Ophiopogon japonicus control regulator based on carbon-based ecological fertilizer loaded composite enzyme preparation and preparation method and application thereof

Abstract

The present application relates to the field of traditional Chinese medicinal material cultivation and enzyme engineering technology, and particularly relates to a kind of ophiopogon japonicus control boom regulator based on carbon-based ecological fertilizer load composite enzyme preparation and its preparation method and application.The regulator comprises the following components in mass fraction:80-95 parts of functional liquid carbon-based ecological fertilizer matrix, 3-15 parts of targeted composite enzyme preparation, 1-3 parts of enzyme stabilizing protection system and 1-2 parts of cofactor activation liquid.The regulator can significantly increase the fresh weight and dry weight of ophiopogon japonicus tuber, the total saponin content and the crude polysaccharide content, has a significant control boom effect, has multiple functions of yield increase, quality improvement and soil improvement, and is suitable for green, efficient and sustainable cultivation of traditional Chinese medicinal materials.

Description

Technical Field

[0001] This invention relates to the fields of Chinese medicinal herb cultivation and enzyme engineering technology, and in particular to a growth regulator for Ophiopogon japonicus based on carbon-based ecological fertilizer loaded with compound enzyme preparation, its preparation method and application. Background Technology

[0002] As a commonly used bulk Chinese medicinal herb, the core medicinal quality of Ophiopogon japonicus depends on the accumulation of polysaccharides, saponins, and flavonoids in its tubers. However, the current large-scale cultivation commonly involves excessive application of nitrogen fertilizer, leading to an imbalance between vegetative and reproductive growth, resulting in excessive vegetative growth ("vigorous vegetative growth"). This not only inhibits tuber development and enlargement, reducing the yield of the medicinal herb, but also weakens the plant's resistance to adverse conditions, ultimately leading to a significant decrease in the content of medicinal components. To combat this excessive vegetative growth, the industry mainly relies on spraying chemically synthesized plant growth regulators such as paclobutrazol. However, these methods have problems such as high residue risks, potential disruption of soil microecological balance, and failure to meet the Good Agricultural Practices (GAP) requirements for input safety. Therefore, there is an urgent need to find green and safe alternative technologies.

[0003] While using conventional organic or carbon-based fertilizers for soil improvement can mitigate the negative effects of excessive chemical fertilizer application to some extent, there are three major bottlenecks in addressing the problem of excessive vegetative growth in *Ophiopogon japonicus*. First, the raw materials for organic fertilizers are complex and may carry heavy metals, antibiotic resistance genes, or pathogenic microorganisms, making their safety difficult to guarantee and posing potential pesticide residue and biological risks. Second, traditional fermentation processes have low degradation efficiency for stubborn components such as lignocellulose in organic materials, resulting in low yields of active small molecules (such as humic acid and phenolic acids), making it difficult to fully realize their potential to stimulate crop growth and regulate secondary metabolism. Finally, existing organic fertilizer products generally lack the ability to precisely intervene in the nitrogen assimilation and distribution pathways of *Ophiopogon japonicus*, failing to address the excessive vegetative growth problem caused by nitrogen metabolism imbalance at its source.

[0004] In recent years, applying biotechnology, such as enzyme preparations, to the creation of agricultural inputs has become a research hotspot, attempting to generate active products through the targeted degradation of specific macromolecules. However, directly applying enzyme preparations to complex organic liquid fertilizer systems faces severe challenges: enzymes are easily inactivated in high-temperature, high-salt, or protease-rich environments; free enzymes lack targeting, making it difficult to effectively accumulate and function in specific parts of the rhizosphere or plant; at the same time, the high cost of large-scale production of enzyme preparations limits their widespread application in agriculture. Therefore, how to achieve the steady-state existence and targeted delivery of enzymes in complex agricultural formulations has become a key bottleneck for the industrialization of this technological route.

[0005] In summary, the existing technological system has not yet established a systematic solution that encompasses ensuring safety at the source and precisely regulating crop metabolism. Addressing the problem of excessive growth caused by excessive nitrogen fertilizer in Ophiopogon japonicus requires a breakthrough in the entire technological chain, integrating "safe raw material purification—efficient generation of active molecules—organic conversion of mineral elements—optimization of rhizosphere microecology—steady-state delivery of functional enzymes." Only by constructing a green technology system that integrates safe pretreatment, targeted biotransformation, and steady-state application can precise intervention in the nitrogen metabolism pathway of Ophiopogon japonicus be achieved, effectively replacing chemical regulation and providing key technological support for the ecological cultivation of medicinal herbs. Summary of the Invention

[0006] The purpose of this invention is to provide a biological regulator that meets the green production standards for Chinese medicinal materials, has a clear mechanism of action, and has a significant effect in controlling excessive growth, thereby solving the problem of excessive growth caused by nitrogen excess in the cultivation of Ophiopogon japonicus and simultaneously improving the yield of tubers, the content of effective ingredients, and the level of soil health.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a growth regulator for Ophiopogon japonicus based on carbon-based ecological fertilizer loaded with a compound enzyme preparation, comprising the following components in parts by weight: 80-95 parts of functionalized liquid carbon-based ecological fertilizer matrix, 3-15 parts of targeted compound enzyme preparation, 1-3 parts of enzyme stabilization and protection system, and 1-2 parts of cofactor activation solution. The functionalized liquid carbon-based ecological fertilizer matrix is ​​made from agricultural and forestry organic waste as raw material, through high-temperature and high-pressure Fenton oxidation treatment, mineral element chelation and compound microbial fermentation.

