avoid fertilizer waste.

[0194] (2) Environmental filter (temperature): Ensures that no large amount of release occurs when the soil temperature does not reach the suitable growth range of crops (such as early spring or cold regions), so as to achieve intelligent matching with the environment and climate.

[0195] (3) Precision targeter (pH / ion): It achieves concentrated release in the active absorption zone of the root system (often accompanied by the secretion of organic acids, which leads to a decrease in pH and changes in ion concentration), thereby improving the utilization rate.

[0196] (4) Artificial control switch (light): Based on the fact that the first three layers all rely on natural conditions, a 'remote control' with human subjective intervention is added to realize the 'on-demand supply' required for smart agriculture.

[0197] Any alteration of the above order (e.g., placing the temperature-sensitive layer on the outermost layer) will result in the loss of system functionality: premature leakage during storage and transportation, inability to achieve precise rhizosphere targeting, and loss of on-demand replenishment capability. Therefore, the 'four-layer order' of this invention is itself an important technical feature, generating a synergistic effect that goes beyond the simple addition of the functions of each layer.

[0198] 3. Synergistic Regulation Theory and Mechanism: "Signal-Guided Cascade Release and Biosynergistic Amplification"; This invention proposes and implements the following operational model: Step 1: Biorecognition Initiation. Healthy roots secrete β-glucanase, degrading the nucleus, and microalgae and plant signal molecules first contact the rhizosphere. Step 2: Environmentally Adaptive Release. Suitable soil temperature triggers the swelling of the thermosensitive layer, accelerating the release of probiotics and enzymes. At this time, microalgae have begun photosynthesis, improving the rhizosphere oxygen environment. Step 3: Establishment and Amplification of the Biosynergistic Network. Plant signals activate plant immunity; carbon sources, oxygen, and fixed nitrogen provided by microalgae support probiotic colonization; probiotics form biofilms and secrete growth-promoting substances, further promoting plant growth; enzymes continuously improve the soil. The three form a positive interaction cycle of "plant-microalgae-probiotics". Step 4: Precise Artificial Intervention. When additional stimulation is needed (such as during flowering), external blue light irradiation triggers the rapid release of reserve active ingredients from the outer layer, achieving "on-demand replenishment".

[0199] Compared to existing controlled-release systems that can only respond to a single signal (such as pH or temperature) or a simple combination of signals, the "signal-guided cascaded release" described in this invention has significant synergistic advantages. The response layers do not work independently or in parallel, but rather unlock sequentially according to the logical order of "biological recognition (rhizosphere enzymes) → physical adaptation (temperature) → chemical initiation (pH / ions) → artificial intervention (light)". This cascaded design avoids ineffective release of active ingredients in non-target environments (such as soil not in contact with roots, or early spring when temperatures are not optimal), ensuring that the release timing is highly synchronized with the crop's physiological needs and the succession process of the rhizosphere microecology, thereby achieving the goal of precise release.

[0200] 4. Optimal formulation of the all-natural intelligent biological soil conditioner:

[0201] Based on the above design, the all-natural intelligent biological soil conditioner of the present invention preferably contains the following components on a dry weight basis (the content range of each component can be adjusted according to actual application, but is not limited thereto):

[0202] Table 9. Bioactive core (total of 100 parts by dry weight).

[0203] Note: The dry weight percentages above represent the relative proportions of each component in the active core. In actual preparation, the proportions can be scaled up proportionally.

[0204] (2) Enzyme-sensitive core (relative to the mass of the active core):

[0205] Sodium alginate aqueous solution: 2.0% (w / v);

[0206] Chondroitin sulfate: 0.5% (w / v);

[0207] Curing agent: 0.1 M CaCl2 solution;

[0208] (3) Temperature-sensitive hydrogel layer (relative to the total mass of gelatin and κ-carrageenan):

[0209] The mass ratio of gelatin to κ-carrageenan is 2.5:1 to 3.5:1 (preferably 3:1).

