Long-acting high-solubility enzymatic hydrolysis polysaccharide solution of non-pulverized pollen and preparation method thereof

Abstract

The present application relates to the technical field of agricultural biological stimulant, in particular to a long-acting high-dissolution-rate non-smashing pollen enzymatic polysaccharide aqueous solution and a preparation method thereof, the preparation method comprising: directly mixing and swelling the bee pollen without smashing with water, and breaking the wall by high-speed homogenization or colloid mill; under the condition of no buffer, the obtained suspension is subjected to constant-temperature enzymolysis by using a composite enzyme, and the pH is periodically adjusted to keep stable; after the enzymolysis, the enzyme is inactivated, and the solid-liquid separation is performed to obtain a clear filtrate; the pH of the filtrate is adjusted to 4.5-5.0, and a compounded preservative is added; finally, sterilization and sterile filling are performed. The present application simplifies the process, avoids the loss of active ingredients caused by smashing and the residue of buffer, and significantly prolongs the shelf life of the product at room temperature through a specific preservative system. The prepared enzymatic solution has a high dissolution rate of active ingredients, and after dilution, it can be used for crop foliar spraying, flushing irrigation and drip irrigation, and seed treatment, and can effectively promote crop growth and improve stress resistance.

Description

Technical Field

[0001] This invention relates to the field of agricultural biostimulant technology, specifically to a long-acting, high-dissolution-rate, non-pulverized pollen enzymatic hydrolysis polysaccharide aqueous solution and its preparation method. Background Technology

[0002] Bee pollen is a natural product formed by bees after collecting plant pollen. It is rich in active ingredients such as polysaccharides, flavonoids, amino acids, and glucose. These ingredients can promote crop root development, enhance stress resistance, and increase yield, making it a high-quality raw material for agricultural biostimulants.

[0003] Existing pollen enzymatic hydrolysate preparation processes suffer from the following significant technical defects, making them unsuitable for large-scale agricultural production: 1. Complex pretreatment: Traditional processes require pollen pulverization, increasing equipment investment and process complexity. Mechanical friction can also lead to the loss of heat-sensitive flavonoids and other active ingredients, and the pulverization process can introduce external impurities, affecting the purity of the finished product. 2. High enzymatic hydrolysis cost: pH adjustment relies on phosphate and acetate buffer solutions, resulting in high buffer procurement costs. Furthermore, residual components may conflict with crop growth requirements, making it unsuitable for low-cost, large-scale agricultural production. 3. Low dissolution rate of active ingredients: Pollen cell walls are dense, and simple enzymatic hydrolysis or conventional water extraction cannot fully dissolve polysaccharides, flavonoids, and pigments, resulting in a light-colored finished product with limited agricultural application effects. 4. Short shelf life: The lack of a targeted preservation system design means the finished product has a shelf life of less than one month at room temperature, requiring refrigerated storage, increasing distribution and storage costs. 5. Poor process convertibility: Existing technical solutions have vague parameters, high equipment dependence, and cannot directly scale up small-scale production results.

[0004] To address the shortcomings of the existing technologies, this invention proposes a long-lasting, high-dissolution-rate, non-pulverized pollen enzymatic hydrolysis polysaccharide aqueous solution and its preparation method, thus solving the aforementioned technical problems. Summary of the Invention

[0005] (a) Purpose of the invention

[0006] This invention aims to overcome the technical problems existing in current bee pollen enzymatic hydrolysis processes, such as complex pretreatment, reliance on buffer systems, limited dissolution efficiency of active ingredients, and insufficient product storage stability. Therefore, the purpose of this invention is to provide a simplified and low-cost method for preparing pollen enzymatic hydrolysate; another purpose of this invention is to provide a highly active pollen enzymatic hydrolysate with a long shelf life obtained by this method.

[0007] (II) Technical Solution

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] On the one hand, a method for preparing a long-lasting, high-dissolution-rate, pollen-enzymatic hydrolysis polysaccharide aqueous solution without pulverization is provided, comprising the following steps:

[0010] (1) Pollen swelling and homogenization:

[0011] Direct water-soluble bee pollen was mixed with distilled water at a mass ratio of 1:6 and allowed to swell at room temperature for 18-22 minutes to obtain a pollen suspension. The pollen suspension was then homogenized to obtain a homogenized pollen suspension with cell wall disruption.

