Preparation method of seabuckthorn fruit polysaccharide and flax protein peptide coupled glycopeptide

Abstract

The application discloses a preparation method of seabuckthorn fruit polysaccharide and flax protein peptide coupled glycopeptide, and the method comprises the following steps: extracting high-purity seabuckthorn fruit polysaccharide and high-activity flax protein peptide; dissolving the two in a buffer solution according to a predetermined molar ratio, adjusting the pH to weak alkalinity, adding a specific catalytic aid to activate the reducing end of the polysaccharide; performing a low-temperature gradient coupling reaction in an inert atmosphere, promoting molecular recognition at low temperature first, then gradually increasing the temperature to complete covalent bonding, and monitoring the viscosity and turbidity online to control the reaction process; after the reaction is terminated, the coupled glycopeptide with uniform structure and good solubility is obtained through ultrafiltration purification, addition of a conformation stabilizer and freeze-drying. Through directional coupling and process precise control, the application significantly improves the reaction selectivity and efficiency, and the product has excellent stability and synergistic activities of antioxidant and immune regulation.

Description

Technical Field

[0001] This invention relates to the field of science, and particularly to a method for preparing a glycopeptide coupled with sea buckthorn fruit polysaccharide and flaxseed protein peptide. Background Technology

[0002] With the rapid development of the biopharmaceutical and functional food industries, the modification and recombination of natural bioactive macromolecules has become a core technological path to enhance product added value. By using covalent bonding or molecular self-assembly to structurally combine plant polysaccharides with natural proteins, not only can the solubility, stability, and bioavailability of individual components be improved, but significant synergistic physiological activities can also be generated through cross-molecular interactions. The precise modification of biomacromolecules relies on a deep understanding of the reaction microenvironment, molecular recognition mechanisms, and structural evolution patterns, which is of paramount strategic importance for developing novel hybrid molecules with specific biological functions.

[0003] Among them, sea buckthorn fruit polysaccharide and flaxseed protein peptides are natural active ingredients with great development potential. The glycopeptide molecules formed by their coupling show great application prospects in the fields of antioxidation, anti-fatigue, and immune regulation. The preparation process of this type of glycopeptide mainly involves the targeted docking between the reducing ends of the polysaccharide chain and the amino residues of the protein peptide, aiming to construct a functional carrier that retains the natural plant activity characteristics and has excellent physicochemical stability. The key to achieving this goal lies in precisely controlling the molar ratio between polysaccharides and peptides, the polar environment of the reaction interface, and the specificity of the catalytic system, thereby guiding the formation of stable covalent linkages between molecules rather than random physical associations.

[0004] However, existing technologies still face numerous challenges in coupling sea buckthorn fruit polysaccharides and flaxseed protein peptides. Traditional preparation methods often employ drastic thermochemical treatments or non-specific cross-linking, leading to damage to the branched structure of sea buckthorn polysaccharides and thermal denaturation of essential amino acids in flaxseed protein peptides, severely inhibiting the bioactivity of the products. Furthermore, the significant differences in molecular weight distribution, surface charge, and steric hindrance between the two result in low coupling efficiency and extremely complex product compositions under conventional reaction systems, making precise molecular-level coupling difficult. In addition, existing processes lack effective control over the conformational distribution of the product molecules, easily leading to irreversible molecular aggregation, resulting in reduced solubility or functional failure of the final glycopeptides in applicable scenarios.

[0005] Therefore, a method for preparing a glycopeptide coupled with sea buckthorn fruit polysaccharide and flaxseed protein peptide is desired. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing sea buckthorn fruit polysaccharide coupled with flaxseed protein peptides, which solves the problems mentioned in the background art.