[0008] Preferably, the conditions for the high-temperature and high-pressure Fenton oxidation treatment are: temperature 170~180℃, pressure 0.8~1.2MPa, and reaction time 1.5~2.5h; the Fe²⁺ content in the Fenton reagent used is... + The concentration is 0.5~1.0 mmol / L, and the H2O2 concentration is 10~15 mmol / L.

[0009] Preferably, the agricultural and forestry organic waste is selected from one or more of straw, edible fungus residue, and soybean meal.

[0010] Preferably, the mineral element chelation process includes: adjusting the pH of the supernatant after high-temperature and high-pressure Fenton oxidation treatment to 5.0~5.5, adding FeSO4, ZnSO4, MnSO4, CuSO4 = 18~22 : 8~10 : 5~7 : 3~5, with a total metal ion concentration of 0.8~1.2 g / L, and chelating at 45~55℃ for 1.5~2.5 h.

[0011] Preferably, the compound microbial fermentation includes: introducing Bacillus subtilis, Aspergillus niger and white rot fungi into the raw material after mineral element chelation, fermenting at 45~50℃ for 4~6 days, and then anaerobic maturing at 30~35℃ for 6~8 days.

[0012] Preferably, the targeted complex enzyme preparation comprises the following components: glutamine synthase 30-40%, glutamate synthase 20-30%, nitrate reductase inhibitory regulatory peptide (Shanghai Qiangyao Biotechnology Co., Ltd.) 10-15%, peroxidase 10-20%, phenylalanine ammonia-lyase 5-10%; total enzyme activity ≥5000 U / g.

[0013] Preferably, the enzyme stabilization protection system is composed of trehalose and β-cyclodextrin mixed at a mass ratio of 1.5~2.5:1.

[0014] Preferably, the cofactor activating solution is Fe²⁺. + Zn² + Mn² + Cu² + An EDTA chelate solution with a ratio of 3~5:1~3:1~3:0.5~1.5 and a total concentration of 0.5~1.0 g / L.

[0015] This invention provides a method for preparing the Ophiopogon japonicus growth regulator, which involves mixing a targeted compound enzyme preparation with an enzyme stabilization and protection system, and then preparing microcapsules by spray freeze-drying; mixing the functionalized liquid carbon-based ecological fertilizer matrix, the microcapsules, and the cofactor activation liquid, and stirring at 35-45°C for 25-35 minutes to obtain the final product.

[0016] This invention provides the application of the aforementioned Ophiopogon japonicus growth regulator in inhibiting excessive growth of the aboveground parts of Ophiopogon japonicus, promoting tuber development, and increasing the content of saponins and polysaccharides in the tubers.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a growth regulator for Ophiopogon japonicus based on a carbon-based ecological fertilizer loaded with a compound enzyme preparation. Through a coupled high-temperature, high-pressure Fenton oxidation and compound microbial fermentation process, it achieves deep degradation and thorough sterilization of agricultural and forestry waste, while simultaneously passivating heavy metals. This ensures the product is free of pathogenic microorganisms and heavy metal contamination, meeting the safety requirements of GAP (Good Agricultural Practices) for traditional Chinese medicinal materials. This regulator precisely intervenes in nitrogen utilization in Ophiopogon japonicus at the metabolic source. Through the GS / GOGAT enzyme system accelerating nitrogen assimilation and NR inhibitory peptides blocking excessive nitrate accumulation, it achieves a metabolic growth control mechanism of "regulating nitrogen with carbon, controlling aboveground growth and promoting belowground growth," effectively inhibiting excessive vegetative growth of the aboveground parts, promoting tuber enlargement, and significantly increasing the content of medicinal components.

[0018] Meanwhile, the carbon-based carrier provides a rich organic carbon skeleton and small molecule active substances, activating the transport of photosynthetic products to the tuberous roots. It also synergistically enhances the synthesis of secondary metabolites such as saponins and polysaccharides by working with enzyme systems such as PAL and POD, thereby improving plant stress resistance and quality. All components are biodegradable, improving the rhizosphere microecological environment after application, exhibiting outstanding synergistic effects and eco-friendliness.

[0019] Field trial results show that the regulator of this invention can significantly increase the fresh weight and dry weight of Ophiopogon japonicus tubers, increase the total saponin content, increase the crude polysaccharide content, and has a significant effect on controlling excessive growth. It has multiple functions of increasing yield and improving quality and soil improvement, and is suitable for green, efficient and sustainable cultivation of Chinese medicinal materials. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 Images of harvested Ophiopogon japonicus fruits in the carbon-based fertilizer group and the control group.

[0022] Figure 2 A comparison chart of harvested Ophiopogon japonicus plants from carbon-based fertilizer and the control group.

[0023] Figure 3 This is a test report for Ophiopogon japonicus, a carbon-based fertilizer.

[0024] Figure 4 This is the test report for Ophiopogon japonicus in the control group. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1: Laboratory Preparation

[0027] This invention provides a growth regulator for Ophiopogon japonicus, comprising the following components in parts by weight: functionalized liquid carbon-based ecological fertilizer matrix: 90g; targeted compound enzyme preparation: 8g; enzyme stabilization and protection system: 1g; cofactor activating liquid: 1g.