[0012] (2) Buffer-free complex enzymatic hydrolysis:

[0013] The homogenized pollen suspension was heated to 43-47℃, and the pH was adjusted to 5.8-6.2 with 0.5mol / L hydrochloric acid or sodium hydroxide. A compound enzyme was then added and incubated at a constant temperature for 3.5-4.5 hours. The compound enzyme, based on the dry weight of the pollen, included 1.2%-1.8% cellulase (activity ≥100,000 U / g), 0.8%-1.2% pectinase (activity ≥50,000 U / g), and 0.4%-0.8% neutral protease (activity ≥200,000 U / g).

[0014] During enzymatic hydrolysis, the pH should be measured every 25-35 minutes. If it deviates from the 5.8-6.2 range, it should be finely adjusted using 0.5 mol / L hydrochloric acid or sodium hydroxide.

[0015] After enzymatic hydrolysis, the liquid is heated to 83-87℃ and kept at that temperature for 18-22 minutes. Then it is cooled to room temperature to complete enzyme inactivation and heat extraction of pigment.

[0016] (3) Solid-liquid separation:

[0017] The cooled liquid is first filtered through a 60-80 mesh filter to remove coarse residue. The first filtrate is then centrifuged (3500-4000 r / min, 10-15 min) or allowed to stand (2.5-3.5 h) to collect the clear filtrate.

[0018] (4) Construction of the anti-corrosion system:

[0019] Adjust the pH of the clarified filtrate to 4.5-5.0 with citric acid, add it to the compound preservative system, and stir until completely dissolved; the compound preservative system, based on the mass of the clarified filtrate before adding the preservative, includes 0.02%-0.03% sodium benzoate, 0.02%-0.03% potassium sorbate, and 0.008%-0.012% disodium EDTA.

[0020] (5) Sterilization and filling:

[0021] After sterilization, the liquid obtained in step (4) is aseptically filled into a sealed container. The sterilization process is pasteurization at 63-67℃ for 28-32 minutes, or sterilization by filtration through a 0.22μm microporous membrane, or autoclaving at 113-117℃ for 13-17 minutes. After filling, the sealed container is inverted and cooled for 0.5-1.5 hours to form a micro-vacuum environment. After cooling, it is stored away from light.

[0022] Preferably, the homogenization process in step (1) is performed using a high-speed homogenizer, wherein the rotation speed of the high-speed homogenizer is set to 12000-25000 r / min and the homogenization time is 2-3 min.

[0023] Preferably, the homogenization process in step (1) is performed using a high-pressure homogenizer, with the pressure set to 20-28 MPa and the homogenization cycle repeated twice.

[0024] Preferably, the homogenization process in step (1) is performed using a colloid mill, and the process is repeated twice, for 3 minutes each time.

[0025] This invention also provides a long-lasting, high-dissolution-rate, non-pulverized pollen enzymatic hydrolysis polysaccharide aqueous solution, prepared by any of the above preparation methods. The aqueous solution contains polysaccharides, flavonoids, and glucose as active ingredients, with a pH of 4.5-5.0. It has a shelf life of ≥6 months under normal temperature and light protection conditions and a shelf life of ≥12 months under refrigeration at 4℃. The finished product is a deep yellow, clear aqueous solution, free of precipitation and odor, with a polysaccharide content of ≥1.2g / 100mL, a flavonoid content of ≥0.3g / 100mL, and a glucose content of ≥0.8g / 100mL.

[0026] The beneficial effects of this invention are:

[0027] Compared with the prior art, the present invention has the following significant advantages:

[0028] 1. Regarding the preparation process, this invention simplifies the process and reduces raw material and equipment costs by eliminating the pulverization step, employing buffer-free enzymatic hydrolysis, and incorporating periodic pH fine-tuning. Simultaneously, the synergistic effect of homogenization and cell wall disruption combined with the complex enzymatic hydrolysis process significantly improves the dissolution efficiency of the active ingredients. Experiments show that the polysaccharide extraction rate using the method of this invention can reach over 28%.

