Preparation method and application of a biomass source plant growth stimulant

Abstract

The application discloses a preparation method and application of a micro-aerobic subcritical treatment for extracting flavonoid plant growth stimulants. A micro-aerobic subcritical weak oxidation environment is constructed to extract high-activity plant growth-promoting flavonoid components in a directional manner, and three coupled reactions of macromolecule degradation, toxin oxidation self-detoxification and flavonoid hydroxyl functional group activation are completed. Residual inhibitors are removed through deep detoxification, and flavonoid phenolic hydroxyl groups are modified through enzymatic hydrolysis, so that the adsorption and transmembrane absorption capacity of roots are promoted. Finally, a plant growth stimulant mainly containing flavonoids is obtained through multi-element chelation and stabilization. Through micro-aerobic precise oxygen control and subcritical treatment, the flavonoids are extracted and enriched in a directional manner. The content of total flavonoids in the product is greater than or equal to 4.5 g / L, which is more than 40% higher than that of a traditional subcritical process. The fresh weight is increased by more than 20% and the total root length is increased by more than 50% in plant growth promotion.

Description

Technical Field

[0001] This invention belongs to the field of plant growth stimulant extraction, specifically relating to a method for preparing and applying a microaerobic subcritical extraction method for flavonoid plant growth stimulants. Background Technology

[0002] Flavonoids are a class of polyphenolic active substances with strong root-promoting, antioxidant, and stress-resistant regulatory functions. They can regulate plant auxin signaling pathways, promote the division of root meristem cells, and enhance the activity of endogenous antioxidant enzymes in roots. They are the main components of high-end plant growth-promoting biostimulants. The current industrial preparation of plant growth-promoting flavonoid products mainly faces the following technical challenges. (1) The raw materials are densely fibrous, and the flavonoid precursors are severely encapsulated, resulting in a very low extraction rate. The lignin cellulose of raw materials such as agricultural and forestry straw and livestock and poultry manure forms a dense network structure, and a large amount of bound flavonoids are encapsulated by fiber bundles. Traditional crushing and ethanol soaking can only slightly damage the surface structure, and the internal flavonoid precursors are difficult to release. Traditional single anaerobic subcritical hydrothermal treatment relies only on high temperature to break biomass macromolecules, without strong physical pre-wall breaking methods. The lignin-bound flavonoid precursors in the raw materials cannot be effectively released, the total flavonoid content in the extract is low, and the plant growth-promoting effect of the product is weak. (2) The system is prone to generating inhibitory impurities, which interfere with rooting activity. Agricultural, forestry and livestock waste contains free tannins, toxic alkaloids and free phenols, which cannot be degraded by conventional extraction. The residual toxins will inhibit the growth of the root meristematic zone and counteract the growth-promoting effect of flavonoids. (3) The hydroxyl sites of naturally extracted flavonoid molecules are not sufficiently exposed, resulting in low absorption and penetration efficiency of crop roots. Ordinary complex enzymes randomly degrade flavonoid molecules, destroying the growth-promoting sites, and cannot achieve directional modification and enhancement of functional groups. (4) Most existing biomass extraction methods use completely closed, oxygen-free systems.

[0003] Therefore, it is necessary to develop a method for extracting plant growth stimulants under micro-oxygen conditions. Summary of the Invention

[0004] To address the problems of expensive raw materials, easy degradation of flavonoid activity, poor active components, highly destructive extraction processes, toxic byproducts in extracts, and low efficacy of finished products in existing biostimulants, this invention provides a method for preparing plant growth stimulants by extracting flavonoids through microaerobic subcritical treatment.

[0005] This invention is achieved through the following technical solution: A method for preparing a plant growth stimulant by extracting flavonoids through microaerobic subcritical treatment includes the following steps: (1) Pretreatment of compound biomass Mix 25-35 parts of livestock and poultry manure (dry matter) and 65-75 parts of plant straw (dry matter) evenly by weight. The raw materials are then removed of impurities, crushed, passed through a 20-40 mesh standard sieve, and dried in a 60-65℃ hot air oven until the moisture content is 10%-15%.