[0028] Wherein: (1) the functionalized liquid carbon-based ecological fertilizer matrix is ​​a dark brown transparent liquid, which is prepared by the following four-stage process: ① High-temperature and high-pressure Fenton catalytic oxidation degradation: Take 500 g of straw, 300 g of mushroom residue, and 200 g of soybean meal, crush them, add 4 L of water, and add Fenton's reagent (Fe²⁺).+ 0.8 mmol / L, H2O2 12 mmol / L), reacted at 175℃ and autogenous pressure (1 MPa) for 2 hours. This process simultaneously achieves: deep cleavage of lignocellulose, generating water-soluble small peptides, oligosaccharides, and organic acids; complete inactivation of pathogenic microorganisms and heat-resistant spores; Fe²⁺ + / Fe³ + with Cd² + Pb² + As³ + Heavy metals precipitate, achieving in-situ passivation.

[0029] ②Active extraction and organic chelation of mineral elements: After cooling, adjust the pH to 5.2, centrifuge to remove residue and heavy metal precipitate; add FeSO4, ZnSO4, MnSO4, and CuSO4 (Fe:Zn:Mn:Cu=4:2:2:1; total metal ion concentration 1 g / L) to the supernatant, chelate at 50℃ for 2 h, so that the metal ions form a stable coordination structure with the organic functional groups (—COOH, —OH, —NH2).

[0030] ③ Directed fermentation by compound microorganisms: Inoculation with Bacillus subtilis (Bacillus subtilis) Aspergillus niger ( ) Aspergillus niger ), white rot fungi ( Phanerochaete chrysosporium (Live bacteria ratio 2:1:1, total ≥1×10) 8 (CFU / mL), aerobic fermentation at 50℃ for 5 days, followed by anaerobic ripening at 35℃ for 7 days, to enrich small molecule organic acids, growth-promoting metabolites and antioxidants.

[0031] The obtained matrix parameters are as follows: total organic carbon (TOC) ≥ 45 g / L; small molecule organic acids (acetic acid, propionic acid, citric acid) ≥ 8 g / L; water-soluble small peptides (<2000 Da) ≥ 5 g / L; pH 5.5~7.5; heavy metal content meets NY 525-2021 standard; no pathogenic bacteria detected.

[0032] (2) The targeted complex enzyme preparation is composed of the following enzymes in the following proportions of activity units and microencapsulated: glutamine synthase (GS, Beijing Solarbio Science & Technology Co., Ltd.): 30%; glutamate synthase (GOGAT): 25%; nitrate reductase (NR) inhibitory regulatory peptide: 15%; peroxidase (POD, Beijing Solarbio Science & Technology Co., Ltd.): 20%; phenylalanine ammonia-lyase (PAL): 10%; total enzyme activity ≥5000 U / g, particle size 50μm.

[0033] (3) The enzyme stabilization and protection system is a mixture of trehalose and β-cyclodextrin at a mass ratio of 2:1; (4) The cofactor activating solution is Fe²⁺+ Zn² + Mn² + Cu² + EDTA chelate solution (total concentration 1.0 g / L).

[0034] The specific preparation method includes the following steps: S1. Prepare functionalized liquid carbon-based ecological fertilizer substrate (as above); S2. The targeted complex enzyme preparation is mixed with an enzyme stabilization protection system and then spray-dried to obtain enzyme microcapsules; S3. Mix the functionalized liquid carbon-based ecological fertilizer matrix, the microcapsules and the cofactor activation liquid, and stir at 40°C for 30 minutes to obtain the liquid product.

[0035] Example 2: Pilot production (scaled up in a 500 L reactor)

[0036] This invention provides a growth regulator for Ophiopogon japonicus, comprising the following components in parts by weight: functionalized liquid carbon-based ecological fertilizer substrate: 90 kg; targeted compound enzyme preparation: 8 kg; enzyme stabilization and protection system: 1 kg; cofactor activating liquid: 1 kg.

[0037] Wherein: (1) the functionalized liquid carbon-based ecological fertilizer matrix is ​​a dark brown transparent liquid, which is prepared by the following four-stage process: ① High-temperature and high-pressure Fenton catalytic oxidation degradation: Take 500 kg of straw, 300 kg of mushroom residue, and 200 kg of soybean meal, crush them, add 4000 L of water, and add Fenton's reagent (Fe²⁺). + The reaction was carried out at 1 mmol / L (H₂O₂ 15 mmol / L) for 2 hours at 175℃ and autogenous pressure (1 MPa). This process simultaneously achieved: deep cleavage of lignocellulose, generating water-soluble small peptides, oligosaccharides, and organic acids; complete inactivation of pathogenic microorganisms and heat-resistant spores; and Fe²⁺… + / Fe³ + with Cd² + Pb² + As³ + Heavy metals precipitate, achieving in-situ passivation.

[0038] ②Active extraction and organic chelation of mineral elements: After cooling, adjust the pH to 5.5, centrifuge to remove residue and heavy metal precipitate; add FeSO4, ZnSO4, MnSO4, and CuSO4 (Fe:Zn:Mn:Cu=4:2:2:1; total metal ion concentration 1 g / L) to the supernatant, chelate at 50℃ for 2 h, so that the metal ions form a stable coordination structure with the organic functional groups (—COOH, —OH, —NH2).