[0029] 2. In terms of product performance, the obtained pollen hydrolysate has a high content of active ingredients (polysaccharides ≥1.2%, flavonoids ≥0.3%) and excellent stability. By constructing a compound preservative system and optimizing the sterilization process, the polysaccharide retention rate is ≥95% after 14 days of heat storage at 54℃, and the shelf life at room temperature away from light can reach more than 6 months, solving the problems of poor storage stability and the need for refrigerated transportation of existing products. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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] It should be noted that the room temperature mentioned in this invention refers to 20℃-25℃. The direct water-soluble bee pollen described in this invention is mold-free, dried bee pollen that has not undergone mechanical pulverization and possesses the following characteristics:

[0032] Physical state: Dry and loose granular bee pollen free from mold and odor, with a particle size range of 10-50μm, free from lumps and visible impurities (such as mud, sand, insect remains, etc.); moisture content ≤12% (GB / T 30359-2021 "Moisture Limit Standard for Bee Pollen").

[0033] Water dispersibility: Take 1g of bee pollen and add it to 10mL of distilled water at 20-25℃. After standing at room temperature for 5min, stir gently. It can be quickly dispersed to form a uniform suspension without obvious precipitation or aggregation. After standing for 30min, the upper layer is still a uniform suspension, and the amount of sediment in the bottom layer is ≤5% of the total mass.

[0034] Feasibility of cell wall disruption: Bee pollen cell walls are structurally intact but loose in texture. After homogenization treatment as described in this invention (high-speed homogenizer 12000-25000r / min or high-pressure homogenizer 20-28MPa), the cell wall disruption rate is ≥85% (observed under a microscope, the percentage of disrupted particles is determined by randomly selecting 100 pollen grains).

[0035] Compatibility requirements: There are no restrictions on the variety of natural bee pollen (such as rapeseed pollen, corn pollen, sunflower pollen, etc.), but it must meet the following requirements: total sugar content ≥20%, flavonoid content ≥3% (dry basis), and no pesticide residue (comply with the requirements of GB2763-2021 "National Food Safety Standard Maximum Residue Limits for Pesticides in Food").

[0036] Example 1: Small-scale preparation (100g pollen standard)

[0037] 1. Pollen swelling and homogenization: Take 100g of direct water-soluble bee pollen, add 600mL of clean distilled water, stir evenly and let stand at room temperature for 20min to swell; pour the suspension into a homogenizer, set the speed to 20000r / min, homogenize for 2.5min to obtain a suspension that has initially changed color.

[0038] 2. Buffer-free compound enzymatic hydrolysis: Transfer the suspension to a constant temperature water bath (temperature control accuracy ±1℃), heat to 45℃, adjust the pH to 6.0 with 0.5mol / L hydrochloric acid, and retest the pH to 5.9; add 1.5g cellulase, 1.0g pectinase, and 0.6g neutral protease sequentially, stir until completely dissolved, and incubate at 45℃ for 4 hours; stir once every 30 minutes and retest the pH during this period, the pH fluctuates between 5.8 and 6.1, no additional fine-tuning is required; after the enzymatic hydrolysis is completed, heat to 85℃, keep warm for 20 minutes, and then cool naturally to room temperature.

[0039] 3. Solid-liquid separation: Use 6 layers of gauze (equivalent to 80 mesh filter) for initial filtration to remove coarse residue; transfer the initial filtrate to a centrifuge and centrifuge at 3500 r / min for 12 min, and collect the dark yellow clear supernatant.

[0040] 4. Construction of the preservative system: Add food-grade citric acid dropwise to the supernatant to adjust the pH to 4.8, then add 0.15g sodium benzoate, 0.15g potassium sorbate and 0.06g disodium EDTA in sequence, and stir for 5 minutes until completely dissolved.

[0041] 5. Sterilization and filling: Transfer the liquid to a clean, sealed glass bottle, heat it in a water bath to 65°C, keep it warm for 30 minutes, and gently shake it twice during the process; fill it aseptically at around 60°C, seal it, invert it to cool for 1 hour, and store it at room temperature away from light.