[0006] The dried, coarsely crushed material is fed into a shredding and puffing device for two-stage physical cell wall breaking treatment: 1) Shredding and kneading stage: The equipment uses rollers to shred at a speed of 300–400 r / min for 12–18 min, mechanically tearing and breaking up the dense biomass clumps, and initially disintegrating the interwoven structure of lignocellulose; 2) Instantaneous puffing stage: The shredded material is fed into a differential pressure drying puffing machine at a pressure of 0.2–0.5 MPa. After holding the pressure for 30–60 s, the pressure is released instantaneously, relying on the instantaneous pressure difference to tear the plant cell walls and open up the internal cavities of the fibers, fully exposing the lignin-flavonoid conjugated binding sites encased within the fibers. After puffing, the material is naturally cooled to room temperature by ventilation, without water washing or organic solvent residue, and the solid material is directly fed into the subsequent subcritical hydrothermal process.

[0007] (2) Microaerobic subcritical treatment Solid materials and deionized water were added to a closed subcritical reactor equipped with online dissolved oxygen monitoring at a solid-liquid ratio of 1:10. A micro-aerobic, weakly oxidizing environment was maintained throughout the process. The reaction was carried out in three stages with gradient heating, pressurization, and precise oxygen control to extract flavonoids: 1) First stage: Temperature 120–155℃, system pressure 100–400 kPa, dissolved oxygen controlled at 0.05–0.1 mg / L, held for 10–15 min; a mild, loose hemicellulose network structure was observed, exposing lignin-flavonoid conjugated binding sites. 2) Second stage: Temperature increased to 160–175℃, system pressure 450–800 kPa, dissolved oxygen maintained at 0.15–0.2 mg / L, held for 15–20 min; trace amounts of oxygen selectively oxidized and broke lignin side chains, releasing free growth-promoting flavonoids, while simultaneously oxidizing and degrading tannins, free phenols, alkaloids, and other root-inhibiting substances. 3) Third stage: Heat to 180-220℃, system pressure 850-1000 kPa, maintain in the slightly aerobic dissolved oxygen range, and hold for 10-20 min; degrade macromolecular impurities and purify free flavonoid components in the liquid phase. After the hydrothermal reaction, rapidly cool to room temperature with water, remove solid residue by plate and frame filter press, and then filter through a 0.22 μm precision ultrafiltration membrane, retaining the liquid phase to obtain an activated crude extract rich in flavonoids.

[0008] (3) Targeted detoxification of microorganisms A compound detoxifying microbial agent was prepared: Bacillus subtilis (≥100 billion CFU / g), Pseudomonas aeruginosa (≥2 billion CFU / g), and Aspergillus niger (≥2 billion CFU / g) were mixed at a viable count ratio of 3:2:1, with a total viable count ≥500 billion CFU / g. 0.5%–1.5% (w / w) of the compound agent was added to the crude extract, and the pH was adjusted to 6.5–7.2. The mixture was then cultured in a constant-temperature shaker at 25–35℃ and 150 r / min for 48–72 h of aerobic fermentation. After fermentation, the bacteria were inactivated in an 85℃ water bath for 30 min, and the mixture was then filtered again through a 0.22 μm ultrafiltration membrane to obtain a detoxified and purified extract. In this step, the microorganisms can directionally degrade harmful compounds in the crude extract, such as free tannins, toxic alkaloids, and volatile phenols, which inhibit crop growth, while simultaneously degrading trace macromolecular impurities, thus initially optimizing the purity of small-molecule active components.

[0009] (4) Enzymatic oxidation targeted modification After detoxification, the extract was subjected to two-stage pH adjustment and stepwise addition of a complex enzyme to target and modify the active sites of flavonoid phenolic hydroxyl groups. Specifically: 1) First stage acidic pretreatment: Adjust the pH of the system to 5.0-5.3, add pectinase and glucose oxidase complex enzyme preparation, pectinase with an enzyme activity of ≥10000 U / g and glucose oxidase with an enzyme activity of ≥5000 U / g, the mass ratio of pectinase to glucose oxidase is 1.5-2:1, and the dosage of pectinase and glucose oxidase complex enzyme preparation is 0.1%-0.30% of the mass of detoxification solution, stir and react at 40-45℃ for 1.5 h; break the heteropolysaccharide branches in the system, release the polysaccharide molecules from the flavonoids, and fully expose the flavonoid phenolic hydroxyl groups.