[0039] ③ Directed fermentation by compound microorganisms: Inoculation with Bacillus subtilis, Aspergillus niger, and white rot fungi (live count ratio 2:1:1, total ≥1×10⁻⁶) 8 (CFU / mL), aerobic fermentation at 50℃ for 5 days, followed by anaerobic ripening at 35℃ for 7 days, to enrich small molecule organic acids, growth-promoting metabolites and antioxidants.

[0040] The obtained matrix parameters are as follows: total organic carbon (TOC) ≥ 48.5 g / L; small molecule organic acids (acetic acid, propionic acid, citric acid) ≥ 8 g / L; water-soluble small peptides (<2000 Da) ≥ 5.7 g / L; pH 5.5~7.5; heavy metal content meets NY 525-2021 standard; no pathogenic bacteria detected.

[0041] (2) The targeted complex enzyme preparation is composed of the following enzymes in the following proportions of activity units and microencapsulated: glutamine synthase (GS, Beijing Solarbio Science & Technology Co., Ltd.): 30%; glutamate synthase (GOGAT): 25%; nitrate reductase (NR) inhibitory regulatory peptide: 15%; peroxidase (POD, Beijing Solarbio Science & Technology Co., Ltd.): 20%; phenylalanine ammonia-lyase (PAL): 10%; total enzyme activity ≥5000 U / g, particle size 50μm.

[0042] (3) The enzyme stabilization and protection system is a mixture of trehalose and β-cyclodextrin at a mass ratio of 2:1; (4) The cofactor activating solution is Fe²⁺ + Zn² + Mn² + Cu² + EDTA chelate solution (total concentration 1 g / L).

[0043] The specific preparation method includes the following steps: S1. Prepare functionalized liquid carbon-based ecological fertilizer substrate (as above); S2. The targeted complex enzyme preparation is mixed with an enzyme stabilization protection system and then spray-dried to obtain enzyme microcapsules; S3. Mix the functionalized liquid carbon-based ecological fertilizer matrix, the microcapsules and the cofactor activating liquid, stir at 40°C for 30 minutes to obtain the liquid product, and then spray dry to make a wettable powder.

[0044] Example 3

[0045] This invention provides a growth regulator for Ophiopogon japonicus, comprising the following components in parts by weight: functionalized liquid carbon-based ecological fertilizer matrix: 90g; targeted compound enzyme preparation: 8g; enzyme stabilization and protection system: 1g; cofactor activating liquid: 1g.

[0046] Wherein: (1) the functionalized liquid carbon-based ecological fertilizer matrix is ​​a dark brown transparent liquid, which is prepared by the following four-stage process: ① High-temperature and high-pressure Fenton catalytic oxidation degradation: Take 500 g of straw, 300 g of mushroom residue, and 200 g of soybean meal, crush them, add 4 L of water, and add Fenton's reagent (Fe²⁺). + 0.8 mmol / L, H2O2 12 mmol / L), reacted at 175℃ and autogenous pressure (1 MPa) for 2 hours. This process simultaneously achieves: deep cleavage of lignocellulose, generating water-soluble small peptides, oligosaccharides, and organic acids; complete inactivation of pathogenic microorganisms and heat-resistant spores; Fe²⁺ + / Fe³ + with Cd² + Pb² + As³ + Heavy metals precipitate, achieving in-situ passivation.

[0047] ②Active extraction and organic chelation of mineral elements: After cooling, adjust the pH to 5.2, centrifuge to remove residue and heavy metal precipitate; add FeSO4, ZnSO4, MnSO4, and CuSO4 (Fe:Zn:Mn:Cu=4:2:2:1; total metal ion concentration 1 g / L) to the supernatant, chelate at 50℃ for 2 h, so that the metal ions form a stable coordination structure with the organic functional groups (—COOH, —OH, —NH2).

[0048] ③ Directed fermentation by compound microorganisms: Inoculation with Bacillus subtilis, Aspergillus niger, and white rot fungi (live count ratio 2:1:1, total ≥1×10⁻⁶) 8 CFU / mL), aerobic fermentation at 50℃ for 12 days.

[0049] The obtained matrix parameters are as follows: total organic carbon (TOC) ≥ 45 g / L; small molecule organic acids (acetic acid, propionic acid, citric acid) ≥ 8 g / L; water-soluble small peptides (<2000 Da) ≥ 5 g / L; pH 5.5~7.5; heavy metal content meets NY 525-2021 standard; no pathogenic bacteria detected.

[0050] (2) The targeted complex enzyme preparation is composed of the following enzymes in the following proportions of activity units and microencapsulated: glutamine synthase (GS, replaced by a product from Novozymes): 30%; glutamate synthase (GOGAT): 25%; nitrate reductase (NR) inhibitory regulatory peptide: 15%; peroxidase (POD, replaced by a product from Novozymes): 20%; phenylalanine ammonia-lyase (PAL): 10%; total enzyme activity ≥5000 U / g, particle size 50μm.

[0051] (3) The enzyme stabilization and protection system is a mixture of trehalose and β-cyclodextrin at a mass ratio of 2:1; (4) The cofactor activating solution is Fe²⁺ + Zn² + Mn² + Cu² + EDTA chelate solution (total concentration 1.0 g / L).