[0007] This invention is achieved by providing a method for preparing a glycopeptide coupled with sea buckthorn fruit polysaccharide and flaxseed protein peptide, comprising the following specific steps: Step 1: Pre-treat the sea buckthorn fruit raw material to extract high-purity sea buckthorn fruit polysaccharide. After drying, the sea buckthorn fruit is crushed, defatted, and decolorized. A crude polysaccharide solution is obtained by hot water extraction combined with enzymatic hydrolysis. This solution is then purified by alcohol precipitation, dialysis, and freeze-drying. Step 2: Grade and separate flaxseed raw material to obtain highly active flaxseed protein peptides. Flaxseed powder is extracted using an alkali-soluble acid precipitation method to obtain flaxseed protein. Subsequently, a compound protease is used for targeted enzymatic hydrolysis, controlling the hydrolysis time and temperature within a specific range. The resulting hydrolysate is then subjected to enzyme inactivation, centrifugation, ultrafiltration, and spray drying to obtain flaxseed protein peptide powder. Step 3: Construct a controllable coupled reaction system. The sea buckthorn fruit polysaccharide and the flaxseed protein peptides are dissolved in a buffer solution at a predetermined molar ratio. The pH of the system is adjusted to a slightly lower value. Step 4: Implement a low-temperature gradient coupling reaction. Under an inert gas protection environment, place the reaction system in a programmable temperature control device. First, maintain the reaction in a lower temperature range for a period of time to promote molecular recognition and initial docking, and then gradually increase the temperature to a medium temperature range to complete covalent bonding. Monitor the viscosity and turbidity changes of the system throughout the reaction to determine the coupling process. Step 5: Purify and conformationally stabilize the coupling product. After the reaction is completed, immediately terminate the reaction and adjust the pH to neutral. Remove unreacted components by ultrafiltration membrane separation. Add a conformational stabilizer to the obtained concentrate and freeze-dry to obtain a structurally uniform and well-soluble sea buckthorn fruit polysaccharide and flax protein peptide coupled glycopeptide product.

[0008] Preferably, in step 1, the temperature of hot water extraction is controlled within a specific range, the extraction time is maintained at a predetermined duration, the enzymatic hydrolysis aid is a composite system of cellulase and pectinase, and the amount added is added according to a preset ratio of substrate mass. The enzymatic hydrolysis process is carried out at a constant stirring rate to ensure that the cell wall is fully degraded and intracellular polysaccharides are released.

[0009] Preferably, in step 2, the pH value of the alkali dissolution stage is adjusted to a specific alkaline range, the final pH value of the acid precipitation stage is controlled near the isoelectric point of the protein, the complex protease is composed of alkaline protease and flavor protease in a specific ratio, the reaction endpoint is dynamically adjusted by online monitoring of the degree of hydrolysis during the enzymatic hydrolysis process, and the molecular weight cutoff of the membrane used for ultrafiltration is set to a predetermined value in order to retain the target peptide and remove small molecule impurities.

[0010] Preferably, in step 3, the buffer solution is a phosphate or carbonate buffer system, the ionic strength of which is maintained within a preset threshold. The molar ratio of sea buckthorn polysaccharide to flaxseed protein peptide is precisely controlled within a specific range after being calculated based on the average molecular weight of the two. The specific catalytic aid is an organic small molecule compound containing a vicinal diol structure, and its concentration is added according to a preset ratio of the total volume of the reaction system to selectively activate the polysaccharide chain ends without affecting the peptide amino groups.

[0011] Preferably, in step 4, the inert gas is nitrogen or argon, the initial temperature range of the programmable temperature control device is set in a lower temperature range, the maintenance time is a predetermined duration, the heating rate is a specific temperature rise per minute, the upper limit of the medium temperature range does not exceed the critical temperature that causes peptide denaturation, the viscosity and turbidity are monitored by online sensors to collect data in real time, and the reaction is determined to have reached coupling equilibrium based on the preset trend.

[0012] Preferably, in step 5, the molecular weight cutoff of the ultrafiltration membrane is determined based on the theoretical molecular weight of the target coupled glycopeptide, the conformation stabilizer is a natural polyol, and its addition concentration is calculated according to a preset ratio of the dry weight of the product. The freeze-drying process adopts a step-by-step cooling and vacuum control strategy to ensure that the internal moisture gradient of the product escapes uniformly and avoids molecular aggregation or conformational collapse caused by rapid dehydration.

[0013] Compared with existing technologies, this invention has the following advantages: By constructing a low-temperature gradient coupling reaction system and a specific catalytic mechanism, this invention achieves directional covalent linkage between the reducing end of sea buckthorn fruit polysaccharide and the amino residues of flaxseed protein peptide, effectively avoiding the damage to active groups caused by traditional thermochemical methods and significantly improving the selectivity and efficiency of the coupling reaction; through refined control of the raw material extraction process, the structural integrity and bioactivity of the starting materials are ensured, providing high-quality precursors for subsequent coupling reactions; the molecular recognition stage and online process monitoring methods introduced during the reaction process ensure the uniformity of molecular weight distribution and structural reproducibility of the coupling products; after conformational stabilization treatment, the final product not only maintains excellent water solubility and dispersion stability, but also exhibits a synergistic enhancement effect in antioxidant and immunomodulatory functions, overcoming the technical defects of existing technologies such as complex product components, large loss of activity, and unstable application performance. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the overall technical solution architecture of a method for preparing a coupling glycopeptide of sea buckthorn fruit polysaccharide and flaxseed protein peptide according to an embodiment of this application. Detailed Implementation