[0010] 2) Second stage: Slightly acidic site-directed modification: Adjust the system pH to 5.5–5.8, add laccase (enzyme activity ≥10000 U / g), the dosage of laccase is 0.10%–0.25% of the detoxification solution mass, and react at a constant temperature of 40–45℃ with stirring for 2 h; laccase directionally catalyzes the mild oxidation modification of flavonoid phenolic hydroxyl groups, improving the water solubility of flavonoids and the transmembrane absorption efficiency of crop roots, and enhancing the meristematic activity of plants. The total enzymatic hydrolysis time is 2.5–3.5 h. After the reaction is completed, raise the temperature to 90℃ and hold for 15 min to completely inactivate the enzyme activity and prevent excessive enzyme degradation that would destroy the flavonoid activity.

[0011] (5) Finished product preparation The preparation and filling of finished products are used to manufacture biostimulant products. Specifically: (1) The enzyme-modified flavonoid stock solution was transferred into a vacuum concentration device and concentrated at a vacuum degree of -0.07 to -0.09 MPa and a low temperature of 50 to 55℃ until the total flavonoid content of the concentrate was ≥4.5 g / L.

[0012] (2) Add trace elements and additives according to the mass percentage of the concentrate: EDTA-Zn 0.1-0.2%, EDTA-Fe 0.05-0.15%, EDTA-Mg 0.1-0.2%, sodium carboxymethyl cellulose 0.1-0.3%, sodium benzoate 0.05-0.1%.

[0013] (3) Stir at room temperature and low speed until uniform, seal and let stand for 24 hours to complete the stable storage of active components; aseptically fill to obtain a biomass source plant growth stimulant with flavonoids as the main active substances.

[0014] Beneficial effects This invention uses inexpensive agricultural waste as raw material, employs microaerobic subcritical treatment, removes harmful inhibitory components through microbial methods, then modifies the functional group structure through enzyme-catalyzed oxidation to enhance biological activity, and finally concentrates it to produce a biomass-based plant growth stimulant rich in flavonoids. Attached Figure Description

[0015] Figure 1 This is a flowchart of the operation process of the present invention; Figure 2 The effect of the biostimulant of the present invention on the growth of pea seedlings; Figure 3 The effect of the biostimulant of the present invention on the total root length of pea seedlings; Figure 4 The effect of the biostimulant of the present invention on the root length of pea seedlings; Figure 5 The effect of the biostimulant of the present invention on the number of root tips in pea seedlings; Figure 6 The effect of the biostimulant of the present invention on the fresh weight of pea shoots; Figure 7 The effect of the biostimulant of the present invention on the number of root nodules in pea seedlings; Figure 8 The effect of the biostimulant of the present invention on the number of branches in pea seedlings. Detailed Implementation

[0016] The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0017] All raw materials used in this invention are commercially available conventional industrial products and agricultural and forestry by-products, which can be purchased in bulk by domestic agricultural input, biochemical reagent, and biological agent companies. The source information of the materials and instruments used in the specific implementation is as follows: Organic substrate for agriculture and forestry: Chicken manure was purchased from Qingdao Kanglilai Biotechnology Co., Ltd.; tomato straw came from the vegetable planting base in Cuijiaji Town, Pingdu City, Qingdao.

[0018] Bacillus subtilis, Pseudomonas, and Aspergillus niger were purchased from Qingdao Weilan Biotechnology Co., Ltd.; pectinase, glucose oxidase, and laccase were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and EDTA-Zn, EDTA-Fe, EDTA-Mg, sodium carboxymethyl cellulose, and sodium benzoate were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0019] The composite biomass pretreatment equipment uses the Yufeida 9RS-10 straw shredder; the extruder uses the Zhongfa Machinery ZF-150 vacuum differential pressure drying extruder; the subcritical reactor uses the Nanjing Zhengxin Instrument Co., Ltd. K-PSA-100 micro intelligent reactor; the vacuum concentrator uses the Changshu Chunlai Machinery Co., Ltd. DTS50-500 vacuum concentrator; and the plant root analyzer uses the Shandong Huode Electronic Technology Co., Ltd. HED-GX02.

