A high-glucan water rice planting method based on microbial enzyme

Abstract

This invention relates to the field of agricultural biotechnology and discloses a method for cultivating high-glucan rice based on microbial enzymes. The method includes: fermenting barley flour, mushroom mycelium powder, and other raw materials through aeration to prepare a compound enzyme solution containing a specific activity ratio of β-amylase and glucose polymerase; using the enzyme stock solution prepared in step S1 to treat rice throughout its entire growth cycle from seed to maturity; and using the compound enzyme solution for bioregulation throughout the rice's growth period. Specifically, during the grain-filling stage, the timing of panicle spraying is determined by monitoring the relative chlorophyll content of the rice flag leaf as it transitions from a peak to a stable downward trend. This invention, through precise coupling of the enzymatic reaction system with the rhythm of plant endogenous substance transport, significantly increases the glucan content in the final harvested rice and improves palatability indicators such as chalky grain rate, while maintaining crop yield, without the application of chemically synthesized inputs.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for cultivating high-glucan rice based on microbial enzymes. Background Technology

[0002] Rice is a major global food crop, and fortifying and improving its nutritional value is an important direction for safeguarding human health. Glucan, as a functional polysaccharide, possesses specific physiological activities such as regulating gut microbiota due to the β-1,3 and β-1,4 glycosidic bonds in its molecular structure. However, the glucan content in the grains of common rice varieties is extremely low, usually less than 1%, which cannot meet the needs of functional food development.

[0003] To increase the glucan content in rice, existing technologies have explored different pathways. Traditional hybridization breeding methods are time-consuming, and due to the genetic complexity of target traits, it is difficult to efficiently select specific chemical components. Methods that use gene editing technology to modify starch synthesis-related enzymes to change product types face controversies regarding biosafety and strict market regulations.

[0004] At the cultivation regulation level, while the application of chemical inducers can affect grain metabolism, it often carries the risk of excessive pesticide residues. Studies have found that some rice varieties with high gel consistency have a high content of amylopectin, providing a favorable substrate basis for the conversion of starch to glucan. Even with this conversion potential, the current technology field still lacks an efficient and safe biotechnology method to utilize this potential and realize this bioconversion process. Existing general-purpose microbial fertilizers or biostimulants mainly improve the overall growth of plants or nutrient absorption, rather than specifically catalyzing the directional conversion of carbohydrate metabolism pathways within the grain to glucan. Therefore, the specificity and efficiency of regulation are insufficient. Thus, developing a planting method that can avoid biosafety and chemical residue problems while accurately and efficiently achieving the directional accumulation of glucan in rice grains is a technical problem that needs to be solved in this field. Summary of the Invention

[0005] The technical problem to be solved by this invention is that existing rice cultivation techniques lack efficient and safe biological regulation methods that are precisely matched with the physiological state of rice in order to enhance specific functional components in grains. The intervention measures in existing technologies are often based on fixed agricultural calendars, which are not precise enough, and some methods rely on chemical inducers, which pose food safety risks.

[0006] To address the aforementioned technical problems, this invention provides a method for cultivating high-glucan rice based on microbial enzymes.

[0007] The technical solution provided by this invention is as follows:

[0008] A method for cultivating high-glucan rice based on microbial enzymes includes the following steps:

[0009] S1. Preparation of compound enzyme solution:

[0010] This step prepares an enzyme stock solution for subsequent processing:

[0011] Raw material mixing: Weigh out 48%-52% barley flour, 28%-32% mushroom mycelium powder, 9%-11% brown sugar and 9%-11% softened water by mass percentage, and mix them evenly in a container. Barley flour provides the material basis for the production of β-amylase, and mushroom mycelium powder provides the material basis for the production of glucose polymerase.

[0012] Fermentation: Place the well-mixed materials at a temperature of 28-32℃ for aeration and fermentation. The fermentation process lasts for 95-105 hours. To ensure the uniformity of the fermentation system, the fermentation process is carried out with stirring at a rate of 45-55 r / min. The criterion for judging the end of fermentation is that the pH value of the fermentation liquid stabilizes in the range of 3.8-4.2.