[0210] Cellulose nanocrystals (CNC) addition amount: 2-5% (preferably 3%) of the total mass of gelatin and κ-carrageenan;

[0211] Gel-sol transition temperature: 28-35℃;

[0212] (4) pH / ion dual-sensing layer (for each solution concentration):

[0213] Chitosan: 0.5% (w / v) soluble in 1% acetic acid;

[0214] Pectin: 0.5% (w / v) containing 1% sodium alginate;

[0215] The mass ratio of chitosan to pectin (controlled during layer-by-layer self-assembly) is 1.5:1 to 2.5:1.

[0216] Crosslinking agent: Genipin 0.2% (w / v), crosslinking at 37℃ for 2 hours;

[0217] (5) Phototriggering and rhizosphere anchoring of the outer layer (concentration of each component):

[0218] Zea prolysin: 2% (w / v);

[0219] Gum arabic: 1% (w / v);

[0220] Riboflavin (vitamin B2): 0.05% (w / v);

[0221] Chitosan oligosaccharide: 0.1% (w / v);

[0222] Solvent: 70% ethanol-water solution;

[0223] The concentrations in the above formulations are the initial concentrations during preparation. In the final product, each component exists in the form of a coating layer. By adjusting the proportions of each layer and the cross-linking conditions, customized release profiles can be achieved for different crops, soil, and climate conditions.

[0224] Additional notes: Microalgae: Probiotics: Enzymes: Signaling molecules = 3:4:1:2 (total dry weight 10 parts), multiplied by 10, it is 30:40:10:20 (total number of parts 100).

[0225] The addition of 2-5% CNC in the temperature-sensitive layer is consistent with the conclusion of Example 5 and supports claim 3.

[0226] The concentrations of the other layers correspond exactly to the concentrations prepared in step S3.

[0227] 5. Unique design and verification of cascading sequence:

[0228] The four-layer response described in this invention is not a simple stacking; its sequence (enzyme → temperature → pH / ion → light) follows a strict logic: the rhizosphere enzyme response ensures that release begins in the healthy root zone (avoiding fertilizer waste); the temperature response matches the crop growing season (avoiding ineffective release in early spring); the pH / ion response is precisely regulated in the rhizosphere acidification zone (peak nutrient absorption); and light triggering acts as an artificial intervention switch (on-demand replenishment). If this sequence is changed, the synergistic effect significantly decreases.

[0229] If the temperature-sensitive layer is placed on the outermost layer (Comparative Example 4), leakage will occur during storage and transportation (temperature fluctuations), and the field shelf life will be shortened by more than 40%.

[0230] If the light triggering layer is placed in the inner layer (Comparative Example 5), blue light cannot penetrate the outer layer, the light response fails, and the yield increases to only 5.2%.

[0231] The above comparative experiments demonstrate that the hierarchical order of the present invention is itself a non-obvious inventive point.

[0232] Table 10. Key differences from existing technologies;

[0233] None of the above differences have been disclosed or revealed by the prior art, and the present invention achieves the outstanding effect of "reducing fertilizer application by 20% while still increasing tomato yield by 18.5%", which is non-obvious.

[0234] Beneficial effects: 1. Intelligent and precise release: Integrating enzyme, temperature, pH / ion, and light four-fold response, the release curve is highly matched with crop needs, and the nutrient utilization rate is increased by more than 50%.

[0235] 2. All-natural and green: All carrier materials are food-grade or organically licensed substances (gelatin, carrageenan, chitosan, corn protein, etc.), eliminating the need for complex toxicological and environmental safety assessments of synthetic or nanomaterials, and allowing for rapid market access.

[0236] 3. Long-lasting and stable bioactivity: The all-natural carrier provides excellent protection. After 24 months of storage at 4°C, the survival rate of probiotics in freeze-dried products is >85%, and the enzyme activity retention is >90%.

[0237] 4. Significant ecological and yield-increasing effects:

[0238] Field trials have confirmed that, even under the stringent condition of reducing fertilizer application (15-25%), the regulator of this invention can still increase tomato yield by 18.5% and strawberry yield by 15.3%. More importantly, this yield increase does not come at the expense of quality or increased disease incidence; on the contrary, it simultaneously achieves: a) a significant reduction in the incidence of diseases (tomato bacterial wilt, strawberry powdery mildew) (reduction >65%); b) a significant improvement in fruit quality (soluble solids) (>20%); and c) a significant increase in soil microbial diversity index. In existing technologies, single-function microbial agents or regulators can usually only achieve one or two of the above effects. However, this invention, through the deep synergy of the "active core" and the "intelligent carrier," simultaneously achieves five goals: reduced fertilizer application, increased yield, improved quality, disease prevention, and soil improvement. Its comprehensive technical effect far exceeds the simple summation of the effects of individual components.

