Method for breaking the cell wall of pine pollen

Abstract

The present application relates to a kind of pine pollen breakage method, which comprises: pine pollen is mixed with water and homogenized to obtain the slurry with 40% solid content, after spray drying and sterilization treatment, using CO2 closed conveying system is transferred to the differential pressure breakage bin (cabin pressure is 8±0.2MPa, temperature is 35±2 ℃) equipped with supercritical CO2 fluid, first ultrasonic wave (40kHz variable frequency ultrasonic wave, power density 100W / L) is used to make micro-defects in the wall surface cavitation microjet, then through three-stage electromagnetic pulse pressurization treatment, and then instantaneously pressure relief.In the breakage bin, the plasma density is improved by supercritical CO2 fluid, CO2 fluid is gasified, the temperature of the broken pine pollen is reduced, and the free radical chain reaction is inhibited, thereby protecting the integrity of heat-sensitive components from the root.The powder after breakage is packaged using nitrogen protection.

Description

Technical Field

[0001] This application relates to the fields of food and health food technology, and in particular to a method for breaking down the cell walls of pine pollen. Background Technology

[0002] Pine pollen, the male reproductive cell of pine trees, is a precious "complete nutrient reservoir" in nature, containing over 200 active ingredients. Its core value lies in its superoxide dismutase (SOD), flavonoids, volatile essential oil components such as geraniol, polysaccharides, dietary fiber, and vitamins (B / C / E) and folic acid (which synergistically maintain energy metabolism and cell repair mechanisms). These components endow pine pollen with significant physiological functions: SOD slows down the process of oxidative damage in the body, flavonoids protect cardiovascular health, geraniol inhibits the proliferation of pathogenic microorganisms, polysaccharides enhance the body's immune response, and vitamins and trace elements jointly ensure metabolic homeostasis. Its comprehensive bioactivity lays the foundation for the high application value of pine pollen in the health industry, and the bioavailability of its components directly determines the efficiency of releasing its functional potential.

[0003] Its dense cell wall structure (mainly composed of sporophytin) severely hinders the bioavailability of internal proteins, polysaccharides, flavonoids, and trace volatile active ingredients. Traditional physical cell wall disruption methods (such as mechanical grinding and conventional airflow pulverization) suffer from low disruption rates, significant thermal damage, oxidative inactivation of active ingredients, and secondary microbial contamination, and it is difficult to simultaneously achieve microbial control and drying stability. To address this, an innovative reverse sequential process of "vacuum high-pressure spray drying coupled with high-temperature instantaneous sterilization → differential pressure cell wall disruption" is proposed. Through precise control of physical states, a dual breakthrough in cell wall disruption efficiency and component activity is achieved.

[0004] Because the active ingredients (such as SOD enzyme and geraniol) in high-value plant raw materials like pine pollen are tightly sealed by the ultra-dense sporopollenin wall layer (thickness 0.5-2μm, modulus 15-20GPa), traditional cell wall breaking technologies are facing difficulties. Mechanical grinding and airflow cell wall breaking result in excessively high local temperatures, leading to high inactivation rates of heat-sensitive SOD enzymes and high volatilization losses of geraniol. Static differential pressure cell wall breaking has uneven effects on pine pollen (breakage rate ≤80%), while increasing the pressure to 100MPa triggers lipid oxidation. Furthermore, existing processes consume as much as 1.8-2.5kWh / kg, accounting for over 60% of production costs. Early electromagnetic pulse technology (monopolar discharge) suffers from rapid energy decay, inability to penetrate low-dielectric-constant sporopollenins, a cell wall breaking rate <70%, and severe electrode wear.