[0015] Example 1 To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0016] In the preparation method of sea buckthorn fruit polysaccharide and flaxseed protein peptide coupled glycopeptide provided in this embodiment, the overall process strictly follows the physicochemical transformation logic and biological enzymatic reaction kinetics, and achieves the directional bonding of macromolecular polysaccharide and medium-molecular peptide segments through precise parameter control. The preparation method specifically includes the following steps: In the method, step 1 involves pretreating the sea buckthorn fruit raw material to extract high-purity sea buckthorn fruit polysaccharides. The specific operation process is as follows: A. Raw Material Screening and Grinding Control: Dried sea buckthorn berries with uniform maturity and no mold were selected as the starting material. First, the dried sea buckthorn berries were fed into a low-temperature airflow mill for grinding. During the grinding process, the airflow pressure within the grinding chamber was adjusted between 0.7 MPa and 0.9 MPa, and the feed rate was controlled between 20 kg and 30 kg per hour to ensure that the heat generated during material collision was immediately dissipated. The outlet temperature of the grinding chamber was consistently maintained below 25 degrees Celsius to prevent degradation of polysaccharide chains due to high temperatures. The ground sea buckthorn berry powder was then sieved through a 300-mesh sieve to ensure uniform particle size distribution.

[0017] B. Deep Degreasing and Decolorization Treatment: The pulverized sea buckthorn fruit powder is placed in an extraction vessel, and 5 to 8 times its weight in petroleum ether is added as a solvent. Under a constant temperature of 35 degrees Celsius, a mechanical stirrer is turned on, and the mixture is stirred at 150 rpm for 3 to 5 hours. During this process, the lipophilicity of the solvent fully dissolves the oils and fat-soluble impurities such as carotenoids in the sea buckthorn fruit powder. After stirring, solid-liquid separation is performed using a plate and frame filter press, and the filter cake is collected. Subsequently, the filter cake is redispersed in a 95% ethanol solution and decolorized through multi-stage circulation at 40 degrees Celsius until the ethanol washing liquid is colorless and transparent. This step not only removes pigment interference but also, through the fixation effect of ethanol, maintains a stable contractile conformation of the polysaccharide molecules, which is beneficial for subsequent extraction.

[0018] C. Hot Water Extraction and Dual-Enzyme-Assisted Degradation: The defatted and decolorized filter cake is added to deionized water at a liquid-to-solid ratio of 1:25 to 1:35, and the pH of the system is adjusted to 5.0 to 5.5. The heating system is turned on, and the temperature is raised to a specific range of 85°C to 90°C, maintaining the extraction time for 4 to 6 hours. During this process, to break the binding of plant cell walls to intracellular polysaccharides, a complex system of cellulase and pectinase is added to the system. The ratio of the complex enzymes is set according to the substrate characteristics, with a mass ratio of cellulase to pectinase of 2:1, and the total addition amount is 1.2% to 1.8% of the substrate dry mass. During hot water extraction, the mechanical stirring rate is maintained at 120 rpm. Continuous shear force helps the enzyme molecules to fully contact the cell wall fibers, ensuring sufficient degradation of the cell wall and release of intracellular polysaccharides.

[0019] D. Obtaining and Concentrating the Crude Polysaccharide Solution: After extraction, the solution is rapidly cooled to room temperature. It is then centrifuged at 8000 rpm for 20 minutes using a high-speed centrifuge to remove residual solids. The resulting supernatant is the crude polysaccharide solution. Using a rotary evaporator or vacuum membrane concentrator, the solution is concentrated to 20% to 25% of its original volume under conditions of a temperature not exceeding 55 degrees Celsius and a vacuum degree not lower than -0.085 MPa.

[0020] E. Purification Process: Anhydrous ethanol is slowly added dropwise to the concentrate until the volume fraction of ethanol in the system reaches 75% to 80%. During this process, polysaccharide molecules precipitate due to polarity changes. The mixture is placed in a cold storage at 4°C and allowed to stand for 12 to 24 hours. The precipitate is collected by centrifugation and washed three times sequentially with absolute ethanol and acetone to remove residual water and solvent. The precipitate is then dissolved in a small amount of deionized water and placed in a dialysis bag with a molecular weight cutoff of 8000 to 14000 Daltons. Dialysis is performed in flowing deionized water for 48 to 72 hours, with the dialysis water changed every 6 hours and the conductivity of the dialysate monitored online. Dialysis is stopped when the conductivity stabilizes and approaches the background value of deionized water. Finally, the dialysate is placed in a freeze dryer and dried for 48 hours at a cold trap temperature of -60°C and a vacuum degree of less than 10 Pa to obtain high-purity purified sea buckthorn fruit polysaccharide.