[0052] The specific preparation method includes the following steps: S1. Prepare functionalized liquid carbon-based ecological fertilizer substrate (as above); S2. The targeted complex enzyme preparation is mixed with an enzyme stabilization protection system and then spray-dried to obtain enzyme microcapsules; S3. Mix the functionalized liquid carbon-based ecological fertilizer matrix, the microcapsules and the cofactor activation liquid, and stir at 40°C for 30 minutes to obtain the liquid product (this method can reduce the cost by 20% and achieve 90% of the original product's growth control effect).

[0053] Example 4

[0054] (a) Application of base fertilizer (before transplanting, to cultivate native microorganisms)

[0055] 1. Application time: Apply 1-2 days before transplanting Ophiopogon japonicus, in conjunction with land preparation.

[0056] 2. Application rate: 20 kg / mu.

[0057] 3. Application method: Spread the liquid carbon-based fertilizer evenly on the planting plot, plow it to a depth of 15-20cm and mix it with the soil. Let it stand for 1-2 days to promote the reproduction of native soil microorganisms and lay the foundation for the recovery of seedlings and root growth after transplanting of Ophiopogon japonicus.

[0058] (ii) Topdressing (divided into 3 applications, totaling 80 kg / mu, can be applied by flood irrigation or drip irrigation, etc.)

[0059] 1. First top dressing (seedling stage, 15-20 days after transplanting): Apply 20 kg / mu of liquid fertilizer and irrigate with plenty of water to supplement nutrients and promote strong seedlings.

[0060] 2. Second topdressing (tillering stage, 40-45 days after transplanting): Apply 35 kg / mu (maximum topdressing amount), with heavy irrigation, taking into account both controlling excessive growth and promoting tuber differentiation.

[0061] 3. Third top dressing (tuberous root enlargement period, 70-75 days after transplanting): Apply 25 kg / mu, flood irrigation, to supplement nutrients, promote tuber enlargement and improve quality.

[0062] (III) Foliar fertilizer application (mid-stage of tuber enlargement)

[0063] 1. Application time: 10-15 days after the third top dressing.

[0064] 2. Method: Dilute the liquid carbon-based fertilizer 600 times, and spray 30-40 kg of the diluted solution per acre, spraying evenly on both sides of the leaves; spray in the evening on sunny days or on cloudy days, and re-spray if it rains within 4 hours.

[0065] Experiment Example 1: Effects of Liquid Carbon-Based Ecological Fertilizer on the Growth and Yield of Ophiopogon japonicus (Trial Design)

[0066] I. Experimental Objective

[0067] Through field comparative experiments, the comprehensive effects of the liquid carbon-based ecological fertilizer (rich in water-soluble small peptides, oligosaccharides, and chelated mineral elements) prepared in Example 2 of this invention on the planting of Ophiopogon japonicus were verified. These effects included promoting soil improvement, cultivating beneficial microorganisms, regulating excessive growth of aboveground parts, promoting tuber differentiation and enlargement, and improving yield and quality, providing a scientific basis for production application.

[0068] II. Materials and Methods

[0069] The experiment was conducted in Huayuan Town, Santai County, Mianyang City, Sichuan Province. The soil was purple soil with a pH of 6.5-7.0, deep soil layer, and good drainage, meeting the requirements for Ophiopogon japonicus cultivation. The tested Ophiopogon japonicus seedlings were robust, free from pests and diseases, and uniform in size.

[0070] The tested fertilizer was the liquid carbon-based ecological fertilizer prepared in Example 2, while the control group used commercially available ordinary compound fertilizer (total nutrients ≥45%). The experiment consisted of two treatments: a treatment group (liquid carbon-based ecological fertilizer) and a control group (conventional compound fertilizer). Each treatment group had two replicate plots, for a total of four plots, arranged in a randomized block design. Each plot covered 1 acre, with a 50cm buffer zone between plots to prevent cross-contamination of fertilizers. The planting density was 100,000 plants per acre, using row sowing and transplanting with a row spacing of 15cm and a plant spacing of 4.5cm.

[0071] III. Fertilization Plan

[0072] The total fertilizer application rate for the treatment group was 100 kg / mu, including 20 kg / mu of basal fertilizer and 80 kg / mu of topdressing. A 600-fold diluted foliar fertilizer was also sprayed once during the mid-stage of tuber enlargement. Topdressing was applied in three stages: 20 kg / mu during the seedling stage (15-20 days after transplanting), 35 kg / mu during the tillering stage (40-45 days after transplanting), and 25 kg / mu during the tuber enlargement stage (70-75 days after transplanting). The total fertilizer application rate for the control group was 80 kg / mu, including 20 kg / mu of basal fertilizer and 60 kg / mu of topdressing, applied in three stages (at the same times as the treatment group), without foliar fertilizer. Apart from fertilization, field management (watering, weeding, pest and disease control, etc.) remained consistent between the two groups.

[0073] IV. Planting Plan for Treatment Groups

[0074] 1. Preparation before planting: Deep plow the land to a depth of 20-25cm, remove weeds and stones, and level the land to make beds 1.2m wide. 1-2 days before transplanting, evenly spread 20kg / mu of liquid carbon-based ecological fertilizer on the bed surface, plow to a depth of 15-20cm to mix with the soil, and let it stand for 1-2 days to activate the native microorganisms.

[0075] 2. Transplanting and planting: Transplanting should be carried out in spring, from March to April, on cloudy days or in the evening when the temperature is 15-25℃. Plant the seedlings on the raised bed at a density of 100,000 plants per acre, at a depth of 5-8cm, lightly press the soil and water thoroughly.