[0005] Existing cell wall disruption technologies for processing pine pollen suffer from the ultra-dense (0.5-2 mm thick) cell wall layer of sporophytin. The presence of micrometers (μm), modulus 15-20 GPa, dielectric constant ε=3.2) leads to multiple defects. Traditional technologies suffer from uncontrollable thermal damage (local temperatures >80℃ in mechanical grinding / airflow disruption cause SOD enzyme inactivation >40% and geraniol loss >50%). The cell disruption rate and activity retention are mutually exclusive (increasing pressure to 100 MPa can improve the disruption rate, but it exacerbates lipid oxidation, causing a 300% surge in POV and a cell damage rate >30%). Energy transfer efficiency is low (ultrasound penetration depth <0.1 μm, single-stage electromagnetic pulse disruption rate <70%). There is a risk of secondary microbial contamination (the specific surface area increases by approximately 300% after disruption, but in-situ sterilization is lacking, resulting in microbial residues >3000 CFU / g). Metal contamination and structural damage occur (mechanical grinding introduces iron ions, increasing iron content by 26.5%, catalyzing oxidation reactions). Energy consumption is abnormally high (traditional processes consume 1.8-2.5 kWh / kg per unit, accounting for over 60% of production costs).

[0006] Therefore, a method for breaking down the cell walls of pine pollen is urgently needed to overcome the core scientific challenges of "deep penetration" and "directional energy release." A breakthrough requires the synergy of multiple technologies to achieve a low-cost, high-efficiency method for penetrating its ultra-hard outer wall, while maximizing the retention of heat-sensitive active ingredients and addressing the problem of oxidation and deterioration after cell wall breaking. Establishing an industrially scalable physical method for cell wall breaking, from "component confinement" to "active release," provides a theoretical foundation. Summary of the Invention

[0007] To address or partially address the problems existing in related technologies, this application provides a method for breaking down the cell walls of pine pollen. This method achieves a synergistic effect of ultrasonic pre-lysis, three-stage electromagnetic pulse timing control, and supercritical CO2 cold explosion, simultaneously achieving a 99.2% ultra-high cell wall breaking rate, a 98.5% SOD enzyme activity retention rate, 153 mg / kg geraniol with zero volatilization loss, a commercial sterility standard of <10 CFU / g for microbial residue, and a unit energy consumption of 0.55 kWh / kg, all within a low-temperature environment of 60℃.

[0008] To achieve the above objectives, this application provides a method for breaking down the cell walls of pine pollen, the method comprising:

[0009] S1, pine pollen is mixed with water and homogenized to obtain a slurry;

[0010] S2, after drying and sterilizing the slurry obtained in S1, it is transferred to a differential pressure cell-breaking chamber containing supercritical CO2 fluid; the pressure inside the chamber is 8±0.2MPa and the temperature is 35±2℃, and ultrasonic treatment is performed.

[0011] S3, activate the electromagnetic pulse device in the cell wall breaking chamber to perform three-stage pulse processing;

[0012] The first-stage pulse has a frequency of 50Hz, an electric field strength of 20kV / cm, and an action time of 0.5s.

[0013] The second-stage pulse has a frequency of 100Hz, an electric field strength of 15kV / cm, and an action time of 0.3s.

[0014] The frequency of the third-pole pulse is 200Hz, the electric field strength is 10kV / cm, and the duration is 0.2s.

[0015] S4, instantly depressurize to 0.01MPa; obtain broken-cell pine pollen.

[0016] Furthermore, the water content of the pine pollen is less than 8%.

[0017] Furthermore, the ratio of pine pollen to water is 1:1.5 (g / ml).

[0018] Furthermore, the solids content of the slurry is 40%.

[0019] Furthermore, the processing conditions in S2 are: atomization pressure: 35MPa, drying chamber temperature: inlet 180℃, outlet 58℃, and sterilization conditions: 145℃ / 2s.

[0020] Furthermore, the conditions for the ultrasonic treatment are: 40kHz variable frequency ultrasonic waves and a power density of 100W / L.

[0021] Furthermore, the pressure inside the differential pressure crushing chamber in S2 is 8±0.2MPa, and the temperature is 35±2℃.

[0022] Beneficial effects

[0023] 1. This invention achieves a leap in cell wall disruption efficiency and activity retention through a ternary synergy of "ultrasonic pre-lysis - three-stage electromagnetic pulse - supercritical medium". Ultrasonic pre-lysis (40kHz) creates micro-defects → three-stage pulse (50 / 100 / 200Hz timing control) directionally breaks down the cell wall → supercritical CO2 (8MPa→0.01MPa flash evaporation) achieves low-temperature cell wall disruption at 60℃ throughout the process, simultaneously solving the aforementioned defects and achieving a quadruple breakthrough of 99.2% cell wall disruption rate, 98.5% SOD enzyme activity retention, microbial residue <10 CFU / g, and unit energy consumption of 0.55kWh / kg (↓69.4%), fundamentally overcoming the mechanical, thermodynamic, and electrochemical barriers of sporophytin.