[0239] To further demonstrate that the synergistic effect of the present invention is not a simple additive effect, a comparative analysis was conducted between the complete four-layer product of the present invention (sample A) and a control product with only a single-layer or double-layer response:

[0240] Table 11. Comparison Table with Tongda;

[0241] The above data shows that the yield increase and disease prevention effects produced by the four-layer synergy exceed the best effect of the three-layer synergy by 53% and 164% respectively, which is a typical "1+1>2" technical effect.

[0242] 5. Sustainable and environmentally friendly: All materials are fully biodegradable in soil within 180 days, with no persistent residues or microplastic risks.

[0243] 6. It overcomes the technical prejudice that 'all-natural materials cannot be used to construct complex, multi-functional intelligent response systems';

[0244] A long-standing technological bias in this field holds that achieving precise responses to multiple signals such as pH, temperature, enzymes, and light requires structurally designable synthetic polymers (such as PNIPAM), metal-organic frameworks, or complex nanomaterials. However, existing technologies, exemplified by CN121609600A, even with multilayer structures, still rely on simple material dissolution or diffusion for functionality, failing to achieve true 'signal gating' cascades. This invention, through innovative material blending (such as gelatin-κ-carrageenan-CNC constructing a precise temperature-sensitive gate, genipin-crosslinked chitosan-pectin constructing a pH / Ca²⁺ dual-response gate, and riboflavin-zein constructing a photosensitizing gate) and specific logical assembly sequences, demonstrates that all-natural materials can not only, but can construct more complex and logically ordered intelligent response systems than synthetic materials. This invention opens up entirely new technological pathways for the development of green agricultural inputs, and its technological contribution transcends the scope of specific products.

[0245] 7. Quantifiable 'super-addition' synergistic effect:

[0246] To mathematically demonstrate that the four-layer cascaded system of this invention produces a nonlinear synergistic amplification effect, rather than a simple summation of the effects of each layer, this study defines a 'synergistic enhancement index':

[0247] SEI=(E full -E best3 ) / (E best3 +E extra laye alone )

[0248] Among them: E full The effect of the complete four-layer system (including blue light triggering) of this invention; E best3 The effect of the three-layer system (enzyme + temperature-sensitive + pH / ion, three layers for best results); E extra layer alone The effect of the outermost layer (light-triggered layer) alone without an inner layer; calculated using tomato yield increase as an example: E full =18.5%; E best3 =12.1%; E extra layer alone =2.4% (product with only light trigger layer, no inner layer, determined by experiment); SEI=(18.5%-12.1%) / (12.1%+2.4%)=6.4% / 14.5%≈0.44;

[0249] For simple addition, the SEI should be close to 0; for synergistic amplification, the SEI > 0. In this invention, the SEI = 0.44, indicating that the four-layer synergy produces an additional 44% super-suppression effect independent of each layer.

[0250] Similarly, based on the disease prevention effect: SEI disease prevention = (65% - 24.6%) / (24.6% + 8.2%) = 40.4% / 32.8% ≈ 1.23, the synergistic amplification effect is more significant.

[0251] The mathematical models and data described above demonstrate that the technical effects of this invention are far beyond what can be expected from the 'structural optimization' or 'material replacement' of multi-layered structures in the prior art, but rather produce significant and unpredictable synergistic qualitative changes.

[0252] 8. Advantages compared to commercially available biostimulants / soil conditioners:

[0253] To demonstrate the practical advancements of this invention, three representative commercially available products were selected for a field comparison (tomatoes, with 20% less fertilizer):

[0254] Product K1: A well-known brand's 'intelligent controlled-release microbial agent' (pH-responsive, carrier is synthetic resin)

[0255] Product K2: A certain imported 'photosensitive bio-fertilizer' (containing TiO2 photocatalytic material)

[0256] Product K3: A certain 'all-natural enzyme fertilizer' (without controlled-release design)

[0257] Table 12. Comparison Results;

[0258] It is evident that this invention is significantly superior to existing commercial products in terms of increased production, disease prevention, and environmental protection. Its comprehensive performance advantages cannot be anticipated by any single existing technology or a simple combination thereof.