[0021] In the above method, step 2 involves grading and separating flaxseed raw materials to obtain highly active flaxseed protein peptides. Specific details are as follows: A. Alkali extraction of flaxseed protein: Hulled flaxseed powder is selected as the raw material, and its protein content must be confirmed by chemical analysis to meet the preset standards. The flaxseed powder is dispersed in deionized water, with a liquid-to-solid ratio controlled at 15:1 to 20:1. Sodium hydroxide solution is slowly added dropwise using a precision dosing pump to adjust the pH of the system to a specific alkaline range of 9.0 to 10.5. The mixture is continuously stirred at 200 rpm for 2 hours in a constant temperature water bath at 50 degrees Celsius. Under these conditions, the carboxyl groups in the protein undergo deprotonation, increasing the charge density and allowing the protein molecules to fully dissolve in the aqueous phase.

[0022] B. Isoelectric Point Precipitation and Purification: The alkali-dissolved suspension is centrifuged at 5000 times the acceleration due to gravity for 30 minutes. The supernatant is collected, and its pH is adjusted using hydrochloric acid solution. The isoelectric point of the protein is determined using an online potentiometer, typically with the endpoint pH controlled within the range of 4.2 to 4.6. At this point, the net charge of the protein molecules approaches zero, solubility is minimized, and flocculation and precipitation occur. The precipitate is collected and washed 3 to 5 times with deionized water to remove impurities and salts.

[0023] C. Targeted Enzymatic Hydrolysis Control: The flaxseed protein obtained from acid precipitation is resuspended in deionized water, with the substrate mass fraction adjusted to 5% to 8%. The pH of the system is adjusted to 8.0, and the temperature is controlled at 55 degrees Celsius. A complex protease, composed of alkaline protease and flavor protease in a 3:1 mass ratio, is added. The enzyme addition amount is 3000 to 5000 enzyme activity units per gram of protein. During enzymatic hydrolysis, alkali solution is added in real time using an automatic burette to maintain pH stability. The degree of hydrolysis is monitored online, and the calculation of the degree of hydrolysis is based on the increase in free amino groups obtained from chemical titration. When the degree of hydrolysis reaches the predetermined target range of 15% to 20%, enzyme inactivation is performed by instantaneously heating to 95 degrees Celsius and maintaining it for 15 minutes, thereby precisely locking the molecular weight distribution of peptides.

[0024] D. Centrifugation and Multistage Ultrafiltration Separation: The hydrolysate after enzyme inactivation is rapidly cooled to 20 degrees Celsius. Centrifugation is then performed at 12,000 rpm to thoroughly remove unhydrolyzed large protein molecules and enzyme residues. The resulting supernatant enters the ultrafiltration system. The ultrafiltration membranes are set with molecular weight cutoffs of 3000 Daltons and 1000 Daltons. First, a 3000 Dalton membrane is used to remove potentially larger molecular weight peptides. Subsequently, a 1000 Dalton membrane is used for concentration and desalting, retaining highly active target peptides with molecular weights between 1000 and 3000 Daltons.

[0025] E. Spray Drying to Powder: The concentrated liquid obtained from ultrafiltration is processed into powder using a pressure spray drying tower. The inlet air temperature is set between 160°C and 175°C, and the outlet air temperature is controlled between 75°C and 85°C. The atomizer speed is adjusted to 18,000 revolutions per minute to ensure fine droplet size. The final flaxseed protein peptide powder has excellent solubility and physiological activity.

[0026] In the method described, step 3 outlines the construction process of the controllable coupled reaction system. This stage is a prerequisite for achieving directed intermolecular connections, and the specific operations include: A. Precise Preparation of Reactant Concentrations: Based on the average molecular weights of sea buckthorn fruit polysaccharide and flaxseed protein peptide obtained in the preceding steps, the required mass ratio of the two to achieve the predetermined molar ratio is calculated using the molar mass conversion relationship. The molar ratio is precisely controlled between 1:1 and 1:3 to ensure that each polysaccharide molecule chain has a sufficient probability of colliding with the peptide segment. Both are dissolved separately in pre-prepared buffer solutions.

[0027] B. Selection of Buffer System and Environmental Conditioning: The buffer solution uses a phosphate buffer system or a carbonate buffer system, with a concentration maintained at 0.05 mol / L to 0.1 mol / L. The ionic strength of the buffer solution is adjusted by adding sodium chloride to ensure that the system conductivity is maintained within a preset threshold of 5 mSiemens / cm to 8 mSiemens / cm. The pH of the system is adjusted to a weakly alkaline range of 7.8 to 8.5 using a precision pH meter. This environment is conducive to the ring-opening of the polysaccharide reduction terminus and the nucleophilic attack of peptide amino groups.