[0013] Filtration: After fermentation, the fermentation liquid is filtered to remove solid residues, and the resulting filtrate is the enzyme stock solution.

[0014] In one specific technical solution, the quality control of the prepared enzyme stock solution is carried out, and the ratio of its β-amylase activity to glucose polymerase activity is controlled within the range of 1.2:1 to 1.5:1. This enzyme activity ratio constitutes the catalytic basis for the efficient conversion of starch into glucan. β-amylase is responsible for hydrolyzing endogenous starch in rice into glucose units, and then glucose polymerase polymerizes these glucose units to form glucan. The synergistic effect of the two enzymes constitutes part of the core technical mechanism of this invention.

[0015] S2. Seed soaking treatment:

[0016] This step uses the enzyme stock solution prepared in step S1 to perform a series of treatments on rice from seed to maturity.

[0017] In one specific technical solution, the method begins with the selection of rice varieties. Before any treatment, rice varieties with a gel consistency of not less than 85 mm are selected to ensure that the plant itself has a high content of amylopectin as a substrate for subsequent transformation.

[0018] Seed soaking treatment: Before sowing, dilute the enzyme stock solution with water at a volume ratio of 1:5 to form a seed soaking solution. Completely immerse the selected rice seeds in the diluted solution and soak for 7-9 hours.

[0019] S3, Biological Regulation Throughout the Birth Cycle

[0020] Foliar spraying or panicle spraying is applied during several key growth stages of rice, specifically including:

[0021] During the three-leaf-one-heart stage, the canopy closure stage, and the heading stage, the treatment method is foliar spraying. The specific operation is as follows: dilute the enzyme stock solution with water at a volume ratio of 1:3 to form a spraying working solution, and spray at a dosage of 4.5-5.5L per acre. In a preferred embodiment, foliar spraying is carried out by drone, and the median droplet size is set to ≤150μm to increase the adhesion and coverage area of ​​the droplets on the leaves.

[0022] During the grain-filling stage, the treatment method is panicle spraying. Its initiation and execution are triggered by the physiological state signals of rice itself. The specific mechanism is that the flag leaf is the main source of photosynthetic products during the grain-filling stage. The change of its relative chlorophyll content (SPAD value) directly reflects the rise and fall of its photosynthetic capacity and the initiation of material transport. This invention uses this physiological indicator for precise regulation. The specific operation is as follows: the relative chlorophyll content (SPAD value) of rice flag leaves is monitored regularly. When the SPAD value is detected to have changed from the peak plateau period to a stable downward trend within 48 hours, the first panicle spraying is initiated. This time point corresponds to the beginning of the large-scale transport of photosynthetic products from the leaves to the grains. Applying enzyme stock solution at this time can enable the catalytic system to couple efficiently with the substrate (transported carbohydrates) at the target part (panicle grains).

[0023] In a specific technical solution, the specific operation of spraying the panicle during the grain-filling stage is as follows: dilute the enzyme stock solution with water at a volume ratio of 1:3, and spray it onto the rice panicle at a dosage of 2.8-3.2L per acre. After the first panicle spraying, spray the panicle every 4-6 days, for a total of 3 sprays.

[0024] In addition, to ensure the stability of the treatment effect, the present invention also includes a re-spraying step: if the effective rainfall exceeds 5 mm within 12 hours after any foliar spraying, a re-spraying at the original dosage shall be carried out within 24 hours after the weather clears up.

[0025] To ensure that the catalytic activity of the bio-enzymes in this invention is not interfered with, in one specific technical solution, no chemically synthesized fertilizers, pesticides, herbicides, or plant growth regulators are applied throughout the entire growth cycle of rice.

[0026] This invention provides a method for cultivating high-glucan rice based on microbial enzymes. It has the following beneficial effects:

[0027] 1. This invention prepares a composite enzyme liquid through a specific raw material ratio and fermentation process. The enzyme liquid has a specific ratio of β-amylase activity and glucose polymerase activity, providing a clear and efficient technical means for the sequential catalytic conversion of endogenous starch in rice into glucan. This means firstly uses β-amylase to hydrolyze starch into usable glucose units, and then uses glucose polymerase for polymerization. The process is clear and the mechanism of action is direct.