[0020] Example 1 1. Compound biomass pretreatment: 25 parts chicken manure (dry matter) and 75 parts tomato straw (dry matter) are mixed evenly, pulverized and passed through a 30-mesh sieve, and dried at 60℃ to a moisture content of 10%. Shredding and puffing pretreatment: Shredding at 350 r / min for 15 min, then feeding the shredded material into a differential pressure drying puffing machine. The differential pressure puffing pressure is 0.4 MPa, held for 50 s, then instantly released for puffing, followed by ventilation and cooling. 2. Micro-aerobic gradient hydrothermal treatment: Solid materials and deionized water are added to a closed subcritical reactor equipped with online dissolved oxygen monitoring at a solid-liquid ratio of 1:10. The micro-aerobic, weakly oxidizing environment is maintained throughout the process. The reaction is carried out in three stages with gradient heating, pressurization, and precise oxygen control. The first stage is 150℃, 400kPa, and dissolved oxygen 0.1mg / L, held for 15 min; the second stage is 175℃, 750kPa, and dissolved oxygen 0.15mg / L, held for 20 min; the third stage is 185℃, 850 kPa, held for 20 min. After cooling, the crude extract is obtained by filtration through a 0.22 μm precision ultrafiltration membrane. 3. Microbial detoxification: Prepare a compound detoxification agent: Bacillus subtilis, Pseudomonas, and Aspergillus niger are compounded at a viable count ratio of 3:2:1, with a total viable count ≥50 billion CFU / g; add 1% (w / w) of the compound agent to the crude extract, adjust the pH of the system to 6.5, and culture in a constant temperature shaker at 30℃ and 150 r / min for 72 h of aerobic fermentation; after the fermentation is completed, inactivate the bacteria in an 85℃ water bath for 30 min, and then filter again through a 0.22 μm ultrafiltration membrane to obtain the detoxified solution.

[0021] 4. Enzymatic oxidation targeted modification: (1) First stage acidic pretreatment: Adjust the pH of the system to 5.0, add 0.2% of pectinase and glucose oxidase compound enzyme preparation (pectinase and glucose oxidase mass ratio is 2:1, pectinase, enzyme activity ≥10000 U / g, glucose oxidase, enzyme activity ≥5000 U / g) to the detoxified and purified extract, stir at 40℃ for 1.5 h; (2) Second stage site-specific modification: Adjust the pH of the system to 5.58, add laccase (enzyme activity ≥10000 U / g), the amount of laccase added is 0.15% of the detoxified liquid mass, stir at 40℃ for 2 h; the total enzymatic hydrolysis time is 3.5 h, after the reaction is completed, raise the temperature to 90℃ and keep warm for 15 min to inactivate the enzyme activity.

[0022] 5. Finished product preparation: The enzyme-treated liquid is transferred to a vacuum concentration device and concentrated at -0.08MPa and 50℃ until the flavonoid content is 4.5g / L. 0.2% EDTA-Zn, 0.15% EDTA-Fe, 0.10% EDTA-Mg, 0.3% sodium carboxymethyl cellulose, and 0.1% sodium benzoate are added according to the mass of the concentrate. The mixture is stirred evenly, allowed to stand at room temperature for 24 hours, and then aseptically filled to obtain 1L of finished biomass-derived plant growth stimulant.

[0023] Table 1 shows the characteristic parameters of the biomass-derived plant growth stimulant in Example 1. Comparative Example 1: Subcritical Hydrothermal Treatment Without Microaerobic Effects All parameters for raw material pretreatment, microbial detoxification, enzymatic oxidation targeted modification, and concentrated finished product preparation are exactly the same as in the previous example, except that trace oxygen supplementation in the system is cancelled, nitrogen replacement is used throughout the reaction vessel to maintain an oxygen-free environment, and the temperature and pressure parameters in the three stages remain unchanged.