[0259] Explanation of the technical solution and its implementation results:

[0260] 1. A detailed description of the structure and mechanism of the intelligent biological soil conditioner of this invention;

[0261] Four-layer core-shell intelligent carrier structure: This product features a unique four-layer concentric spherical shell structure, forming a complete "multi-gated" release system from the inside out. The specific structure is shown in Table 13.

[0262] Table 13. Composition of the four-layer core-shell smart carrier structure:

[0263]

[0264] (2) Mechanism of “signal-guided synergistic amplification”;

[0265] The core mechanism of this invention is a dynamic, enhanced cyclical process, which can be divided into four logical stages: Stage 1 (Biorecognition Initiation): Healthy plant roots secrete specific β-glucanases, which degrade the first layer (enzyme-sensitive core), allowing engineered microalgae and plant-derived signaling molecules located in the core to be released into the rhizosphere. Stage 2 (Environmental Adaptation and Initial Network Establishment): When the soil temperature reaches the optimal growth range for crops (e.g., >28℃), the second layer (thermosensitive hydrogel layer) swells, accelerating the release of probiotic communities and ultra-stable enzyme complexes. At this time, the previously released microalgae improve the rhizosphere oxygen environment through photosynthesis and secrete polysaccharides, providing a carbon source for probiotic colonization; plant signaling molecules activate the plant's own stress resistance and growth pathways. Stage 3 (Biosynergistic Amplification): Colonized probiotics form a biofilm and continuously secrete growth-promoting substances, further promoting plant root development; the developed roots secrete even more beneficial substances, feeding back into the microbial community; the enzyme complex continuously improves the soil's physicochemical properties. This forms a positive and self-reinforcing ecological cycle among plants, microalgae, and probiotics. The fourth stage (precise artificial intervention): During the critical period of crop nutrient demand or the early stage of stress, external blue light can be used to irradiate the rhizosphere area, triggering an instantaneous response of the fourth layer (phototriggered layer), which quickly releases the stored active ingredients, achieving precise agronomic management of "on-demand replenishment".

[0266] 2. Detailed description of in vitro intelligent response performance data:

[0267] (1) Temperature-sensitive release characteristics data: To verify the "switching" performance of the temperature-sensitive layer, an in vitro release test was conducted. The product of this invention (sample A) and the control product embedded in pure sodium alginate (sample B) were placed at different temperatures to determine the cumulative release rate of the model substance. The key data are as follows:

[0268] Table 14. Comparison of cumulative release rates of the product of this invention and the product encapsulated with pure sodium alginate at different temperatures:

[0269] Conclusion: The product of this invention exhibits a sharp change in release rate around 28-30℃, while the control group does not show this phenomenon. This fully demonstrates the successful construction of the gelatin-κ-carrageenan-based thermosensitive layer and its precise design of the critical response temperature.

[0270] (2) Enzyme response degradation and release data: To verify the core's targeted release capability, tests were conducted in a simulated rhizosphere enzyme environment.

[0271] Morphological observation (SEM): In the initial state, the surface of the microspheres of this invention is smooth and intact. After soaking in simulated rhizosphere solution containing β-glucanase (5 U / mL) for 12 hours, numerous pores and obvious cracks with a diameter of approximately 1-5 μm were observed on the surface of the microspheres, indicating a tendency for structural disintegration. However, after soaking in the control solution without the enzyme for 72 hours, the microspheres still maintained structural integrity.

[0272] Comparison of cumulative release rates:

[0273] Enzyme-containing environment: Within 12 hours, the cumulative release rate of active ingredients rapidly reaches 78%.

[0274] In an enzyme-free environment, the release rate is only 12% within 12 hours; even when extended to 72 hours, the release rate is still less than 15%.

[0275] Conclusion: The release of the product of this invention is highly dependent on the presence of a specific enzyme, and it is only released in large quantities when the target biological signal (the enzyme secreted by healthy roots) appears, thus achieving precise targeting based on biorecognition and greatly reducing ineffective release.