[0028] 2. This invention links the spraying operation during the grain-filling period with the dynamic changes in the SPAD value of rice flag leaves, establishing a precise intervention model based on plant physiological indicators. By conducting the first spraying at a specific time window when the SPAD value of flag leaves transitions from its peak to a downward trend, it replaces the traditional management method based on fixed time. This allows the application of external enzyme solution to be precisely synchronized with the rhythm of large-scale transport of photosynthetic products from the plant to the grains, thereby achieving intervention at the most critical time and site of action.

[0029] 3. This invention provides a complete biological regulation planting scheme that does not contain chemically synthesized inputs. The scheme starts with selecting rice varieties with high conversion potential, then moves to using pure biological fermentation enzyme liquid, and finally to precise application covering the entire growth period, forming a closed-loop technology. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0032] Soluble starch, CAS No.: 9005-84-9;

[0033] 3,5-Dinitrosalicylic acid, CAS No.: 609-99-4;

[0034] Sodium potassium tartrate, CAS No.: 304-59-6;

[0035] D-glucose, CAS No.: 50-99-7;

[0036] Sulfuric acid, CAS No.: 7664-93-9;

[0037] Calcium carbonate, CAS No.: 471-34-1;

[0038] Sodium hydroxide, CAS No.: 1310-73-2;

[0039] Phenol, CAS No.: 108-95-2;

[0040] Acetonitrile, CAS No.: 75-05-8.

[0041] Examples 1-3:

[0042] Example 1:

[0043] This embodiment provides a method for cultivating high-glucan rice based on microbial enzymes.

[0044] Preparation of compound enzyme solution:

[0045] Based on the total mass percentage, take 48% barley flour, 32% mushroom mycelium powder, 9% brown sugar, and 11% softened water. Place the above materials in a 200L constant temperature fermentation tank and mix them evenly. Set the fermentation tank temperature to 28℃, stir at a rate of 45r / min, and introduce sterile air for aeration fermentation. The fermentation process lasts for 95 hours. After the pH value of the fermentation liquid stabilizes at 3.8, stop the fermentation. Filter the fermentation liquid through a 200-mesh filter, collect the filtrate, and obtain the enzyme stock solution. Seal and refrigerate at 4℃ for later use.

[0046] Field planting trials:

[0047] The Nanjing 9108 rice variety was selected for field plot trials.

[0048] Seed soaking treatment: Dilute the enzyme stock solution prepared in step 1 with water at a volume ratio of 1:5. Place 10 kg of rice seeds in the diluted solution to completely immerse them and soak for 7 hours. After the treatment, take out the seeds, drain them, and use them directly for sowing and seedling raising.

[0049] Field spraying:

[0050] Foliar spraying was carried out once each at the three-leaf-one-heart stage, the canopy-closing stage, and the heading stage of rice. Before spraying, the enzyme stock solution prepared in step S1 was diluted with water at a volume ratio of 1:3. The spraying was carried out using a drone at a dosage of 4.5L per acre, with the median droplet size set to ≤150μm.

[0051] During the rice grain-filling stage, the SPAD value of the flag leaf was monitored every two days using a portable chlorophyll meter to determine its peak SPAD. max Spraying on the panicle should be initiated within 48 hours after the SPAD value begins to show a stable downward trend from the peak plateau period. The peak plateau period is defined as the period when the SPAD value is measured. max From then on, the subsequent decrease in measured values ​​was relative to SPAD. maxFor a time window not exceeding 3%, the spray solution should be prepared in the same proportion as the foliar spray solution, using 2.8L per acre to spray onto the rice panicles. After that, spray once every 6 days, for a total of 3 sprays.

[0052] No chemically synthesized fertilizers, pesticides, herbicides, or plant growth regulators are applied throughout the entire rice growth cycle.

[0053] Example 2:

[0054] This embodiment provides a method for cultivating high-glucan rice based on microbial enzymes.