[0042] Example 2: Pilot-scale preparation (500g pollen standard)

[0043] (1) Pollen swelling and homogenization: Take 500g of direct water-soluble bee pollen, add 3000mL of reverse osmosis pure water, transfer to a 10L stainless steel reactor, turn on low-speed stirring (30r / min), and swell at room temperature for 22min; pump the suspension into a laboratory high-pressure homogenizer, set the pressure to 22MPa, and homogenize twice to obtain a dark yellow suspension.

[0044] (2) Buffer-free compound enzymatic hydrolysis: Reflux the suspension to the reactor, heat it to 45°C in the jacket, adjust the pH to 6.0 with 5% dilute hydrochloric acid and stabilize for 10 min; add 7.5 g (1.5%) of cellulase, 5.0 g (1.0%) of pectinase and 3.0 g (0.6%) of neutral protease according to the dry weight of pollen, and stir for 20 min until completely dissolved; hydrolyze at 45°C for 3.5 h, during which time the mixture is automatically stirred for 5 min every 30 min and the pH is monitored online. The pH fluctuates between 5.8 and 6.1 and no fine adjustment is required; after the enzymatic hydrolysis is completed, raise the temperature to 85°C and keep it at that temperature for 20 min, and then cool it to room temperature by purging with cooling water.

[0045] (3) Solid-liquid separation: Use a 60-mesh filter bag for initial filtration to remove coarse residue; transfer the initial filtrate to a high-speed centrifuge and centrifuge at 4000 r / min for 10 min to collect about 2800 mL of clear filtrate; mix the filter residue with water at a ratio of 1:1.2, stir at 80℃ for 10 min, filter again, and combine the filtrate with the clear filtrate to obtain a final filtrate volume of about 3000 mL.

[0046] (4) Construction of the preservative system: Adjust the pH of the filtrate to 4.7 with citric acid solution. Based on the mass of the clarified filtrate before adding the preservative (about 3000g), add 0.75g sodium benzoate (0.025%), 0.75g potassium sorbate (0.025%), and 0.3g disodium EDTA (0.01%) in sequence. Turn on the reactor and stir for 15min until completely dissolved.

[0047] (5) Sterilization and filling: pasteurize at 65℃ for 30 minutes, stirring once every 10 minutes during the process; after sterilization, aseptically fill into 500mL light-proof glass bottles while hot, seal and invert to cool for 1 hour, and store at room temperature away from light.

[0048] Example 3: Large-scale preparation (10kg pollen standard)

[0049] (1) Pollen swelling and homogenization: Take 10 kg of direct water-soluble bee pollen, add 60 kg of reverse osmosis pure water, put it into a 500 L raw material storage tank, start stirring (25 r / min), and swell at room temperature for 22 min; the suspension is circulated and homogenized twice by a continuous high pressure homogenizer (pressure 25 MPa, processing capacity 1 t / h) to obtain a homogenized and cell-wall-broken suspension.

[0050] (2) Buffer-free compound enzymatic hydrolysis: The suspension was pumped into a 1000L jacketed enzymatic hydrolysis reactor, the jacket was heated to 45℃, and the pH was adjusted to 6.1 with 5% dilute hydrochloric acid and stabilized for 15 min; 150g (1.5%) of cellulase, 100g (1.0%) of pectinase and 60g (0.6%) of neutral protease were added according to the dry weight of pollen, and stirred for 30 min until dissolved; the enzymatic hydrolysis was carried out at a constant temperature of 45℃ for 3.5 h, and the pH was monitored online (fluctuation 5.8-6.2), and the acid / alkali was automatically added for fine adjustment; after the enzymatic hydrolysis was completed, the temperature was raised to 85℃ and kept at that temperature for 20 min, and then cooled to room temperature with cooling water.

[0051] (3) Solid-liquid separation: The mixture is continuously filtered through an 80-mesh plate and frame filter press (pressure 0.25MPa), and the filter residue is collected for later use. The initial filtrate is transferred to a horizontal screw centrifuge (4000r / min), and after centrifugation, approximately 65L of clear filtrate is collected.

[0052] (4) Construction of anti-corrosion system: Adjust the pH of the filtrate to 4.8 using the online citric acid dosing system, add 0.025% sodium benzoate, 0.025% potassium sorbate and 0.01% disodium EDTA, and start the reactor to homogenize and stir (50r / min) for 20min to ensure uniform dissolution.