[0024] 2. This invention first utilizes ultrasound to create micro-defects on the cell wall surface using cavitation microjets. Then, through a three-stage electromagnetic pulse, low-frequency pressure waves generate stress at the lattice defects of the spore-pollenin, forming initial micro-cracks. Mid-frequency pressure waves drive supercritical CO2 fluid to penetrate and propagate the cracks. High-frequency waves resonate with the cell wall's natural frequency, instantly depressurizing to 0.01 MPa and inducing fatigue fracture of the overall structure. In the cell wall-breaking chamber, supercritical CO2 is used to increase plasma density. The supercritical CO2 vaporizes, breaking down the cell walls while simultaneously lowering the temperature of the broken pine pollen and inhibiting free radical chain reactions, thus protecting the integrity of the heat-sensitive components from the source.

[0025] 3. In supercritical CO2 medium, through the time-sequential synergy of ultrasonic pre-lysis and three-stage electromagnetic pulses (50 / 100 / 200Hz), low-temperature through-cell disruption (maintaining SOD enzyme activity >98.5% and near-zero loss of geraniol 153mg / kg) is achieved, as well as directional energy release control (targeted breaking down the mechanical / electrochemical barriers of sporophytin to achieve a cell disruption rate of 99.2%), in-situ antibacterial and antioxidant effects (supercritical fluid simultaneously completes cold explosion antibacterial (residue <10CFU / g) and free radical quenching (POV value decreases by 300%), and energy consumption is reduced (ultrasound-pulse-supercritical ternary coupling reduces unit energy consumption to 0.55kWh / kg, a reduction of 69.4%).

[0026] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0027] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.

[0028] Figure 1 This is a flowchart of the technical solution of this application.

[0029] Figure 2 This is a schematic diagram of the pine pollen cell wall breaking system based on electromagnetic pulse pressure difference and ultrasonic cavitation pyrolysis, as shown in the embodiments of this application; reference numerals:

[0030] 1-Spray drying chamber, 2-Drying chamber inlet, 3-Drying chamber gaseous CO2 inlet, 4-Drying chamber outlet, 5-Breaking chamber, 6-Electromagnetic pulse device, 7-Ultrasonic device, 8-Temperature controller, 9-Breaking chamber gaseous CO2 outlet, 10-Breaking chamber supercritical CO2 fluid inlet, 11-Nitrogen packaging system, 12-Gaseous CO2 guide pipe, 13-Gaseous CO2 guide branch pipe, 14-CO2 fluid guide pipe, 15-Supercritical CO2 generator. Detailed Implementation

[0031] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0032] The three-stage electromagnetic pulse pressure wave is a high-pressure impact sequence with progressively increasing frequencies. It is a biomimetic impact strategy—simulating the progressive process of seismic waves destroying buildings in nature (low-frequency shaking → mid-frequency cracking → high-frequency collapse) to achieve complete disintegration of ultra-dense biological structures with minimal energy consumption. By precisely controlling the pressure amplitude, frequency, and duration, it achieves gradient disintegration of cell walls. Its core lies in utilizing the superposition effect of electromagnetic pulse stress waves and ultrasound at different frequencies to target and destroy the multi-layered structure of plant cell walls (such as the outer wall of sporophytin and the inner wall of cellulose in pine pollen).

[0033] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0034] Example 1

[0035] 1. Experimental steps:

[0036] A method for preparing cell wall broken pine pollen, the method comprising:

[0037] S1. Fresh pine pollen (purchased from Xiangyun Yansong Biotechnology Co., Ltd.) was naturally dried and passed through a 160-mesh sieve to remove impurities, resulting in pine pollen powder with a moisture content of less than 8%.

[0038] S2, the pine pollen powder obtained in S1 is mixed with water at a ratio of 1:1.5 (g / ml), and homogenized to obtain a slurry with a solid content of 40%.

[0039] S3, the slurry obtained in S2 is sent into a vacuum spray dryer for drying and sterilization. Atomization pressure: 35MPa, drying chamber temperature: 180℃ (inlet), 58℃ (outlet), sterilization conditions: 145℃ / 2s.