[0028] C. Introduction and Activation of Specific Catalytic Auxiliary Agents: Specific catalytic auxiliary agents are introduced into the reaction system. These agents are small organic molecule compounds containing a vicinal diol structure, added at a concentration of 0.5% to 1.2% of the total volume of the reaction system. The mechanism of action of the catalytic auxiliary agent lies in the fact that its vicinal diol structure can form a temporary, highly active cyclic transition state with the aldehyde or hemiacetal structure at the end of the polysaccharide chain, thereby reducing the activation energy of the subsequent reaction with the amino group of the peptide. This process achieves selective activation of the reducing end of the polysaccharide without destroying the peptide bonds or other amino acid side chain groups within the peptide. After thorough mixing, an intermediate system with directional reaction capability is formed.

[0029] In the method, step 4 is the core implementation step, namely, carrying out a low-temperature gradient coupling reaction. This process is conducted in a highly controlled physical environment: A. Inertization of the reaction environment: Place the prepared reaction system in a reactor equipped with a jacket and a precision stirrer. Turn on the vacuum pump to remove air from the reactor, then purge with high-purity nitrogen or argon. Repeat this purging process three times to ensure the dissolved oxygen content in the system is reduced to a minimum. Throughout the reaction, maintain a slight positive pressure environment inside the reactor to prevent external air infiltration that could lead to non-specific oxidative degradation of polysaccharides or peptides.

[0030] B. Programmed Low-Temperature Gradient Temperature Control Strategy: First Stage: Initial Recognition Stage. The programmed temperature control device sets the system temperature within a low range of 25°C to 30°C and maintains this temperature for 3 to 5 hours. During this stage, due to the low thermal energy, molecules primarily recognize and dock through hydrogen bonds, van der Waals forces, and electrostatic attraction. Polar groups in the polysaccharide chains approach hydrophilic residues on the peptide surface, forming non-covalently bonded physical complexes, providing space for subsequent chemical bonding. Second Stage: Linear Heating Stage. The temperature control device steadily raises the system temperature to the target reaction temperature at a specific rate of 0.2°C to 0.5°C per minute. This slow heating process helps maintain the microscopic order of the system, avoiding violent molecular collisions and aggregation caused by sudden temperature changes. Third Stage: Intermediate-Temperature Covalent Bonding Stage. The temperature is maintained within a moderate range of 50°C to 65°C, and the reaction continues for 8 to 12 hours. The upper temperature limit is strictly set below the critical temperature that will not cause irreversible denaturation of the peptide's secondary or tertiary structure. During this period, the aforementioned activated polysaccharide reducing terminal undergoes a condensation reaction with the free amino group of the peptide (mainly the N-terminal amino group or the side chain amino group of the lysine residue), removing one molecule of water and forming a stable covalent link, thereby producing a coupled glycopeptide.

[0031] C. Online Process Monitoring and Endpoint Determination: Viscosity and turbidity data of the system are collected in real time throughout the reaction using online sensors. Viscosity is monitored using a falling ball or online rotational viscometer, while turbidity is monitored using a light scattering sensor. Due to the significant increase in molecular weight and conformational stretching after the coupling of polysaccharides and peptides, the kinetic viscosity of the system exhibits a characteristic upward trend, while turbidity fluctuates due to changes in molecular polarity and microscopic solubility. The rate of change of viscosity over time is calculated. When this rate of change is less than a preset small threshold for 60 consecutive minutes, and the turbidity curve tends to flatten, the reaction is considered to have reached coupling equilibrium.

[0032] In the method, step 5 involves purifying and conformationally stabilizing the coupling product to ensure high product quality. A. Reaction Termination and pH Neutralization: Once the reaction endpoint is detected, immediately activate the circulating cooling water system to rapidly reduce the temperature inside the reactor to below 10 degrees Celsius to terminate all chemical reactions. Add dilute hydrochloric acid or citric acid solution to restore the pH of the system to a neutral range of 6.8 to 7.2, ensuring the chemical properties of the product remain stable.

[0033] B. Ultrafiltration Membrane Purification and Separation: Cross-flow ultrafiltration technology is used to treat the reaction solution. The molecular weight cutoff of the ultrafiltration membrane is determined based on the theoretical molecular weight of the target coupled glycopeptides. Typically, a membrane module with a molecular weight cutoff slightly higher than the sum of the upper limits of sea buckthorn fruit polysaccharide and flaxseed protein peptides is selected (e.g., 30,000 to 50,000 Daltons). At an operating pressure of 0.15 MPa to 0.25 MPa, equal-volume filtration is performed by continuously adding purified water to completely remove unreacted residual peptides, monosaccharides, catalysts, and salt ions.