[0053] (5) Sterilization and filling: The filtrate is sterilized by a continuous pasteurizer (65℃, 30min residence time), and then filled into 200L light-proof ton containers by an aseptic filling machine. After sealing, the containers are inverted and cooled for 1.5h to form a micro-vacuum, and then transferred to a light-proof warehouse for storage.

[0054] Example 4: Large-scale preparation (500kg pollen standard)

[0055] (1) Pollen swelling and homogenization: Take 500 kg of direct water-soluble dried bee pollen, add 3000 kg of reverse osmosis pure water, put it into the raw material storage tank, turn on low speed stirring (30 r / min), and swell at room temperature for 22 min; continuously send the suspension into a high pressure homogenizer, set the pressure to 22 MPa, and homogenize twice to obtain the homogenized suspension.

[0056] (2) Buffer-free compound enzymatic hydrolysis: Pump the suspension into the jacketed enzymatic hydrolysis reactor, heat to 45°C, adjust the pH to 6.1 with 5% industrial grade dilute hydrochloric acid, and stabilize for 10 min; add 7.5 kg of cellulase, 5.0 kg of pectinase and 3.0 kg of neutral protease in sequence, stir for 20 min until completely dissolved, and enzymatically hydrolyze at 45°C for 3.5 h, retesting the pH every 30 min and fine-tuning; after the enzymatic hydrolysis is completed, heat to 85°C, keep warm for 20 min, and cool to room temperature by purging with cooling water.

[0057] (3) Solid-liquid separation: The coarse residue is removed by primary filtration through an 80-mesh plate and frame filter press (pressure 0.25MPa); the primary filtrate is transferred to a centrifuge and centrifuged at 4000r / min for 10min to collect the clear filtrate.

[0058] (4) Construction of anti-corrosion system: Add citric acid to the filtrate in batches, adjust the pH to 4.7, add 0.75 kg of sodium benzoate, 0.75 kg of potassium sorbate and 0.3 kg of disodium EDTA, stir for 30 min to dissolve, and add reverse osmosis pure water to make up to 3000 kg.

[0059] (5) Sterilization and filling: Sterilize by a continuous pasteurizer (65℃, 30min), fill into food-grade light-proof ton containers by an aseptic filling machine, seal and invert to cool for 1 hour, then transfer to a light-proof warehouse for storage.

[0060] Comparative Example 1: Traditional Grinding Process

[0061] Pollen was pulverized to 80 mesh and mixed with distilled water at a ratio of 1:6. The mixture was homogenized for 2 minutes at 8000 rpm using a conventional homogenizer. The pH was adjusted to 5.8-6.2 with phosphate buffer (0.05 mol / L). The same proportion of compound enzyme as in Example 1 was added for enzymatic hydrolysis. After enzyme inactivation, 0.06% sodium benzoate was added as a preservative. The remaining steps were the same as in Example 1.

[0062] Comparative Example 2: Cell wall disruption process without homogenization

[0063] The pollen was mixed directly with distilled water at a ratio of 1:6 without any homogenization treatment, and the rest was the same as in Example 1 (without buffer solution and compound preservative).

[0064] Comparative Example 3: Buffer Enzymatic Digestion Process

[0065] High-pressure homogenization of pollen without pulverization (same as in Example 1), pH adjusted to 5.8-6.2 with phosphate buffer (0.05mol / L), the rest is the same as in Example 1 (compounded preservative, no pulverization).

[0066] The performance of the aqueous solutions obtained in Examples 1-4 and Comparative Examples 1-3 was tested.

[0067] I. Active ingredient testing

[0068] (1) Polysaccharide content test

[0069] The polysaccharide content was tested according to GB / T15672-2009 "Test of Total Sugar Content in Edible Fungi". A standard curve of glucose from 0 to 1.0 mg / mL was plotted. The finished solution was filtered through a 0.22 μm filter membrane and diluted 10 times. 1 mL of the sample solution was taken and the color was developed using the phenol-sulfuric acid method. The absorbance was measured at 490 nm. The polysaccharide content (calculated as glucose, unit: g / 100 mL) was calculated by substituting the values ​​into the standard curve.