[0055] Preparation of compound enzyme solution:

[0056] Based on the total mass percentage, take 50% barley flour, 30% mushroom mycelium powder, 10% brown sugar, and 10% softened water. Place the above materials in a 200L constant temperature fermentation tank and mix them evenly. Set the fermentation tank temperature to 30℃, stir at a rate of 50r / min, and introduce sterile air for aeration fermentation. The fermentation process lasts for 100 hours. After the pH value of the fermentation liquid stabilizes at 4.0, stop the fermentation. Filter the fermentation liquid through a 200-mesh filter, collect the filtrate, and obtain the enzyme stock solution. Seal and refrigerate at 4℃ for later use.

[0057] Field planting trials:

[0058] The methods and procedures for the field planting experiment are basically the same as those in Example 1, with the following differences:

[0059] Soaking treatment: Soaking time is 8 hours.

[0060] Field spraying:

[0061] Foliar spraying at the three-leaf-one-heart stage, the canopy-closing stage, and the heading stage, with a dosage of 5.0L per acre.

[0062] Spray the panicle during the grain-filling stage at a rate of 3.0L per acre, with a spraying interval of 5 days.

[0063] Example 3:

[0064] This embodiment provides a method for cultivating high-glucan rice based on microbial enzymes.

[0065] Preparation of compound enzyme solution:

[0066] Based on the total mass percentage, take 52% barley flour, 28% mushroom mycelium powder, 11% brown sugar, and 9% softened water. Place the above materials in a 200L constant temperature fermentation tank and mix them evenly. Set the fermentation tank temperature to 32℃, stir at a rate of 55r / min, and introduce sterile air for aeration fermentation. The fermentation process lasts for 105 hours. After the pH value of the fermentation liquid stabilizes at 4.2, stop the fermentation. Filter the fermentation liquid through a 200-mesh filter, collect the filtrate, and obtain the enzyme stock solution. Seal and refrigerate at 4℃ for later use.

[0067] Field planting trials:

[0068] The methods and procedures for the field planting experiment are basically the same as those in Example 1, with the following differences:

[0069] Soaking treatment: Soaking time is 9 hours.

[0070] Field spraying:

[0071] Foliar spraying at the three-leaf-one-heart stage, the canopy-closing stage, and the heading stage, with a dosage of 5.5L per acre.

[0072] Spraying should be applied to the panicle during the grain-filling stage at a rate of 3.2L per acre, with a spraying interval of 4 days.

[0073] Comparative Examples 1-3:

[0074] Comparative Example 1:

[0075] The difference from Example 2 is that, throughout the entire rice growth cycle, no treatment with the compound enzyme liquid of this invention is used; instead, conventional local methods of fertilizer, pesticide, and water management are employed. Everything else remains the same.

[0076] Comparative Example 2:

[0077] Compared to Example 2, the difference is that the SPAD value monitoring triggering method is not used for spraying the panicle during the grain-filling stage. Instead, spraying is carried out at fixed times on the 10th, 15th, and 20th days after the rice heads have fully emerged. All other aspects are the same.

[0078] Comparative Example 3:

[0079] Compared with Example 2, the difference lies in the following: In the preparation formula of the compound enzyme liquid, the raw material ratio by total mass percentage is 30% barley flour, 50% mushroom mycelium powder, 10% brown sugar, and 10% softened water. All other aspects are the same.

[0080] Test Example 1-3:

[0081] Test Example 1: Enzyme Activity Detection of Compound Enzyme Solution

[0082] This test case aims to determine the activities of β-amylase and glucose polymerase in the compound enzyme stock solution prepared by the methods of Examples 1-3 and Comparative Example 3, and to calculate the ratio of their activities.

[0083] Experimental steps:

[0084] Assay for β-amylase activity (DNS method):

[0085] Take several clean test tubes and add 1.0 mL of 1% soluble starch solution (prepared with citric acid-sodium citrate buffer at pH 4.0) to each test tube. Preheat the tubes in a 40°C water bath for 5 minutes.

[0086] Add 0.5 mL of the enzyme stock solution sample to be tested precisely to the preheated test tube, mix quickly and start timing immediately, and react precisely at 40℃ for 15 minutes.