[0034] C. Precise addition of conformation stabilizer: Add a conformation stabilizer to the obtained glycopeptide concentrate. The stabilizer is selected from one or more natural polyols, such as trehalose, sorbitol, or mannitol. The concentration is precisely calculated at 3% to 5% of the product's dry weight. The polyol molecules form multi-point hydrogen bonds with the hydroxyl or amide groups on the glycopeptide molecular chain, constructing a stable hydration film on the molecular surface. This prevents excessive folding, aggregation, or even conformational collapse within or between molecules due to moisture loss during drying.

[0035] D. Stepped Freeze-Drying Strategy: The concentrated liquid containing stabilizers is dispensed into sterile trays. The freeze-drying process consists of three controlled stages: Stage 1: Rapid Pre-freezing. The material temperature is lowered to below -45°C at a rate of 5°C per minute and maintained for 4 hours to ensure that the water in the system forms fine and uniform ice crystals. Stage 2: Sublimation Drying. The vacuum degree is slowly adjusted to 15 Pa, and the heating plate temperature is increased in stages, 5°C each time, until the material temperature reaches approximately 0°C. Rapid temperature rise is strictly prohibited during this process to ensure that the internal moisture gradient escapes evenly and avoids bottle spraying. Stage 3: Desorption Drying. The temperature is further increased to 25°C to 30°C, maintaining a high vacuum degree, until the residual moisture in the product is below 3%. The final product is a homogeneous, well-soluble, and loosely porous sea buckthorn fruit polysaccharide and flaxseed protein peptide coupled glycopeptide.

[0036] Example 2 Based on Example 1, this embodiment has made engineering adjustments to some process parameters and stoichiometric logic to meet specific bioactivity requirements.

[0037] In step 1, the extraction process of sea buckthorn fruit polysaccharides was optimized. To further improve the branching degree of the polysaccharides, the liquid-to-solid ratio was adjusted to 40:1 during the hot water extraction stage. The proportion of hemicellulase was increased in the complex enzyme system, with the mass ratio of cellulase, pectinase, and hemicellulase set at 4:2:1. The stirring rate during the enzymatic hydrolysis process was adjusted in a stepwise manner, initially at 180 rpm to promote rapid mixing, and then reduced to 80 rpm to reduce shearing and breakage of the polymer chains. The alcohol precipitation process employed a gradient concentration precipitation method, first adjusting the ethanol concentration to 40% to remove high-molecular-weight impurities, and then increasing it to 80% to precipitate the target polysaccharides, resulting in a 5 percentage point increase in the purity of the refined polysaccharides.

[0038] In step 2, the preparation of flaxseed protein peptides focuses on obtaining components with a narrower molecular weight distribution. The pH of the alkaline dissolution process is strictly controlled at 9.5, and ultrasonic-assisted extraction is employed with an ultrasonic power set to 400-600 watts for 20 minutes, utilizing cavitation to improve protein dissolution efficiency. During enzymatic hydrolysis, an online feedback control logic based on conductivity changes is introduced, maintaining a constant pH by adding sodium hydroxide solution in real time. The ultrafiltration stage uses a ceramic membrane module with a molecular weight cutoff of 2000 Daltons, and by increasing the circulation rate, the recovery rate of the target peptides is ensured to reach over 90%.

[0039] In step 3, the stoichiometric ratio of the reactants was recalculated when constructing the coupled reaction system. Considering the number of reducing end groups on the sea buckthorn fruit polysaccharide chains, the molar ratio of polysaccharide to peptide was adjusted to 1:1.5. A borate buffer system was used instead of the original buffer solution to facilitate the formation of a more stable complex intermediate during the reaction. The specific catalyst was changed to an oligosaccharide alcohol with a multi-hydroxyl structure, and its addition was fine-tuned to 0.8% of the total system mass. Monitoring the surface tension of the system ensured that the reactants were highly dispersed in the solution.