[0070] (2) Flavonoid content test

[0071] The flavonoid content was tested according to Y / T 1295-2007 Determination of Flavonoid Content in Plants. A standard curve of 0~0.2 mg / mL rutin was plotted. The finished solution was diluted 20 times and operated according to the colorimetric method. The absorbance was measured at 510 nm, and the flavonoid content (calculated as rutin, unit: g / 100mL) was calculated.

[0072] (3) Glucose content test

[0073] The glucose content was tested according to GB / T18932.22-2003 "Test Method for Fructose, Glucose and Sucrose Content in Honey - Liquid Chromatography Differential Refractive Index Detection". The finished solution was filtered, diluted and injected, and the glucose content was calculated by external standard method (unit: g / 100mL).

[0074] (4) Polysaccharide extraction rate test

[0075] The polysaccharide extraction rate was tested according to NY / T2742-2015 "Test of soluble sugars in fruits and their products - 3,5-dinitrosalicylic acid colorimetric method". The total mass of polysaccharides in pollen raw materials was tested using the acid extraction-phenol sulfuric acid method. The extraction rate = (total mass of polysaccharides in the finished product / total mass of polysaccharides in the pollen raw materials) × 100%.

[0076] Table 1: Test data on polysaccharide, flavonoid, and glucose content and polysaccharide extraction rate of Examples 1-4 and Comparative Examples 1-3

[0077]

[0078] The polysaccharide, flavonoid, and glucose contents, as well as the polysaccharide extraction rate in Examples 1-4 of this invention, were significantly higher than those in the three comparative groups. The core reason for this is:

[0079] 1. The non-pulverizing process avoids the heat loss of heat-sensitive flavonoids and polysaccharides during mechanical pulverization (pulverization friction raises the temperature to above 40°C, which can easily lead to denaturation of active ingredients), and at the same time reduces the interference of impurities introduced by pulverization on enzymatic hydrolysis;

[0080] 2. High-pressure homogenization (20,000 r / min) results in more thorough cell wall disruption. Compared to ordinary homogenization (8,000 r / min) or no homogenization treatment, it can effectively break down the dense cell wall structure of pollen, allowing the complex enzyme to fully contact the internal substrate and improving the dissolution rate of active ingredients.

[0081] 3. By eliminating phosphate buffer, the complexation and precipitation reactions between metal ions (PO4³⁻, Na⁺) in the buffer and polysaccharides and flavonoids are avoided, reducing the loss of active ingredients. Comparative Example 3, due to buffer complexation, had the lowest extraction rate among all groups. Furthermore, the detection data deviation of Examples 1-4 was ≤3%, indicating that the process of this invention has strong convertibility from small-scale to pilot-scale to large-scale production, good batch consistency, and is suitable for the needs of large-scale agricultural production.

[0082] II. Finished Product Stability Testing

[0083] (1) Thermal storage stability: According to GB / T19136-2003 "Determination of thermal storage stability of pesticides", 20 mL of the finished product solution was placed in a sealed glass bottle with a stopper and placed in a constant temperature incubator at 54℃±2℃ for 14 days. After cooling to room temperature, the appearance was observed and the polysaccharide content retention rate was tested (retention rate = polysaccharide content after thermal storage / initial polysaccharide content × 100%).

[0084] (2) Cold storage stability: According to GB / T19137-2003 "Determination of cold storage stability of pesticides", 20 mL of finished product liquid was placed in a sealed glass bottle and placed in a refrigerator at 0℃±2℃ for 7 days. After returning to room temperature, the appearance was observed and the polysaccharide content retention rate was tested.

[0085] (3) Stability at room temperature: The finished liquid is sealed and protected from light and placed in a room temperature environment of 25℃±5℃. The appearance is observed every month, and the retention rate of active ingredients is tested after 6 months.