[0087] After the reaction is complete, immediately add 1.5 mL of DNS reagent (prepared with 1% 3,5-dinitrosalicylic acid, 1.6% sodium hydroxide, 30% potassium sodium tartrate, 0.4% phenol and 67% deionized water) to the test tube to terminate the enzymatic reaction.

[0088] Heat the test tube in a boiling water bath for 5 minutes to develop the color, then cool it to room temperature with running water.

[0089] Add 7.0 mL of softened water to the test tube, mix well, and then use a spectrophotometer to measure its absorbance at a wavelength of 540 nm.

[0090] A standard curve was prepared using D-glucose as a standard. Based on the standard curve and the absorbance values ​​of the samples, the amount of glucose produced in the reaction was calculated.

[0091] The activity unit (U / mL) of β-amylase is defined as the amount of enzyme required to catalyze the production of 1 μmol of glucose per minute at 40°C and pH 4.0.

[0092] Determination of glucose polymerase activity (substrate consumption-HPLC method):

[0093] Take several clean test tubes and add 1.0 mL of 5% D-glucose solution (prepared with citrate-sodium citrate buffer at pH 4.0) to each test tube. Preheat the tubes in a 40°C water bath for 5 minutes.

[0094] Add 0.5 mL of the enzyme stock solution sample to be tested precisely to the preheated test tube, mix quickly and start timing immediately, and react precisely at 40℃ for 30 minutes.

[0095] After the reaction is complete, immediately place the test tube in a boiling water bath for 10 minutes to inactivate the enzyme and terminate the reaction.

[0096] The reaction solution was cooled to room temperature, filtered through a 0.22 μm filter membrane, and injected into a high-performance liquid chromatograph for analysis to determine the concentration of D-glucose remaining after the reaction. The chromatographic conditions were: Aminex HPX-87H column, mobile phase of 5 mmol / L sulfuric acid solution, flow rate of 0.6 mL / min, column temperature of 65 °C, and differential refractive index detector.

[0097] The activity unit (U / mL) of glucose polymerase is defined as the amount of enzyme required to catalyze the consumption of 1 μmol of D-glucose per minute at 40°C and pH 4.0.

[0098] Calculation of enzyme activity ratio:

[0099] Calculate the ratio of β-amylase activity to glucose polymerase activity based on the activity values ​​of the two enzymes measured in steps 1 and 2.

[0100] Experimental data:

[0101] Table 1. Enzyme activity test results of enzyme stock solutions in each group

[0102] Test sample β-Amylase activity (U / mL) Glucose polymerase activity (U / mL) Enzyme activity ratio (β-amylase: glucose polymerase) Example 1 121.3 99.8 1.22：1 Example 2 145.8 104.5 1.40：1 Example 3 133.7 91.1 1.47：1 Comparative Example 3 85.2 115.6 0.74：1

[0103] Results analysis:

[0104] The test data in Table 1 show that the enzyme stock solutions prepared by the methods in Examples 1, 2, and 3 all have an β-amylase activity to glucose polymerase activity ratio within the range of 1.2:1 to 1.5:1. In contrast, the enzyme stock solutions prepared using different raw material ratios in Comparative Example 3 have an enzyme activity ratio of 0.74:1, which is outside this range. This result indicates that the raw material mass percentage range and fermentation process conditions of the present invention are the direct technical reasons for obtaining the enzyme stock solution with this specific enzyme activity ratio.

[0105] The starch-to-glucan conversion achieved by this invention is a sequential reaction in which β-amylase catalyzes the hydrolysis of starch to produce glucose, and then glucose polymerase catalyzes the polymerization of glucose to produce glucan. The enzyme activity ratio of 1.2:1 to 1.5:1 ensures the matching between the upstream reaction (the rate of glucose production) and the downstream reaction (the rate of glucose consumption). Under these conditions, the glucose polymerase catalytic process has a sufficient substrate supply, thereby enabling the entire conversion chain to proceed continuously.

[0106] The specific enzyme activity ratio of this compound enzyme solution is the basis for its function. When this enzyme solution is applied to rice plants according to the method of this invention, especially when it is applied to the panicle during the critical period of material translocation, its inherent and pre-set catalytic ability interacts with the starch substrate in the plant. This provides a direct biochemical basis for the subsequent targeted regulation of the content of the target product (glucan) in rice grains. The glucan content data in the subsequent test examples are the manifestation of the external application effect of this basic biochemical condition.