[0040] In step 4, the temperature control curve for the low-temperature gradient coupling reaction was redesigned. Initial identification stage: The temperature was set at 20 degrees Celsius, and the duration was extended to 8 hours. By reducing the rate of molecular thermal motion, the physical encapsulation effect of the long polysaccharide chain on the peptide was enhanced, increasing the probability of effective intermolecular contact. Heating stage: The heating rate was slowed to 0.1 degrees Celsius per minute. This quasi-static heating method maximized the preservation of the molecular docking conformation formed in the previous stage. Covalent bonding stage: The target temperature was set at 58 degrees Celsius, and the reaction time was shortened to 10 hours. Utilizing this "low-temperature duration compensation" strategy, the impact of browning reaction on product color was further reduced while ensuring the efficiency of covalent bond formation. In the online monitoring system, a dynamic light scattering module was introduced to track the changes in the hydrodynamic radius of particles in the solution in real time, serving as an auxiliary indicator for measuring the degree of molecular coupling.

[0041] In step 5, nanofiltration was introduced into the purification process. A nanofiltration desalination step was added after ultrafiltration. Through the selective permeation principle of the nanofiltration membrane, monovalent and divalent salt ions in the system were thoroughly removed, reducing the ash content of the finished product to below 0.5%. A trehalose and mannitol compound system was used as the conformation stabilizer in a 2:1 ratio. Vacuum control during the freeze-drying process was more precise; the vacuum pump speed was controlled by frequency conversion, resulting in a non-linear, stepwise decrease in pressure as the material temperature increased. The resulting coupled glycopeptides exhibited extremely high resolubility in cold water, and their antioxidant activity was verified by in vitro free radical scavenging experiments, demonstrating a significant additive effect.

[0042] Example 3 This embodiment aims to verify the process stability and parameter consistency in a large-scale industrial production environment.

[0043] In the industrial implementation of step 1, the sea buckthorn fruit is pulverized using a large-scale low-temperature ultrafine pulverization system with a designed capacity of 200 kg per hour. The extraction vessel volume is increased to 5 cubic meters. During the hot water extraction process, external circulation heating technology is employed, and the temperature fluctuation of the fluid inside the vessel is precisely controlled within ±0.5 degrees Celsius using a plate heat exchanger. The enzymatic hydrolysis-assisted process uses a multi-point dosing method to ensure rapid and uniform diffusion of the enzyme preparation within the large reaction volume. The alcohol precipitation process utilizes a horizontal centrifuge for continuous solid-liquid separation.

[0044] In step 2, flaxseed protein extraction employed a continuous countercurrent extraction system. Flaxseed powder and alkaline aqueous solution flowed counter-currently within the system, significantly improving extraction efficiency and reducing solvent consumption. Directional enzymatic hydrolysis was carried out in an enzyme reaction vessel equipped with a high-precision mass flow meter. The amount of alkali added was controlled via a PID algorithm linked to an online pH meter, ensuring a constant chemical potential throughout the reaction process. The spray drying tower was equipped with a waste heat recovery system, significantly reducing the unit energy consumption of the drying process.

[0045] In step 3, due to the large volume of materials, a high-shear mixing emulsifier was used for pre-configuration of the reaction system. The dissolution process of sea buckthorn fruit polysaccharide and flaxseed protein peptides was completed within 30 minutes. The addition of specific catalysts was completed in the mixing pipeline via an online metering pump. The ionic strength and pH value of the system were monitored in real time and automatically corrected by a central control system.

[0046] In the industrial-scale reaction of step 4, the protection against inert gases is even more stringent. The reactor is equipped with an online oxygen content detector, which automatically triggers a nitrogen replenishment program when the oxygen volume fraction exceeds 0.01%. The programmed temperature control device employs a multi-segment slope control mode. In the lower temperature range (28 degrees Celsius), microbubbles generated by bottom aeration are used for auxiliary stirring, which enhances mass transfer while avoiding excessive mechanical shear that could damage the molecular structure. During the heating phase, fuzzy control logic is used to regulate the jacket hot water flow rate to ensure that there are no local hot spots within the large reactor. In the medium temperature range (62 degrees Celsius), the rheological parameters of the system are monitored. Using online acoustic vibration frequency monitoring technology, the propagation and attenuation characteristics of sound waves in the reaction medium are analyzed, and the viscosity changes of the system are inverted to determine the endpoint of the coupled reaction.

[0047] In step 5, large-scale purification employed a hollow fiber ultrafiltration system with a membrane area of ​​50 square meters. A high membrane flow rate was maintained using a circulation pump, effectively mitigating concentration polarization. Conformation stabilizers were homogenized in a mixing tank. Freeze-drying utilized a large-scale silo freeze dryer, with the material layer thickness strictly controlled to within 15 millimeters. Multiple thermocouple temperature sensors embedded within the material were used to precisely monitor the movement of the sublimation interface. The final product was then pulverized by airflow and passed through an 80-mesh sieve before being directly vacuum-packaged. Quality testing showed that the chemical composition ratio, molecular weight distribution, and functional activity indicators of the industrially produced glycopeptide product were highly consistent with the laboratory-scale test results, demonstrating the strong engineering reproducibility of this preparation method.