[0024] Test and pot experiment results: (1) The total flavonoid content was 2.0 g / L, which is 55% lower than that of the embodiments of the present invention; (2) There is no weak oxidation self-detoxification in the hydrothermal stage, and the tannin residue in the crude extract is 2.5 times that of the present invention.

[0025] Table 2 shows the characteristic parameters of the biomass-derived plant growth stimulant in Comparative Example 1. Example 2 1. Compound biomass pretreatment: Mix 30 parts chicken manure (dry matter) and 70 parts tomato straw (dry matter) evenly, crush and pass through a 20-mesh sieve, and dry at 60℃ to a moisture content of 10%. Shredding and puffing pretreatment: Shred at 400 r / min for 18 min, then feed the shredded material into a differential pressure drying puffing machine. The differential pressure puffing pressure is 0.5 MPa, and after holding the pressure for 60 s, the pressure is instantly released for puffing, followed by ventilation and cooling. 2. Microaerobic gradient hydrothermal treatment: Solid materials and deionized water are added to a closed subcritical reactor equipped with online dissolved oxygen monitoring at a solid-liquid ratio of 1:10. The microaerobic and weakly oxidizing environment is maintained throughout the process. The reaction is carried out in three stages with gradient heating, pressurization, and precise oxygen control. The first stage is 155℃, 400kPa, and dissolved oxygen 0.1mg / L, held for 15 min; the second stage is 175℃, 800kPa, and dissolved oxygen 0.2mg / L, held for 20 min; the third stage is 190℃, 900 kPa, held for 20 min. After cooling, the crude extract is obtained by filtration through a 0.22 μm precision ultrafiltration membrane. 3. Microbial detoxification: Prepare a compound detoxification agent: Bacillus subtilis, Pseudomonas, and Aspergillus niger are compounded at a viable count ratio of 3:2:1, with a total viable count ≥50 billion CFU / g; add 1.5% (w / w) of the compound agent to the crude extract, adjust the pH of the system to 6.5, and culture in a constant temperature shaker at 25℃ and 150 r / min for 72 h of aerobic fermentation; after the fermentation is completed, inactivate the bacteria in an 85℃ water bath for 30 min, and then filter again through a 0.22 μm ultrafiltration membrane to obtain the detoxified solution.

[0026] 4. Enzymatic oxidation targeted modification: (1) First stage acidic pretreatment: Adjust the pH of the system to 5.0, add 0.2% of the mass of the detoxified and purified extract of pectinase and glucose oxidase compound enzyme preparation (mass ratio of pectinase to glucose oxidase is 2:1, pectinase, enzyme activity ≥10000 U / g, glucose oxidase, enzyme activity ≥5000 U / g), stir at 40℃ for 1.5 h; (2) Second stage site-specific modification: Adjust the pH of the system to 5.58, add laccase (enzyme activity ≥10000 U / g), the amount of laccase added is 0.20% of the mass of the detoxified liquid, stir at 40℃ for 2 h; the total enzymatic hydrolysis time is 3.5 h, after the reaction is completed, raise the temperature to 90℃ and keep warm for 15 min to inactivate the enzyme activity.

[0027] 5. Finished product preparation: The enzyme-treated liquid is transferred to a vacuum concentration device and concentrated at -0.08MPa and 50℃ until the flavonoid content is 5.0g / L. 0.2% EDTA-Zn, 0.15% EDTA-Fe, 0.10% EDTA-Mg, 0.3% sodium carboxymethyl cellulose, and 0.1% sodium benzoate are added according to the mass of the concentrate. The mixture is stirred evenly, allowed to stand at room temperature for 24 hours, and then aseptically filled to obtain 1L of finished biomass-derived plant growth stimulant.

[0028] Table 3 shows the characteristic parameters of the biomass-derived plant growth stimulant in Example 2. Test case Field trials of the biostimulant products from Example 1 and Comparative Example 1 were conducted on peas. The products were diluted 2000 times for foliar spraying and 1000 times for drip irrigation, while the amount of conventional fertilizer was kept consistent. The tested pea variety was Zhongwan 6. The trials were conducted from May 20268 to June 2026 in a greenhouse in Kutao Village, Zhonghan Street, Laoshan District, Qingdao.