[0076] 3. Topdressing Management: Topdressing is carried out using flood irrigation, in three stages according to the above plan. Topdressing during the seedling stage promotes seedling establishment; topdressing during the tillering stage (maximum amount) aims to control excessive growth and promote underground tuber differentiation; topdressing during the tuber enlargement stage promotes tuber enlargement.

[0077] 4. Foliar fertilization: 10-15 days after the third topdressing (mid-stage of tuber enlargement), dilute liquid carbon-based fertilizer 600 times and spray 35 kg per acre. Spray evenly on both sides of the leaves. Choose a sunny evening or a cloudy day for spraying. If it rains, re-spray.

[0078] 5. Water management: Keep the soil moist after transplanting, drain water promptly after irrigation during the growing season, and take precautions against waterlogging during the rainy season.

[0079] 6. Weed and pest control: Manual weeding should be carried out regularly, with agricultural control methods given priority. Low-toxicity biological pesticides should be used when necessary.

[0080] V. Control Group Planting Program

[0081] The control group received 20 kg / mu of ordinary compound fertilizer (15-15-15) as base fertilizer, which was tilled and mixed before transplanting. Topdressing was applied in three applications, totaling 60 kg / mu, at the same time as the treatment group, using conventional broadcasting or furrow application. Foliar spraying was not performed. Other field management (watering, weeding, and pest and disease control) was the same as the treatment group.

[0082] VI. Measurement Indicators and Methods

[0083] At harvest time (180-200 days after transplanting, when the leaves turn yellow), 15 plants of *Ophiopogon japonicus* were randomly selected from each plot. Soil and fibrous roots were removed, and fresh weight was measured. The fresh samples were then blanched at 105℃ for 30 minutes and dried at 80℃ to constant weight, and the dry weight was measured. Each plot was measured twice, and the average value was taken. The dried samples were sent for testing to determine the content of effective components such as saponins and polysaccharides. Data from each replicate were recorded, and the mean and standard deviation were calculated. A t-test or analysis of variance was used to compare the significance of differences between the two groups.

[0084] VII. Experimental Implementation Steps

[0085] Preliminary preparations: complete site preparation, basic soil physicochemical property testing, and seedling selection.

[0086] Base fertilizer application and transplanting: Apply base fertilizer according to groups, and transplant uniformly after allowing to stand.

[0087] Growing season management: Implement topdressing, foliar spraying, water and pest and weed management according to the plan, and keep detailed records.

[0088] Sample collection and testing: Samples are collected during the harvest period for fresh weight and dry weight determination and quality testing.

[0089] Data processing and analysis: Summarize the data, conduct statistical analysis, and write the experimental report.

[0090] VIII. Precautions

[0091] Strictly manage the isolation zones between cells to prevent cross-contamination of fertilizer and water. Perform all management operations synchronously as much as possible to minimize time errors. Process samples promptly after collection to avoid water loss affecting measurement results.

[0092] Table 1 Comparison of Fresh Weight of Ophiopogon japonicus from Mianyang

[0093] Table 2 Comparison of Dry Weight of Ophiopogon japonicus from Mianyang

[0094] This experiment used the planting of Ophiopogon japonicus in Huayuan Town, Santai County, Mianyang as the core scenario, strictly adhering to a planting density of 100,000 plants per mu. Two comparative units were set up: a conventional planting control group (CK) and a liquid carbon-based ecological fertilizer treatment group. Yield measurements were conducted at harvest time. Fifteen Ophiopogon japonicus plants from each group were randomly selected as samples, and fresh weight and dry weight were measured twice. For the control group (CK), the first fresh weight measurement was 192.21g, and the second was 146.00g, with corresponding dry weights of 64.71g and 49.64g. For the carbon-based fertilizer treatment group, the first fresh weight was 252.19g, and the second was 245.79g, with dry weights of 86.07g and 78.98g.

[0095] Calculations showed that the average fresh weight of the 15 *Ophiopogon japonicus* plants in the control group was 169.11g and the average dry weight was 57.18g, while the average fresh weight of the carbon-based fertilizer treatment group reached 248.99g and the average dry weight was 82.53g. The data from repeated measurements of the two groups showed minimal deviation, indicating good experimental repeatability and strong data reliability. Based on a planting density of 100,000 plants per mu (approximately 667 square meters), the yield per mu (approximately 0.067 hectares) in the carbon-based fertilizer treatment group was approximately 1659.93 kg / mu (approximately 72.53 kg / mu), an increase of 47.23% compared to the control group's 1127.4 kg / mu (approximately 72.53 kg / mu); the dry weight yield per mu was approximately 550.2 kg / mu (approximately 72.53 kg / mu), an increase of 44.33% compared to the control group's 381.2 kg / mu (approximately 14.33 kg / mu). Comprehensive analysis shows that, under the purple soil planting environment of Huayuan Town, Santai County, Mianyang, the application of liquid carbon-based ecological fertilizer can significantly promote the growth and development of Ophiopogon japonicus tubers by cultivating native soil microorganisms, improving soil physicochemical properties, and synergistically controlling excessive growth and promoting tuber development. This not only greatly increases the fresh and dry weight yield of Ophiopogon japonicus, but also effectively improves the economic benefits of planting. It provides an efficient and feasible technical path for the large-scale planting of Ophiopogon japonicus in the local area, and verifies the application value and promotion potential of liquid carbon-based ecological fertilizer in Ophiopogon japonicus planting.