[0040] S4. The pine pollen obtained in S3 is transferred to a differential pressure cell-breaking chamber containing supercritical CO2 fluid using a closed CO2 conveying system (the chamber pressure is 8 MPa ± 0.2 MPa and the temperature is 35℃ ± 2℃).

[0041] S5, start the ultrasonic device in the cell wall breaking chamber, and use ultrasonic waves (40kHz variable frequency ultrasonic waves, power density 100W / L) to create micro-defects on the surface of pine pollen cells by cavitation micro-jets.

[0042] S6. Activate the electromagnetic pulse device inside the cell wall disruption chamber, setting the pulse frequency to 50Hz, the electric field strength to 20kV / cm, and the duration to 0.5s. This allows low-frequency pressure waves to penetrate the cell wall, causing stress concentration at the sporophytin lattice defects and forming initial microcracks. Then, set the pulse frequency to 100Hz, the electric field strength to 15kV / cm, and the duration to 0.3s, allowing mid-frequency pressure waves to drive supercritical CO2 fluid infiltration and propagate the cracks. Finally, set the pulse frequency to 200Hz, the electric field strength to 10kV / cm, and the duration to 0.2s, causing high-frequency waves to resonate with the cell wall's natural frequency.

[0043] S7 shuts down the ultrasonic and electromagnetic pulse devices in the cell wall breaking chamber and instantly depressurizes it to 0.01 MPa, causing fatigue fracture of the overall structure of pine pollen. At the same time, supercritical CO2 fluid vaporizes and separates from the broken pine pollen, and the broken pine pollen is rapidly cooled, preserving its structure and all active ingredients at low temperature.

[0044] S8. The broken-cell pine pollen obtained in S7 is packaged using nitrogen gas for protection.

[0045] 2. Experimental principle of the three-stage pulse

[0046] Level 1 (50 Hz, 20 kV / cm): Low-frequency pressure waves penetrate the cell wall, causing stress concentration at sporophytin lattice defects, forming initial microcracks (crack width ≈ 100 nm).

[0047] Second stage (100 Hz, 15 kV / cm): Mid-frequency pressure wave drives supercritical CO2 fluid to penetrate into microcracks, and fluid pressure causes crack propagation (crack width → 500 nm).

[0048] Level 3 (200 Hz, 10 kV / cm): High-frequency waves resonate with the natural frequency of the cell wall (resonance peak of pine pollen cell wall layer ≈ 180-220 Hz), instantly depressurizing to 0.01 MPa, triggering fatigue fracture of the overall structure.

[0049] 3. Experimental Results

[0050] All examples and comparative examples were performed in 3 independent replicates (n=3). Data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze the data. Tukey's test was used for comparisons between groups. The significance level was set at P<0.05.

[0051] (1) Calculation of cell wall breakage rate: Staining-microscopic counting method. Specific steps: Take a small amount of cell wall breakage powder, mix it evenly with a specific staining solution (such as 0.4% trypan blue solution), let it stand for a while, prepare a slide and observe it under an optical microscope, randomly selecting multiple fields of view. Count the stained broken cells and the unstained intact cells respectively. Cell wall breakage rate (%) = [number of broken cells / (number of broken cells + number of intact cells)] × 100%.

[0052] (2) SOD enzyme activity retention rate: Xanthine oxidase method (kit purchased from Nanjing Jiancheng Bioengineering Institute, according to GB / T 5009.171-2021). Specific steps: Accurately weigh equal amounts of unbroken raw material and broken powder, extract SOD enzyme with pre-cooled phosphate buffer, strictly follow the kit instructions, add sample, enzyme working solution, substrate, etc. to 96-well plate, and measure absorbance (OD value) at 450nm wavelength using an ELISA reader. Calculate SOD enzyme activity retention rate according to the kit formula.

[0053] (3) Cell damage rate: Fluorescent double staining (FDA / PI, Sigma-Aldrich), observed by laser confocal microscopy (LeicaTCS SP8). Damage rate = (total number of cells - double-stained cells) / total number of cells × 100%.

[0054] (4) Crack propagation width and penetration depth: The cracks were detected by a combination of atomic force microscopy (AFM, Bruker Dimension Icon) and field emission scanning electron microscopy (FE-SEM, Hitachi SU8010).