[0086] Table 2: Stability test data of finished products from Examples 1-4 and Comparative Examples 1-3

[0087]

[0088] The stability of Examples 1-4 of this invention under hot storage, cold storage, and room temperature storage is significantly better than that of Comparative Examples 1-3. The core reason is that:

[0089] 1. Precise pH control + compound preservative system: Citric acid adjusts the pH of the finished product to 4.5-5.0, providing the optimal antibacterial pH environment for sodium benzoate and potassium sorbate. Disodium EDTA chelates metal ions in water, preventing the aggregation of active ingredients caused by ions, while enhancing the antibacterial efficiency of the preservative. In contrast, Comparative Example 1 uses a single preservative, while Comparative Examples 2-3 have too many undissolved particles in the finished product due to process defects, which easily become aggregation nuclei and cause stratification and precipitation.

[0090] 2. Homogenization and cell disruption + solid-liquid separation: High-pressure homogenization makes the pollen particles uniform in size (≤10μm), combined with 80-mesh filtration + centrifugation at 3500r / min, effectively removing coarse residue and undissolved particles, avoiding particle sedimentation during storage; while Comparative Example 2 did not undergo homogenization treatment, and the pollen particles were not broken and easily settled quickly; Comparative Example 3 had the worst stability due to the complexation of buffer solution, resulting in flocculent precipitation.

[0091] 3. Synergistic effect of enzyme inactivation and sterilization: While inactivating enzymes and extracting pigments at 85℃, microorganisms in the feed solution are initially killed. Subsequent pasteurization further reduces the microbial load and reduces the deterioration of the finished product caused by microbial metabolism during storage.

[0092] III. Long-term test of the corrosion resistance of finished products

[0093] The testing methods refer to the food microbiology testing standards: 1. Total bacterial count: Refer to GB4789.2-2016 "Food Microbiology Test - Determination of Total Bacterial Count", nutrient agar medium, incubate at 37℃±1℃ for 48h, counting unit: CFU / mL; 2. Molds and yeasts: Refer to GB4789.15-2016 "Food Microbiology Test - Mold and Yeast Count", Bengal red agar medium, incubate at 28℃±1℃ for 72h, counting unit: CFU / mL; 3. Testing time points: the day the finished product is prepared (initial), 1 month, 3 months, and 6 months after storage at room temperature and protected from light.

[0094] Table 3: Long-term test data on the anti-corrosion effect of Examples 1-4 and Comparative Examples 1-3

[0095]

[0096] Data Result Analysis:

[0097] 1. After 6 months of storage at room temperature and away from light, the total bacterial count in Examples 1-4 of this invention was ≤55 CFU / mL and the number of molds and yeasts was ≤12 CFU / mL, both of which met the microbial limit requirements for agricultural biological agents (total bacterial count ≤100 CFU / mL and number of molds and yeasts ≤50 CFU / mL), demonstrating that the compound preservative system (0.02%-0.03% sodium benzoate + 0.02%-0.03% potassium sorbate + 0.008%-0.012% disodium EDTA) has a significant long-lasting antibacterial effect.

[0098] 2. The number of microorganisms in Comparative Examples 1-3 approached the limit after 3 months of storage and seriously exceeded the limit after 6 months. The core reasons are: Comparative Example 1 is sodium benzoate alone, which has a poor inhibitory effect on molds and yeasts; Comparative Examples 2-3 have a lot of undissolved particles in the finished product, which provides a carrier for microorganisms to attach and metabolize. At the same time, the pH of the finished product in Comparative Example 3 fluctuates slightly due to buffer complexation, which reduces the activity of preservatives.

[0099] 3. The number of microorganisms in Examples 1-4 increased slowly over 6 months with little difference between batches, indicating that the process has good repeatability and stability and that the preservative system is highly compatible with the finished product system.

[0100] Based on the above Examples 1-4, Comparative Examples 1-3, and test data, it can be concluded that the long-lasting, high-dissolution-rate, pollen-hydrolyzed polysaccharide aqueous solution of the present invention has the following advantages:

[0101] 1. The dissolution rate of active ingredients is significantly improved, with polysaccharide extraction rate ≥28%, polysaccharide content ≥1.2g / 100mL, flavonoid content ≥0.3g / 100mL, and glucose content ≥0.8g / 100mL;

[0102] 2. The finished product exhibits good stability; after 14 days of heat storage at 54℃ and 7 days of cold storage at 0℃, no stratification or precipitation occurs; and after 6 months of storage at room temperature away from light, the retention rate of active ingredients is ≥90%.