[0096] Depend on Figure 1 It can be seen that the basal fertilizer treatment group (left image) has more tubers, which are fuller and thicker, and the overall volume and weight are significantly greater than those of the control group. The tubers are white in color, uniform in shape, and have fewer fibrous roots, showing a better tuber development state. The control group (right image) has fewer tubers, which are thinner and smaller, with a relatively slender shape. Some tubers are shriveled, and the overall growth is weaker than that of the carbon-based fertilizer treatment group.

[0097] Combined with yield data, this visual difference directly corresponds to the quantitative results: the average fresh weight of the 15 Ophiopogon japonicus plants in the carbon-based fertilizer treatment group was 248.99g and the dry weight was 82.53g, which increased by 47.23% and 44.33% respectively compared with the control group. This fully demonstrates that liquid carbon-based ecological fertilizer can effectively promote the enlargement of Ophiopogon japonicus tubers and increase the yield per plant, while inhibiting excessive growth of the above-ground parts and allowing more nutrients to accumulate in the underground tubers, ultimately achieving a simultaneous improvement in yield and quality. It also provides intuitive physical support for the yield value per mu calculation mentioned above.

[0098] Figure 2 The morphology of the whole plant of Ophiopogon japonicus after harvest was shown in the control group (CK, conventional planting) and the carbon-based fertilizer treatment group, which intuitively presented the significant effect of liquid carbon-based ecological fertilizer on the root development and plant growth of Ophiopogon japonicus.

[0099] ① Comparison of morphological features

[0100] Control group (above): Above-ground parts: leaves are relatively sparse, plant growth is weak, and plant height is slightly low.

[0101] Underground part: There are fewer tubers, and the individual tubers are relatively thin and long. The overall shape is shriveled, and the distinction between tubers and fibrous roots is low. The tubers are not plump enough.

[0102] Carbon-based fertilizer treatment group (see below): Above-ground parts: The leaves are denser and stronger, and the plant growth is significantly better than the control group, demonstrating a stronger nutrient supply and growth control effect.

[0103] Underground part: The number of tubers increased significantly, the individual tubers were plump and robust, spindle-shaped, the boundary between tubers and fibrous roots was clear, and the overall weight and density were significantly higher than the control group, indicating more complete tuber development.

[0104] ② Optimization of nutrient distribution: The control group showed weaker above-ground growth and smaller underground tubers, reflecting low efficiency in nutrient allocation to the underground tubers. In contrast, the carbon-based fertilizer group suppressed ineffective above-ground growth through its growth-regulating effect, promoting the accumulation of more nutrients in the underground tubers, ultimately resulting in a state of "lush foliage and strong roots".

[0105] ③ Core Conclusions

[0106] This image, through a direct comparison of physical samples, quantitatively verifies the application effect of liquid carbon-based ecological fertilizer in Ophiopogon japonicus cultivation: Promoting tuber development: Significantly increasing the number of Ophiopogon japonicus tubers, improving tuber plumpness and individual plant weight, is the core reason for the increased yield.

[0107] Optimize plant growth: ensure robust growth of the above-ground parts while inhibiting ineffective excessive growth, achieving growth regulation of "controlling the top and promoting the bottom".

[0108] Figure 3 and Figure 4 These two test reports (Nos. N202404001 and N202404002) correspond to the quality test results of the control group (CK) and the carbon-based fertilizer treatment group in the Ophiopogon japonicus planting experiment in Huayuan Town, Santai County, Mianyang. The core focus is on pesticide residues, active ingredients, and heavy metal indicators. The comparison visually demonstrates the optimizing effect of liquid carbon-based ecological fertilizer on the quality of Ophiopogon japonicus. The specific analysis is as follows: Table 3 Comparison of Core Indicators and Key Conclusions

[0109] 1. Detailed analysis of key dimensions

[0110] (1) Pesticide residue indicators: Safety significantly improved

[0111] Paclobutrazol, a commonly used plant growth regulator in Ophiopogon japonicus cultivation, had a residue level of 1.09 mg / kg in the control group, while the carbon-based fertilizer group showed a reduction to 0.37 mg / kg, a decrease of over 66%. Uniconazole was undetectable in both groups, meeting the food safety standards of GB 23200.121-2021. This difference stems from the fact that carbon-based fertilizer improves soil microecology and enhances plant resistance, reducing the need for pesticide application and residue accumulation, thus ensuring the medicinal safety of Ophiopogon japonicus from the source.

[0112] (2) Effective ingredient indicators: The efficacy and quality of the medicine have been greatly improved.

[0113] The core medicinal components of Ophiopogon japonicus, total saponins and crude polysaccharides, are the core standards for measuring its quality. The total saponins in the carbon-based fertilizer group of Ophiopogon japonicus reached 19.5%, which is 20.5% higher than the 16.1% in the control group, far exceeding the pharmacopoeia requirements; The crude polysaccharide content was 16.23 g / 100 g, which was 27.3% higher than that of the control group (12.75 g / 100 g). The extract content was 88.6%, an increase of 8.4% compared to the control group's 81.7%, reflecting a higher dissolution efficiency of the active ingredients. This indicates that the liquid carbon-based ecological fertilizer not only did not affect the efficacy, but also promoted the synthesis and accumulation of the active ingredients of Ophiopogon japonicus by optimizing nutrient supply (chelated mineral elements and water-soluble carbon sources), thereby enhancing its medicinal value.