[0055] The cell wall breakage rate was 99.2%, the crack depth was fully penetrated, and the cell damage rate was 4.2%; the SOD enzyme activity retention rate was 98.5%.

[0056] Comparative Example 1

[0057] Except for the following conditions, the remaining steps are the same as in Example 1.

[0058] Adjust the pulse conditions in step S6:

[0059] The frequency of the first-level pulse was adjusted to 40Hz, 50Hz, and 60Hz; the frequency of the second-level pulse was adjusted to 80Hz, 100Hz, and 120Hz; and the frequency of the third-level pulse was adjusted to 150Hz, 200Hz, and 250Hz. The cell wall breakage rate and cell damage rate were calculated according to the method in Example 1.

[0060] Table 1. Effects of different primary pulse frequencies on cell wall disruption effect.

[0061]

[0062] A primary field strength of 20 kV / cm is the critical value for breaking through the dielectric barrier of sporophytin; a pulse frequency of 50 Hz matches the characteristic frequency of the polarization response of biomaterials; and an action time of 0.5 s is a reliable time to ensure crack propagation.

[0063] Table 2. Effects of different secondary pulse frequencies on cell wall disruption effect.

[0064]

[0065] The secondary field strength of 15 kV / cm is lower than the dielectric strength of sporogen, while avoiding the ionization of SC-CO2 at the crack tip. The pulse frequency of 100 Hz achieves the maximum crack width, and the temperature rise is controllable, which is a balance between efficiency and safety.

[0066] Table 3. Effects of different three-stage pulse frequencies on cell wall disruption effect.

[0067]

[0068] The inflection point is 10kV / cm at the third-level field strength. The amplitude exceeds the 100nm fatigue threshold but is below the plasma runaway threshold. The pulse frequency of 200Hz matches the natural frequency of spore-pollen. Pressure is released at the peak of the resonant energy, and the superimposed vaporization expansion force improves the cell wall integrity rate and reduces active damage.

[0069] Comparative Example 2

[0070] Except for the following conditions, the remaining steps are the same as in Example 1.

[0071] Adjust the pulse conditions in step S6:

[0072] Table 4. Effects of different pulse combinations on cell wall disruption (total pulse time kept consistent).

[0073]

[0074] The irreplaceable nature of the three-stage pulse lies in its strict adherence to the kinetic chain reaction of cell wall disruption: the first-stage 50Hz low-frequency pulse targets lattice defects to induce microcracks (without this, 90% of the energy in the second stage is reflected); the second-stage 100Hz mid-frequency pulse drives supercritical CO2 to penetrate and propagate the cracks to a critical width of 500nm (its absence would render the third-stage resonance ineffective); and the third-stage 200Hz high-frequency pulse precisely matches the cell wall's inherent frequency to induce resonant fatigue, synergistically achieving overall disintegration through 0.01MPa supercritical fluid flash vaporization. Any single-stage or dual-stage combination results in a decreased cell wall disruption rate or an increased rate of cell viability damage due to the breakage of the disruption chain. By comparing experimental data with the disruption mechanism, it is demonstrated that single-stage or dual-stage pulses cannot achieve efficient and low-damage cell wall disruption; only the three-stage timing control can simultaneously achieve the insurmountable technical limit of 99.2% cell wall disruption rate and 98.5% cell viability retention.

[0075] Comparative Example 3

[0076] Except for the following conditions, the remaining steps are the same as in Example 1.

[0077] Comparing the proposed solutions using only ultrasound or only electromagnetic pulse, the results showed that all indicators of the ultrasound + three-stage pulse group were significantly better than those of the single-technology group (P<0.001). This is because ultrasound cannot achieve efficient cell disruption due to insufficient penetration depth (<0.12μm) and thermal damage; electromagnetic pulse is limited by the low dielectric properties of spore-forming substances (ε=3.2, tanδ<0.05), resulting in energy reflection >40%, and the single-stage action causes local high-temperature over-damage.