[0276] 3. Detailed data on field application effects:

[0277] To comprehensively verify the application effects of the regulator of this invention in different crop systems, a comparative experiment was conducted on greenhouse cultivation of tomatoes and strawberries. The core effect data and conclusions are as follows:

[0278] (1) Tomato greenhouse experiment:

[0279] A one-season field trial was conducted in tomato greenhouses in Changchun, Jilin Province.

[0280] Experimental design: Three treatments were set up, with three replicates for each treatment. T1: Conventional fertilization (CK); T2: Conventional fertilization + commercially available compound microbial agent (10 kg / ha); T3: Conventional fertilizer reduction of 20% + sample A of this invention (2 kg / ha).

[0281] Management: During the initial flowering and fruit enlargement stages of tomatoes, the root zone was irradiated twice using a portable 470nm LED lamp, for 10 minutes each time.

[0282] Table 15. Evaluation of the experimental effects of the product of this invention applied to tomato greenhouse cultivation (including tomato yield, disease incidence, microbial diversity, and relative abundance of dominant bacteria):

[0283] Key findings: 1. Reduced fertilizer input and increased yield: Under the premise of reducing fertilizer input by 20%, the application of the product of this invention achieved the highest yield (18.5% increase), proving that it can significantly improve nutrient utilization efficiency.

[0284] 2. Superior disease prevention: The disease incidence rate of the treatment (T3) of this invention is the lowest (4.4%), and its prevention effect is significantly better than that of commercially available fungicides, proving that its comprehensive disease resistance is stronger through multiple biological synergies.

[0285] 3. Soil improvement: The product of this invention can significantly improve the diversity of soil microbial communities (Shannon diversity index 9.25) and the proportion of beneficial bacteria, indicating that it has a healthy function of continuously improving soil microecology.

[0286] (2) Strawberry greenhouse experiment:

[0287] A verification experiment was conducted in a strawberry greenhouse in Changchun, Jilin.

[0288] Experimental design: Three treatments were set up, with three replicates for each treatment. T1: Conventional fertilization (CK); T2: Conventional fertilization + commercially available compound microbial agent (12 kg / ha); T3: Conventional fertilizer reduction of 15% + sample A of this invention (3 kg / ha).

[0289] Management: During the strawberry budding and fruit coloring stages, the root zone was irradiated twice using a portable 470 nm LED lamp for 15 minutes each time.

[0290] Table 16. Core effects of the product of this invention applied to strawberry field experiments:

[0291] Key conclusions:

[0292] 1. Improved quality and increased yield: Under the condition of reducing fertilizer by 15%, the product of this invention not only significantly increases strawberry yield by 15.3%, but also significantly improves fruit sugar content and firmness, and extends shelf life.

[0293] 2. Highly Effective Disease Control: It exhibits excellent control of strawberry powdery mildew, significantly outperforming conventional commercially available inoculants. The incidence of powdery mildew is reduced by 45.6% compared to fertilization with commercially available inoculants.

[0294] 3. Improved soil health and growth: Significantly enhanced rhizosphere soil microbial diversity, with a Shannon diversity index of 9.18, creating a better rhizosphere environment for healthy strawberry growth.

[0295] Overall conclusion: Field trial results show that the all-natural multi-component intelligent responsive biological soil conditioner of this invention can achieve a quadruple synergistic effect of "reducing fertilizer use, increasing yield, preventing disease, and improving soil" in both fruit tomatoes and strawberries, verifying the reliability of the invention and its good agricultural ecological value.

[0296] Definitions and explanations:

[0297] To clarify the scope of protection and technical contributions of this invention, the following terms are defined:

[0298] 1. 'Cascaded Response': In this invention, it specifically refers to the conditionally dependent release of the four-layer intelligent carrier system in a strict sequential order of 'enzyme signal → temperature signal → pH / ion signal → light signal'. That is, the activation of the subsequent response layer is predicated on the perception of the signal of the previous response layer, rather than each layer responding to its own signal independently and in parallel. This definition differs from the concepts of 'multi-layer simultaneous response' or 'single signal response' in the prior art.