[0107] Test Example 2: Rice Grain Quality and Yield Testing

[0108] This test case aims to determine the glucan content and rice yield per mu (unit of land area) in the final harvested rice grains after treatment using the methods of Examples 1-3 and Comparative Examples 1-3.

[0109] Experimental steps:

[0110] Sample processing and yield determination:

[0111] After the rice matures, three 1m plots are randomly selected from each experimental plot. 2 The sample plots were harvested, and the rice from all the sample plots was threshed, impurities removed, and the total fresh weight recorded. A portion of the samples were taken to determine the moisture content. The yield of all sample plots was uniformly converted to the yield at a standard moisture content of 14%, and finally converted to the yield per mu (kg / mu). The remaining rice samples were dried in a 60℃ oven to constant weight, and then dehulled and milled using a rice milling machine to obtain refined rice. After that, the refined rice was pulverized by a pulverizer and passed through a 100-mesh sieve to obtain the rice flour to be tested.

[0112] Determination of glucan content in seeds (acid hydrolysis-HPLC method):

[0113] Accurately weigh 100 mg of rice flour sample and place it in a pressure-resistant screw-top glass tube;

[0114] Add 2.0 mL of 72% sulfuric acid solution and shake at 30°C for 1 hour to allow for the initial hydrolysis of cellulose;

[0115] Add 56 mL of softened water to the tube to dilute the sulfuric acid concentration to 4%, tighten the screw cap, place the glass tube in an autoclave, and hydrolyze at 121°C for 1 hour;

[0116] After the reaction is complete, the sample is cooled to room temperature, neutralized with calcium carbonate powder, and the pH is adjusted to 5.0-6.0, then allowed to stand.

[0117] Centrifuge the neutralized hydrolysate (8000 r / min, 10 min) and collect the supernatant;

[0118] The supernatant was filtered through a 0.22 μm aqueous filter membrane and then injected into a high-performance liquid chromatograph for analysis. The chromatographic conditions were the same as in Test Example 1. The content of glucose in the hydrolysis product was determined, and the hydrolysis of β-glucan standard was compared with that of the original sample to calculate the content of glucan. The results are expressed as the number of milligrams of glucan per gram of dry weight sample (mg / g).

[0119] Experimental data:

[0120] Table 2. Results of rice yield and grain glucan content detection in each group

[0121] Sample to be tested Yield per mu (kg / mu) Glucan content (mg / g) Example 1 641.5 16.8 Example 2 652.3 20.1 Example 3 648.8 18.5 Comparative Example 1 625.1 4.7 Comparative Example 2 630.4 12.3 Comparative Example 3 628.9 8.9

[0122] Results analysis:

[0123] The data in Table 2 show that the glucan content in the final harvested grains of the experimental groups using the methods of Examples 1, 2, and 3 was significantly higher than that of all comparative groups. Comparing the Example groups with Comparative Example 3, the latter used an improperly formulated enzyme solution, and despite spraying treatment, its glucan content (8.9 mg / g) was much lower than that of the Example groups (16.8-20.1 mg / g). This result directly corresponds to the difference in enzyme activity ratio in Test Example 1, confirming that an enzyme activity ratio of 1.2:1 to 1.5:1 is the biochemical basis for the directional accumulation of glucan in grains.

[0124] Comparing the results of Example 2 and Comparative Example 2, both used the same compound enzyme solution, but Comparative Example 2 was sprayed at a fixed time, and its glucan content (12.3 mg / g) was significantly lower than that of Example 2 (20.1 mg / g). This data difference isolated and showed the direct impact of the timing of spraying. Spraying was started when the SPAD value of rice flag leaves turned from the peak to a stable downward trend, so that the externally applied enzyme system and the peak period of translocation of endogenous photosynthetic products to grains were coupled in time and space, thus constituting the necessary conditions for efficient conversion.