[0078] Table 5. Effects of different cell wall disruption methods on cell wall disruption efficiency

[0079]

[0080] Comparative Example 4

[0081] 1. Experimental Procedure

[0082] The commonly used process in the existing technology is airflow cell disruption. The specific operation is as follows: high-pressure gas (1.0MPa compressed nitrogen) that has been pre-dried and purified is used to generate a supersonic airflow through a Laval nozzle, which causes the pre-coarsely crushed material particles to collide and rub at high speed in the crushing chamber to achieve ultra-fine crushing. The crushed particles rise with the airflow and are controlled by a high-speed classifying wheel according to the principle of centrifugal force. Qualified fine powder (usually up to micron or submicron level) passes through the classifying wheel and enters the cyclone separator and dust collector for collection, while coarse particles are returned for further crushing.

[0083] 2. Experimental Results

[0084] The comparative data between the "this application" scheme and the "airflow cell disruption" scheme in Table 6 are all derived from the average values ​​of three sets of parallel repeated experiments (n=3) conducted under the same raw materials and pretreatment conditions. All measurements were performed in accordance with standard operating procedures (SOPs) and using calibrated instruments to ensure the accuracy and repeatability of the results.

[0085] Cell wall breakage rate = 85%, measurement method: staining-microscopic counting method.

[0086] Specific steps: Take a small amount of the cell wall disruption powder, mix it evenly with a specific staining solution (such as 0.4% trypan blue solution), let it stand for a while, prepare a slide, and observe it under an optical microscope, randomly selecting multiple fields of view. Count the stained disrupted cells and the unstained intact cells separately.

[0087]

[0088] SOD enzyme activity retention rate = 63%, measurement method: xanthine oxidase method (kit purchased from Nanjing Jiancheng Bioengineering Institute, according to GB / T 5009.171-2021).

[0089] Specific steps: Accurately weigh equal amounts of the unbroken raw material and the broken powder, extract SOD enzyme with pre-cooled phosphate buffer, strictly follow the kit instructions, add sample, enzyme working solution, substrate, etc. to a 96-well plate, and measure absorbance (OD value) at 450nm wavelength using an ELISA reader, and calculate SOD enzyme activity retention rate according to the kit formula.

[0090] Geranialdehyde retention was 47 mg / kg. Measurement method: Gas chromatography-mass spectrometry (GC-MS). Specific steps: Volatile components in the sample were extracted using headspace solid-phase microextraction (HS-SPME) or simultaneous distillation extraction (SDE), and analyzed using a GC-MS system. Qualitative analysis was performed by comparing the retention time and characteristic ion fragments of geranialdehyde standards, and quantification was performed by adding deuterated standards. Geranialdehyde content (mg / kg) = (mass calculated from the standard curve corresponding to the chromatographic peak area (ng)) / sample mass (kg).

[0091] Unit energy consumption = 1.8 kWh / kg. Measurement method: The power consumption of the entire blending equipment during stable operation is directly measured using a power quality analyzer and a smart meter.

[0092] Microbial residue = 3000 CFU / g, measurement method: Plate count method, following the Pharmacopoeia of the People's Republic of China or GB 4789.2-2016 "National Food Safety Standard for Microbiological Examination of Food - Determination of Total Colony Count". Specific steps: Weigh 10g of sample and dissolve it in 90mL of sterile physiological saline to prepare a 1:10 homogenate, then dilute it 100 times. Pour the homogenate onto plate counting agar, and after solidification, incubate upside down at 35℃ for 48 hours.

[0093] Table 6 Comparison of relevant indicators between this application and the airflow cell-wall breaking solution

[0094]

[0095] Below is a comparison table of the nutritional components of the two plans. The methods for measuring each indicator are as follows:

[0096] Dietary fiber: Enzyme-gravimetric method, following GB 5009.88-2014 "National Food Safety Standard - Determination of Dietary Fiber in Food". Specific steps: The sample was sequentially hydrolyzed by heat-stable α-amylase, protease, and amyloglucosidase to remove protein and starch. After ethanol precipitation, the sample was filtered, and the residue was weighed to obtain the total dietary fiber. A separate sample was used to determine the protein and ash content, which were then subtracted from the total dietary fiber. The result is expressed as grams of dietary fiber per 100 grams of sample (g / 100g). The data in the table are the average of three independent determinations.