[0114] (3) Heavy metal indicators: Simultaneous optimization of soil and product safety

[0115] According to the GB 5009 series of food safety standards, the levels of copper, arsenic, cadmium, mercury, and lead in both groups met the standards, but the carbon-based fertilizer group showed significantly lower levels of all heavy metals compared to the control group. The lead content decreased from 0.34 mg / kg to 0.13 mg / kg, a reduction of 61.8%. The arsenic content decreased from 0.089 mg / kg to 0.049 mg / kg, a reduction of 44.9%. The copper content decreased from 2.6 mg / kg to 1.7 mg / kg, a reduction of 34.6%. This confirms the soil-improving effect of carbon-based fertilizers—by passivating heavy metals in situ and improving soil physicochemical properties, it reduces the absorption and accumulation of heavy metals by Ophiopogon japonicus, thus enhancing the ecological safety of the product from the source of planting.

[0116] In summary, the carbon-based fertilizer treatment group achieved a yield increase of over 40% while simultaneously reducing pesticide residues, increasing effective components, and optimizing heavy metal content, thus achieving the triple goals of "high yield + high quality + safety." The liquid carbon-based ecological fertilizer, through a synergistic mechanism of "cultivating beneficial bacteria and improving soil, controlling excessive growth and promoting root development, and enhancing efficiency and quality," optimized the growth environment of Ophiopogon japonicus and improved the plant's own metabolic capacity, making it a core technology for achieving a dual improvement in the quality and yield of Ophiopogon japonicus. These test results fully verify the application value of carbon-based fertilizer in the large-scale cultivation of Ophiopogon japonicus, providing reliable technical support for improving the quality and efficiency of the local Ophiopogon japonicus industry.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A growth regulator for Ophiopogon japonicus based on a carbon-based ecological fertilizer loaded with a compound enzyme preparation, characterized in that, It comprises the following components in parts by weight: 80-95 parts of functionalized liquid carbon-based ecological fertilizer matrix, 3-15 parts of targeted compound enzyme preparation, 1-3 parts of enzyme stabilization and protection system, and 1-2 parts of cofactor activation solution. The functionalized liquid carbon-based ecological fertilizer matrix is ​​made from agricultural and forestry organic waste as raw material, through high-temperature and high-pressure Fenton oxidation treatment, mineral element chelation and compound microbial fermentation.

2. The ophiopogon japonicus growth regulator according to claim 1, characterized in that, The conditions for the high-temperature and high-pressure Fenton oxidation treatment are: temperature 170~180℃, pressure 0.8~1.2 MPa, and reaction time 1.5~2.5 h; the Fe²⁺ content in the Fenton reagent used is... + The concentration is 0.5~1.0 mmol / L, and the H2O2 concentration is 10~15 mmol / L.

3. The ophiopogon japonicus growth regulator according to claim 1, characterized in that, The agricultural and forestry organic waste is selected from one or more of straw, edible fungus residue, and soybean meal.

4. The ophiopogon japonicus growth regulator according to claim 1, characterized in that, The mineral element chelation process includes: adjusting the pH of the supernatant after high-temperature and high-pressure Fenton oxidation treatment to 5.0~5.5, adding FeSO4, ZnSO4, MnSO4, CuSO4 = 18~22 : 8~10 : 5~7 : 3~5, with a total metal ion concentration of 0.8~1.2 g / L, and chelating at 45~55℃ for 1.5~2.5 h.

5. The ophiopogon japonicus growth regulator according to claim 1, characterized in that, The compound microbial fermentation includes: introducing Bacillus subtilis, Aspergillus niger and white rot fungi into the raw material after mineral element chelation, fermenting at 45~50℃ for 4~6 days, and then anaerobic maturing at 30~35℃ for 6~8 days.

6. The ophiopogon japonicus growth regulator according to claim 1, characterized in that, The targeted complex enzyme preparation comprises the following components: glutamine synthase 30-40%, glutamate synthase 20-30%, nitrate reductase inhibitory regulatory peptide 10-15%, peroxidase 10-20%, phenylalanine ammonia-lyase 5-10%; total enzyme activity ≥5000 U / g.

7. The ophiopogon japonicus growth regulator according to claim 1, characterized in that, The enzyme stabilization and protection system is composed of trehalose and β-cyclodextrin mixed at a mass ratio of 1.5~2.5:

1.

8. The ophiopogon japonicus growth regulator according to claim 1, characterized in that, The cofactor activating solution is Fe²⁺ + Zn² + Mn² + Cu² + An EDTA chelate solution with a ratio of 3~5:1~3:1~3:0.5~1.5 and a total concentration of 0.5~1.0 g / L.

9. A method for preparing the Ophiopogon japonicus growth regulator as described in any one of claims 1 to 8, characterized in that, The targeted compound enzyme preparation is mixed with an enzyme stabilization and protection system and then spray-dried to form microcapsules; the functionalized liquid carbon-based ecological fertilizer matrix, the microcapsules, and the cofactor activation liquid are mixed and stirred at 35~45℃ for 25~35 minutes to obtain the final product.

10. The application of the ophiopogon japonicus growth regulator as described in any one of claims 1 to 8 in inhibiting excessive growth of the aboveground parts of ophiopogon japonicus, promoting tuber development, and increasing the content of saponins and polysaccharides in the tubers.

Images