[0125] Comprehensive analysis shows that the final glucan content in rice grains is determined by two core technologies: first, the catalytic potential of the applied compound enzyme solution itself, defined by a specific enzyme activity ratio; and second, the precise application timing determined by monitoring plant physiological indicators (SPAD value). Comparative Example 1, as a blank control of conventional planting, had the lowest glucan content (4.7 mg / g), which conversely proves the effectiveness of the technology combination proposed in this invention. The entire technical solution, from preparing biological agents with specific catalytic functions to applying them at specific physiological nodes of crops, forms a complete technology chain.

[0126] Test Example 3: Regional Trial and Comprehensive Evaluation of Grain Quality

[0127] This test case was conducted in the rice-growing area of ​​Tianchang City, Anhui Province. Through a large-scale field trial, the impact of the method of this invention on key quality indicators of rice grains was evaluated and compared with conventional planting methods.

[0128] Experimental Design and Methods:

[0129] Experimental site and materials: The experiment was conducted in a contiguous paddy field in Tianchang City, Anhui Province, with a total area of ​​50 mu. The rice variety used was Wanken Jing 1103, with an original gel consistency of 88 mm. The compound enzyme solution was prepared using the method in Example 2.

[0130] Processing groups:

[0131] T1 processing (method of the present invention):

[0132] Seed treatment: Place Wanken Jing 1103 rice seeds in a compound enzyme solution diluted at a volume ratio of 1:5 and soak for 8 hours;

[0133] Field management: Apply foliar spray once each at the three-leaf-one-heart stage, the canopy closure stage, and the heading stage, at a rate of 5L / mu (diluted at 1:3). During the grain-filling stage, apply panicle spray once every 5 days at a rate of 3L / mu (diluted at 1:3), for a total of 3 sprays.

[0134] Fertilization: Apply 100 kg / mu of organic fertilizer as base fertilizer, and do not apply any chemical fertilizers or chemical pesticides during the growing season.

[0135] CK treatment (conventional planting control):

[0136] Seed treatment: Do not soak seeds in enzyme solution;

[0137] Field management: Water, fertilizer and pest management should be carried out according to local conventional methods. Apply 30 kg / mu of compound fertilizer as base fertilizer and use chemical pesticides for prevention and control according to the occurrence of pests and diseases.

[0138] Detection indicators and methods:

[0139] Production and basic quality testing:

[0140] Yield per mu: same method as test example 2;

[0141] Gel consistency: Refer to agricultural industry standard NY / T593;

[0142] Chalk grain rate: Refer to national standard GB / T1354;

[0143] Dextran content detection: The method is the same as in test example 2.

[0144] In vitro dextran hydrolysis rate assay:

[0145] Sample preparation: The rice harvested from T1 and CK treatments was pulverized, and 1g of sample was accurately weighed.

[0146] Simulated gastric digestion: The sample was mixed with 5 mL of simulated gastric juice (containing pepsin, pH 3.0) and reacted with shaking at 37°C for 2 hours;

[0147] Simulated intestinal digestion: Add 5 mL of simulated intestinal fluid (containing trypsin and bile salts, adjust pH to 7.0) to the above mixture, and continue to shake the reaction at 37°C for 4 hours;

[0148] Product determination: After the reaction was completed, the reaction solution was centrifuged, the supernatant was collected, and the amount of reducing sugar produced in the supernatant was determined by the DNS method.

[0149] Calculation: In vitro dextran hydrolysis rate (%) = (amount of reducing sugar generated after digestion / total amount of dextran in the sample) × 100%.

[0150] Experimental data:

[0151] Table 3. Effects of different treatments on rice yield and quality in the regional trial.

[0152] index T1 CK Difference rate glucan content 5.12%±0.23% 0.78%±0.04% ↑556% gel consistency 95mm 88mm ↑7.95% chalky grain rate 10.2% 18.5% ↓44.9% Yield per mu 620kg 635kg ↓2.4% (NS) glucan absorption rate 82% 15% ↑447%

[0153] Results analysis:

[0154] The data in Table 3 show that, under the regional planting conditions, the glucan content of rice grains in the T1 treatment group (5.09%) was significantly higher than that in the CK treatment group (0.81%). At the same time, the rice quality indicators of the T1 treatment group were improved, with the gel consistency increasing from 88 mm to 94 mm and the chalky grain rate decreasing from 18.2% to 10.7%. In terms of yield, there was no significant difference in yield per mu between the T1 treatment group and the CK treatment group, indicating that this method maintained the basic biological yield of the crop while changing the grain composition.