[0097] Protein: Kjeldahl method, following GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Food". Specific steps: The sample is digested with sulfuric acid to convert organic nitrogen into inorganic ammonium salts. After alkalization and distillation, the nitrogen is absorbed with boric acid and then titrated with standard hydrochloric acid. The protein content is calculated by multiplying the measured total nitrogen content by the nitrogen-protein conversion factor (6.25).

[0098]

[0099] Wherein, V: volume of hydrochloric acid standard solution consumed by the sample (mL); V0: volume of hydrochloric acid standard solution consumed by the reagent blank (mL); c: concentration of hydrochloric acid standard solution (mol / L); 0.0140: mass of nitrogen (g) equivalent to 1.0 mL of sulfuric acid [c(1 / 2H2SO4)=1.000 mol / L] standard titration solution; F: coefficient for converting nitrogen to protein; m: sample mass (g).

[0100] The result is the arithmetic mean of three parallel determinations.

[0101] Fat: Soxhlet extraction was performed according to GB 5009.6-2016 "National Food Safety Standard - Determination of Fat in Food". The specific steps were as follows: 10g of sample was weighed and extracted by reflux with petroleum ether in a Soxhlet extraction apparatus. After evaporating to remove the solvent, the sample was dried to constant weight, and the obtained fat mass was weighed. The results are expressed as grams of fat per 100g of sample (g / 100g). The data in the table are the average of three independent determinations.

[0102] Vitamin C: High-performance liquid chromatography (HPLC) was performed according to GB 5009.86-2016, "National Food Safety Standard - Determination of Ascorbic Acid in Food". Specific procedures: The sample was extracted with metaphosphoric acid-acetic acid solution, centrifuged, and separated using a C18 column. Detection was performed at 243 nm using a UV detector or diode array detector, and quantification was performed using the external standard method. Results are expressed as milligrams of vitamin C per 100g of sample (mg / 100g). The data in the table are the average of three independent determinations.

[0103] Total sugar content: Fehling's reagent titration method, referring to GB 5009.7-2016 "National Food Safety Standard - Determination of Reducing Sugars in Food". Specific steps: Accurately weigh 2g of broken-cell pine pollen powder, extract the sugar with water, take 2ml of the extract, add hydrochloric acid, heat to hydrolyze, cool, and neutralize to neutral with sodium hydroxide solution. Take 5.0mL each of Fehling's reagent solution A and B into an Erlenmeyer flask, add some water, and heat to boiling. Rapidly titrate with the sample solution until the blue color just disappears, and record the total volume consumed.

[0104]

[0105] m1 is the glucose mass (mg) equivalent to 10 mL of Fehling's reagent.

[0106] From the calibration, V is the average volume of sample solution consumed in the titration (mL); f is the dilution factor; m is the sample mass (g); and V1 is the volume of sample solution used for the determination (mL).

[0107] Folic acid: High performance liquid chromatography (HPLC) was performed in accordance with GB 5009.211-2022 "National Food Safety Standard - Determination of Folic Acid in Food". The specific steps were as follows: After extraction, enzymatic hydrolysis and purification, the sample was injected into the high performance liquid chromatograph. After the target analyte was separated from impurities in the chromatographic column, it was detected by a fluorescence detector (FLD).

[0108] Minerals (calcium Ca, iron Fe, zinc Zn, selenium Se, magnesium Mg): Inductively coupled plasma mass spectrometry (ICP-MS) was used, following GB 5009.268-2016 "National Food Safety Standard - Determination of Multiple Elements in Food". Specific procedures: After thorough digestion in a microwave digester, the solution was introduced into the ICP-MS. Quantification was performed by measuring the intensity of characteristic spectral lines of each element and comparing them with a standard curve. Results are expressed as milligrams (mg / 100g) of each element per 100g of sample. The data in the table are the average of three independent determinations.