[0103] 3. Excellent long-lasting anti-corrosion properties; microbiological indicators meet the requirements for agricultural biological agents after 6 months of storage at room temperature; shelf life ≥ 6 months.

[0104] 5. The process has strong convertibility from pilot-scale to intermediate-scale to large-scale production, good batch consistency, and requires no crushing or buffer solution, simplifying the process and reducing costs.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a long-lasting, high-dissolution-rate, non-pulverized pollen enzymatic hydrolysis polysaccharide aqueous solution, characterized in that, The preparation method includes the following steps: (1) Pollen swelling and homogenization: Direct water-soluble bee pollen and distilled water were mixed at a mass ratio of 1:6 and swollen at room temperature for 18-22 min to obtain a pollen suspension; the pollen suspension was homogenized to obtain a homogenized pollen suspension with cell wall disruption. (2) Buffer-free compound enzymatic hydrolysis: The homogenized pollen suspension was heated to 43-47℃, and the pH was adjusted to 5.8-6.2 with 0.5mol / L hydrochloric acid or sodium hydroxide. The compound enzyme was added and enzymatic hydrolysis was carried out at a constant temperature for 3.5-4.5h. The compound enzyme, based on the dry weight of pollen, included 1.2%-1.8% cellulase (activity ≥100,000 U / g), 0.8%-1.2% pectinase (activity ≥50,000 U / g), and 0.4%-0.8% neutral protease (activity ≥200,000 U / g). After the enzymatic hydrolysis was completed, the solution was heated to 83-87℃, kept at this temperature for 18-22min, and then cooled to room temperature. (3) Solid-liquid separation: The cooled liquid is initially filtered through a 60-80 mesh filter to remove coarse residue; the initial filtrate is centrifuged (3500-4000 r / min, 10-15 min) or allowed to stand (2.5-3.5 h) to collect the clear filtrate. (4) Construction of the preservative system: Adjust the pH of the clarified filtrate to 4.5-5.0 with citric acid, add the compound preservative system, and stir until completely dissolved; the compound preservative system, based on the mass of the clarified filtrate before adding the preservative, includes 0.02%-0.03% sodium benzoate, 0.02%-0.03% potassium sorbate, and 0.008%-0.012% disodium EDTA; (5) Sterilization and filling: After sterilization, the liquid obtained in step (4) is aseptically filled into a sealed container, cooled and stored away from light; the sterilization process is pasteurization at 63-67℃ for 28-32 minutes, or filtration sterilization using a 0.22μm microporous membrane, or autoclaving at 113-117℃ for 13-17 minutes.

2. The method for preparing a long-lasting, high-dissolution-rate, non-pulverized pollen enzymatic hydrolysis polysaccharide aqueous solution according to claim 1, characterized in that, During the isothermal enzymatic hydrolysis process described in step (2), the pH is measured every 25-35 minutes. If the pH deviates from the range of 5.8-6.2, it is finely adjusted using 0.5 mol / L hydrochloric acid or sodium hydroxide.

3. The method for preparing a long-lasting, high-dissolution-rate, non-pulverized pollen enzymatic hydrolysis polysaccharide aqueous solution according to claim 1, characterized in that, The homogenization process in step (1) is performed using a high-speed homogenizer, a high-pressure homogenizer, or a colloid mill. When using a high-speed homogenizer, the rotation speed of the high-speed homogenizer is set to 12000-25000 r / min, and the homogenization time is 2-3 min. When using a high-pressure homogenizer, the pressure setting for the high-pressure homogenizer is 20-28 MPa, and the homogenization is performed twice. When using a colloid mill, the treatment is performed twice, each time for 3 minutes.

4. The method for preparing a long-lasting, high-dissolution-rate, non-pulverized pollen enzymatic hydrolysis polysaccharide aqueous solution according to claim 1, characterized in that, After aseptic filling in step (5), the sealed container is inverted and cooled for 0.5-1.5 hours to form a micro-vacuum environment.

5. A long-lasting, high-dissolution-rate, non-pulverized pollen enzymatic hydrolysis polysaccharide aqueous solution, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.

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