[0299] 2. 'All-natural components': In this invention, it specifically refers to all carrier materials being derived from naturally occurring biological macromolecules (including but not limited to gelatin, carrageenan, chitosan, pectin, alginate, zein, gum arabic, genipin, riboflavin, etc.) and not subject to chemical synthesis modification (except for genipin, which is a naturally extracted cross-linking agent). All materials are listed as permitted inputs or processing aids by the Chinese Organic Product Standard (GB / T19630) or the EU Organic Agriculture Regulation (EC834 / 2007). Synthetic biologically modified microorganisms are not considered 'carrier materials', but their metabolites and the microorganisms themselves meet the requirements of organic agriculture for microbial agents.

[0300] 3. 'On-demand replenishment': In this invention, it specifically refers to the artificial, proactive release achieved through external blue light irradiation when natural conditions (enzymes, temperature, pH / ions) are insufficient to trigger complete release, or when crops face sudden stress. This function is one of the core features that distinguishes this invention from all existing technologies that rely on natural conditions for release.

[0301] Industrial applicability:

[0302] The preparation method provided by this invention has clear process steps, and the equipment used (fermentation tank, microfluidic instrument, freeze dryer, etc.) are all conventional industrial equipment. The raw materials are all readily available commercial natural products or can be obtained through cultivation, thus possessing the feasibility for industrial-scale production. Field trials have verified the product's significant effects, making it suitable for field crops, cash crops, and facility agriculture, with broad application prospects. Furthermore, the gelatin, carrageenan, chitosan, and zein used in this invention are all commercially available food industry additives with low costs; engineered microalgae and probiotics can be produced on a large scale through conventional fermentation processes. Therefore, the product of this invention not only has excellent effects but also controllable overall production costs, possessing the market potential to replace some chemical inputs on a large scale. This technical solution is applicable to food crops, cash crops, fruits and vegetables, and the remediation of degraded soil.

[0303] Example 3:

[0304] A method for preparing the above-mentioned all-natural multi-component intelligent responsive bio-soil conditioner includes the following steps:

[0305] S1. Preparation of bioactive core freeze-dried powder: The engineered microalgae and the synthetic probiotic group are cultured and harvested separately, mixed with stable enzyme complex and plant-derived signaling molecules, and then freeze-dried.

[0306] S2. Construction of enzyme-sensitive core: Using microfluidic technology, the lyophilized active core powder obtained in S1 was dispersed in an aqueous solution of sodium alginate and chondroitin sulfate, and then added dropwise to a calcium chloride solution to solidify and form primary microspheres;

[0307] S3. Layer-by-layer self-assembly intelligent response layer:

[0308] Thermosensitive coating: The microspheres obtained from S2 are immersed in a composite aqueous solution of gelatin, κ-carrageenan and cellulose nanocrystals at 50-60℃, and cooled to room temperature to form a gel coating layer;

[0309] pH / ion-sensitive layer coating: Through layer-by-layer self-assembly technology, chitosan solution and pectin-calcium alginate mixture are alternately deposited on the surface of the temperature-sensitive layer, and finally immersed in genipin solution for cross-linking.

[0310] Phototriggered and anchored outer coating: Microspheres were immersed in an ethanol-water solution containing zein, gum arabic, riboflavin and chitosan oligosaccharide, and then dried to form a film;

[0311] S4. Post-processing of the product: Freeze-dry the coated microspheres to obtain the final powder product;

[0312] Furthermore, in step S3, the mass ratio of gelatin to κ-carrageenan is 2.5:1 to 3.5:1, and the amount of cellulose nanocrystals added in step S3 is 2%-5% of the total mass of gelatin and κ-carrageenan; the mass ratio of chitosan to pectin in step S3 is 1.5:1 to 2.5:1; and the photosensitizer in step S3 is riboflavin with a concentration of 0.01%-0.1% w / v.

[0313] Example 4:

[0314] Application of an all-natural, multi-component, intelligently responsive bio-soil conditioner as described in Example 1 in improving crop yield and stress resistance, restoring degraded soil, or for use in organic agricultural production.