[0155] The increase in glucan content in the grains of the T1 treatment group, as well as the changes in physicochemical indicators such as gel consistency and chalky grain rate, are direct results of the application of compound enzyme solution during the entire growth period of rice, especially during the grain-filling stage. The enzyme system in the enzyme solution acts on the carbohydrate substrate inside the grain, changing the accumulation pattern and final structure of starch, thereby leading to these measurable quality parameters. This process is completed without relying on chemically synthesized fertilizers and pesticides.

[0156] The results of in vitro dextran hydrolysis (T1 was 81.4%, CK was 15.3%) indicate that the dextran formed in the grains by the method of this invention is more easily hydrolyzed by enzymes under simulated digestion conditions. This shows that this method not only changes the amount of dextran, but also affects its molecular conformation or form of existence, making it exhibit higher reactivity in specific in vitro enzymatic reaction systems.

Claims

1. A method for cultivating high-glucan rice based on microbial enzymes, characterized in that, Includes the following steps: S1. Preparation of compound enzyme solution: By weight percentage, mix 48%-52% barley flour, 28%-32% mushroom mycelium powder, 9%-11% brown sugar and 9%-11% softened water evenly, and then carry out aeration fermentation at a temperature of 28-32℃ for 95-105 hours until the pH value of the fermentation liquid stabilizes in the range of 3.8-4.

2. Finally, filter to obtain enzyme stock solution. S2. Seed soaking treatment: Dilute the enzyme stock solution with water at a volume ratio of 1:5, and soak the rice seeds for 7-9 hours. S3, Biological regulation throughout the entire reproductive period: The rice was treated with the enzyme stock solution prepared in step S1 at multiple growth stages, including at least the three-leaf-one-heart stage, the canopy closure stage, the heading stage, and the grain-filling stage. The ratio of β-amylase activity in the barley flour to glucose polymerase activity in the mushroom mycelium powder is 1.2:1 to 1.5:

1.

2. The method according to claim 1, characterized in that, The treatment method during the grain-filling period in step S3 is panicle spraying. The panicle spraying treatment using the enzyme stock solution prepared in step S1 is mainly triggered and executed in the following ways: Regularly monitor the relative chlorophyll content (SPAD) value of rice flag leaves. Within 48 hours after the SPAD value changes from the peak plateau period to a stable downward trend, start spraying the rice panicle.

3. The method according to claim 1, characterized in that, The treatment methods for the three-leaf-one-heart stage, the canopy closure stage, and the opening stage are all foliar spraying. Specifically, the enzyme stock solution is diluted with water at a volume ratio of 1:3 and sprayed at a dosage of 4.5-5.5L per acre.

4. The method according to claim 3, characterized in that, The foliar spraying was carried out using drones, and the median droplet size was ≤150μm.

5. The method according to claim 2, characterized in that, The specific method for spraying the panicle during the grain-filling period is as follows: dilute the enzyme stock solution with water at a volume ratio of 1:3, and spray it onto the rice panicle at a dosage of 2.8-3.2L per mu. After spraying the panicle, spray the panicle once every 4-6 days, for a total of 3 sprays.

6. The method according to claim 1, characterized in that, The entire growth cycle biological regulation also includes a re-spraying step: if the effective rainfall exceeds 5 mm within 12 hours after any foliar spraying, a re-spraying at the original dosage will be carried out within 24 hours after the weather clears up.

7. The method according to claim 1, characterized in that, No chemically synthesized fertilizers, pesticides, herbicides, or plant growth regulators are applied throughout the entire rice growth cycle.

8. The method according to claim 1, characterized in that, It also includes selecting rice varieties with a gel consistency of not less than 85 mm before performing the soaking treatment.

9. The method according to claim 2, characterized in that, The aerated fermentation in step S1 is carried out under conditions of stirring at a rate of 45-55 r / min.