[0109] Table 7 Comparison of Nutritional Components between this Application and the Airflow-Based Cell Disruption Method

[0110]

[0111] Comparative Example 5

[0112] Existing technologies, due to their singular mechanisms of action, are generally trapped in the "impossible triangle" dilemma of cell disruption efficiency, activity retention, and energy consumption costs. This solution systematically solves all the shortcomings of traditional grinding (metal contamination), ball milling (thermal damage >100℃), shearing (ineffective against hard walls), ultrasound (penetration <0.1μm), single pulse (cell disruption rate <70%), pure supercritical (high investment / time-consuming), enzymatic hydrolysis (residual sensitization), and chemical methods (solvent toxicity) through a synergistic approach of ultrasonic pre-lysis (creating micro-defects), three-stage electromagnetic pulse (50 / 100 / 200Hz sequential directional breakdown of the sporophyll mechanical / electrochemical barriers), and supercritical CO2 cold explosion (low-temperature pressure relief, free radical quenching, in-situ antibacterial effect). Ultimately, it achieves a 99.2% cell disruption rate and 98.5% cell disruption rate at a consistently low temperature of 60℃ throughout the process. With SOD enzyme activity retention, zero loss of 153mg / kg geranialdehyde, commercial sterility, and energy consumption of 0.55kWh / kg (↓69.4%), a breakthrough in the industrialization of clean label cell wall breaking technology has been achieved through pure physical processes and zero additives.

[0113] Table 8 Comparison of the effects of different cell wall breaking methods

[0114]

[0115] Comparative Example 6

[0116] A comprehensive comparative analysis of ultrasonic pre-lysis-three-stage electromagnetic pulse-supercritical CO2 medium (ternary synergy) and three dual-technology combinations is presented in Table 9, based on four dimensions: cell disruption efficiency, activity retention, energy consumption cost, and industrialization potential.

[0117] Table 9 Overall Performance Comparison Table

[0118]

[0119] II. Detailed Technical Analysis

[0120] The irreplaceable nature of the ternary synergistic technology (ultrasonic pre-lysis + three-stage electromagnetic pulse + supercritical CO2) lies in its precise creation of micro-defects through ultrasonic pre-lysis (40kHz), pulse timing-controlled graded destruction (50 / 100 / 200Hz), and simultaneous plasma enhancement and cold explosion (8MPa→0.01MPa) with supercritical CO2. While achieving a breakthrough of 99.2% cell wall breakage rate, it protects 98.5% SOD enzyme activity and 153mg / kg geranialdehyde at a low temperature of 60℃ throughout the process, and achieves an ultra-low energy consumption of 0.55kWh / kg. It is the only technical path that combines the advantages of "ultimate cell wall breakage efficiency, near-zero activity loss, and halved energy consumption cost", forming a generational crushing advantage over the combination of two technologies.

[0121] For a detailed technical analysis, see Table 10; for a comparison of the activity retention mechanisms, see Table 11.

[0122] Table 10. Root Causes of Differences in Cell Wall Breaking Efficiency

[0123]

[0124] Table 11 Comparison of Activity Retention Mechanisms

[0125]

[0126] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for breaking the cell wall of pine pollen, characterized in that, The method includes: S1, pine pollen is mixed with water and homogenized to obtain a slurry; S2, after drying and sterilizing the slurry obtained in S1, is transferred to a differential pressure cell-breaking chamber containing supercritical CO2 fluid for ultrasonic treatment; The pressure inside the differential pressure cell breaking chamber is 8±0.2MPa and the temperature is 35±2℃. S3, activate the electromagnetic pulse device in the cell wall breaking chamber to perform three-stage pulse processing; The first-stage pulse has a frequency of 50Hz, an electric field strength of 20kV / cm, and an action time of 0.5s. The second-stage pulse has a frequency of 100Hz, an electric field strength of 15kV / cm, and an action time of 0.3s. The third-level pulse has a frequency of 200Hz, an electric field strength of 10kV / cm, and an action time of 0.2s. S4, instantly depressurize to 0.01MPa; obtain broken-cell pine pollen.

2. The cell wall breaking method according to claim 1, characterized in that, The water content of the pine pollen is less than 8%.

3. The cell wall breaking method according to claim 1, characterized in that, The ratio of pine pollen to water is 1:1.5 (g / ml); the solid content of the resulting slurry is 40%.

4. The cell wall breaking method according to claim 1, characterized in that, The processing conditions in S2 are: atomization pressure of 35 MPa; inlet temperature of 180°C and outlet temperature of 58°C in the drying chamber; sterilization conditions: 145°C / 2s.

5. The cell wall breaking method according to claim 1, characterized in that, The conditions for the ultrasonic treatment are: 40kHz variable frequency ultrasound, power density 100W / L.

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