[0029] After washing the roots of the pea seedlings obtained from the experiment, a plant root analyzer was used to measure and analyze the root length, root tip, fresh weight, root nodules, and number of branches of the pea seedlings based on image recognition technology.

[0030] As can be seen from the attached figures, compared with Comparative Example 1, Example 1 showed an increase of 52.5% in total root length, 30.8% in root length, 53.4% ​​in the number of root tips, 20% in fresh weight, 34.7% in the number of root nodules, and 54.7% in the number of branches.

Claims

1. A method for preparing flavonoid plant growth stimulants by microaerobic subcritical extraction, characterized in that, Includes the following steps: (1) Pretreatment of compound biomass: livestock and poultry manure and plant straw are compounded according to mass parts, coarsely crushed and sieved, and dried at low temperature; the dried coarsely crushed material is sent to a shredding and puffing equipment, first mechanically shredding and tearing the fiber bundles, and then using a pressure differential drying puffing machine for instantaneous puffing to fully expose the lignin-flavonoid conjugated binding sites, and after cooling, puffed solid material is obtained. (2) Micro-aerobic subcritical treatment: A micro dissolved oxygen environment is maintained in a closed subcritical reactor, and the reaction is carried out in three stages with gradient temperature and pressure increase. The crude extract is obtained by cooling membrane filtration. (3) Microbial targeted detoxification: The residual toxins are mineralized by aerobic fermentation using a compound detoxification agent, and then detoxified by ultrafiltration after inactivation; (4) Enzymatic oxidation targeted modification: The pH of the system is controlled in two stages, and the complex enzyme is added and the enzyme activity is inactivated by high temperature; (5) Preparation of finished product: Low temperature vacuum concentration, multi-component chelate stabilizer preparation, aseptic filling after maturation, to obtain plant growth stimulant.

2. The preparation method according to claim 1, characterized in that, Step (1) Mix livestock and poultry manure and plant straw in a dry matter mass ratio of 15-35:65-75, coarsely crush and pass through a 10-mesh sieve, and dry at a low temperature of 55-65℃ until the moisture content is 10%-25%; shredding process parameters: shredding speed 300-400 r / min, shredding for 12-18 min; pressure difference puffing pressure 0.2-0.5 MPa, pressure held for 30-60 s and then instantaneously released.

3. The preparation method according to claim 1, characterized in that, Step (2) Three-stage microaerobic subcritical treatment parameters: First stage: 120~155℃, 100~400 kPa, dissolved oxygen 0.05~0.1 mg / L, keep warm for 10~15 min; Second stage: 160~175℃, 450~800kPa, dissolved oxygen 0.15~0.2 mg / L, keep warm for 15~20 min; Third stage: 180~220℃, 850~1000kPa, maintain the temperature in the slightly aerobic dissolved oxygen range for 10~20 min.

4. The preparation method according to claim 1, characterized in that, Step (3) The compound detoxifying agent is composed of Bacillus subtilis, Pseudomonas, and Aspergillus niger in a viable count ratio of 3:2:1, with a total viable count ≥2.0×10⁻⁶. 9 CFU / g.

5. The preparation method according to claim 1, characterized in that, Step (4) Enzymatic oxidation targeted modification is divided into two stages: the first stage is at pH 5.0-5.3, where pectinase and glucose oxidase are added to remove polysaccharide encapsulation; the second stage is at pH 5.5-5.8, where laccase is added to directionally modify the flavonoid hydroxyl groups.

6. The preparation method according to claim 1, characterized in that, Step (5) The total flavonoid content of the concentrated liquid is ≥4.5 g / L; the additives include chelated zinc, chelated boron, chelated magnesium, and sodium carboxymethyl cellulose, which form a chelated stable structure with the flavonoid phenolic hydroxyl groups to prevent flavonoid oxidation and inactivation.

7. A biomass-derived plant growth stimulant prepared by the method described in any one of claims 1 to 6.

8. Application of a biomass-based plant growth stimulant in crop seedling root promotion, transplanting seedling recovery, root system repair in saline-alkali land, root vitality enhancement in continuous cropping soil, and stress resistance and yield increase in fruits and vegetables.

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