Claims

1. A multi-component, intelligently responsive bio-soil conditioner with all-natural components, characterized in that, include: The bioactive core includes engineered microalgae, synthetic probiotics, ultrastable enzyme complexes, and plant-derived signaling molecules, including glycosylated signaling substances extracted from salicylic acid hyperaccumulating white willow. The four-layer core-shell structured intelligent carrier system encapsulating the bioactive core consists of, from the inside out: an enzyme-sensitive core, a temperature-sensitive hydrogel layer, a pH / ion dual-sensitive layer, and a light-triggered and rhizosphere-anchored outer layer.

2. The regulator according to claim 1, characterized in that, The thermosensitive hydrogel layer is composed of gelatin, κ-carrageenan and cellulose nanocrystals, and its gel-sol transition temperature is controlled between 28-35℃ by adjusting the mass ratio of gelatin to κ-carrageenan.

3. The regulator according to claim 2, characterized in that, The mass ratio of gelatin to κ-carrageenan is 2.5:1 to 3.5:1, and the amount of cellulose nanocrystals added is 2%-5% of the total mass of gelatin and κ-carrageenan.

4. The regulator according to claim 1, characterized in that, The pH / ion dual-sensing layer is composed of chitosan, pectin, and calcium alginate cross-linked by the natural cross-linking agent genipin, wherein the mass ratio of chitosan to pectin is 1.5:1 to 2.5:

1.

5. The regulator according to claim 1, characterized in that, The light-triggered and rhizosphere anchoring outer layer is composed of zein, gum arabic, photosensitizer, and root chemotactic agent; the photosensitizer is riboflavin or curcumin; and the root chemotactic agent is chitosan oligosaccharide.

6. The regulator according to claim 1, characterized in that, The engineered microalgae is the gene-edited Synechocystis XL-1 strain, whose genome integrates the nitrogen fixation gene cluster nifH and the phytase gene phyA, and overexpresses the endogenous strigolactone synthesis key gene MAX.

7. The regulator according to claim 1, characterized in that, The synthetic probiotic group is composed of Bacillus subtilis EA-1, Acinetobacter AB-3 and Bacillus amyloliquefaciens BA-5 in a live bacteria ratio of 5:3:

2.

8. The regulator according to claim 1, characterized in that, The ultrastable enzyme complex includes the thermostable xylanase Xyn10B-DM4, the broad-spectrum phosphatase PhoA-M2, and the highly efficient laccase LacS-EC1, all obtained through directed evolution.

9. A method for preparing a multi-component, intelligently responsive bio-soil conditioner according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Preparation of bioactive core freeze-dried powder: The engineered microalgae and the synthetic probiotic group are cultured and harvested separately, mixed with stable enzyme complex and plant-derived signaling molecules, and then freeze-dried. S2. Construction of enzyme-sensitive core: Using microfluidic technology, the lyophilized active core powder obtained in S1 was dispersed in an aqueous solution of sodium alginate and chondroitin sulfate, and then added dropwise to a calcium chloride solution to solidify and form primary microspheres; S3. Layer-by-layer self-assembly intelligent response layer: Thermosensitive coating: The microspheres obtained from S2 are immersed in a composite aqueous solution of gelatin, κ-carrageenan and cellulose nanocrystals at 50-60℃, and cooled to room temperature to form a gel coating layer; pH / ion-sensitive layer coating: Through layer-by-layer self-assembly technology, chitosan solution and pectin-calcium alginate mixture are alternately deposited on the surface of the temperature-sensitive layer, and finally immersed in genipin solution for cross-linking. Phototriggered and anchored outer coating: Microspheres were immersed in an ethanol-water solution containing zein, gum arabic, riboflavin and chitosan oligosaccharide, and then dried to form a film; S4. Post-processing of the product: Freeze-dry the coated microspheres to obtain the final powder product; Furthermore, in step S3, the mass ratio of gelatin to κ-carrageenan is 2.5:1 to 3.5:1, and the amount of cellulose nanocrystals added in step S3 is 2%-5% of the total mass of gelatin and κ-carrageenan; the mass ratio of chitosan to pectin in step S3 is 1.5:1 to 2.5:1; and the photosensitizer in step S3 is riboflavin with a concentration of 0.01%-0.1% w / v.

10. The application of an all-natural, multi-component, intelligently responsive bio-soil conditioner as described in any one of claims 1-8 in improving crop yield and stress resistance, restoring degraded soil, or in organic agricultural production.

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