[0048] Example 4 This embodiment focuses on the effects of different catalyst concentrations on the structure and stability of the coupled reaction products.

[0049] While keeping the parameters of Example 1 unchanged in steps 1 and 2, this example sets up three parallel experimental groups in step 3. Group 1: The concentration of the specific catalyst was set to 0.3% of the total system volume. Group 2: The concentration of the catalyst was set to 0.9%. Group 3: The concentration of the catalyst was set to 1.5%.

[0050] In the low-temperature gradient coupling reaction of step 4, all experimental groups followed the same temperature control protocol: an initial temperature of 26 degrees Celsius maintained for 4 hours, followed by a temperature increase to 60 degrees Celsius at a rate of 0.3 degrees Celsius per minute and maintenance for 9 hours. The results showed that the first group, due to its low catalyst concentration, had limited activation of the polysaccharide reduction terminus, resulting in a lower coupling degree in the final product. Centrifugation analysis after step 5 revealed a significant number of unbound peptides. While the third group exhibited extremely high catalytic efficiency, the excessive catalytic agent increased the load on the ultrafiltration membrane during subsequent purification steps, and a small amount of non-specific cross-linking was observed in the product conformational characterization.

[0051] The second group (0.9% concentration) exhibited the best balance of technical parameters. Its product molar mass distribution was the most concentrated. High-performance liquid chromatography combined with multi-angle laser light scattering analysis showed that sea buckthorn fruit polysaccharide and flaxseed protein peptides successfully achieved a 1:1 or 1:2 covalent linkage. In step 5, an improved conformational stabilization strategy was adopted for the second group of products. In addition to adding 4% of the product dry weight of natural polyol, trace amounts of vitamin E acetate were also introduced as antioxidant protection for the fat-soluble side chains. During the freeze-drying stage, pressure swing desorption drying technology was used, that is, the vacuum degree was periodically changed at the end of the drying process, utilizing the "pressure pump" effect to remove the residual trace amounts of bound water inside the material.

[0052] The final product is a milky white, loose powder, and its protein content, polysaccharide content, and the proportion of bound glycopeptides all meet the design requirements. The product exhibits excellent solubility and stability in both acidic (pH 3.0) and alkaline (pH 9.0) environments, and after simulated gastrointestinal digestion experiments, it still retains more than 70% of its molecular integrity, demonstrating its potential high bioavailability.

[0053] Example 5 This embodiment further refines the influence of microscopic parameter changes on product conformation when the programmed temperature control device performs a low-temperature gradient coupling reaction.

[0054] In step 4, a detailed microscopic experiment was conducted on the heating rate. Experimental scheme A: A uniform temperature rise scheme was adopted, with the rate set at 0.4 degrees Celsius per minute. Experimental scheme B: A stepped nonlinear temperature rise scheme was adopted. That is, when rising from 25 degrees Celsius to 40 degrees Celsius, the rate was 0.6 degrees Celsius per minute; when rising from 40 degrees Celsius to 55 degrees Celsius, as the molecules approach the glass transition region, the rate decreased to 0.2 degrees Celsius per minute; when rising from 55 degrees Celsius to 62 degrees Celsius, the rate was finely adjusted to 0.3 degrees Celsius per minute.

[0055] Conformational analysis of the product obtained in step 5 (using circular dichroism spectroscopy and infrared spectroscopy) revealed that the peptide secondary structures (such as alpha helices and beta folds) of the product from experimental scheme B were more intact. This is because, within the critical temperature range of 40°C to 55°C, the slow energy input prevented thermal expansion mismatch between the long polysaccharide chain and the peptide backbone, thereby reducing the damage to fragile covalent bonds caused by shear forces.

[0056] In the online monitoring stage of step 4, this embodiment introduces near-infrared spectroscopy online analysis technology. An optical probe is installed on the sidewall of the reactor to acquire the absorption spectrum of the system in the 1000 nm to 2500 nm wavelength range in real time. Characteristic peak information related to amide bonds, hydroxyl groups, and specific covalent linkages is extracted using chemometric methods. When the rate of change of the intensity of the characteristic peak reaches zero and tends to be constant, the system automatically determines this as the reaction endpoint and automatically initiates the cooling termination procedure in step 5.

[0057] In step 5, the drying process employed microwave vacuum drying as an alternative or auxiliary to freeze drying. After the moisture content dropped to 10%, the microwave source was turned on, with the power set to 0.5 to 1.0 watts per gram of material. The penetrating heating characteristics of microwaves effectively broke down the capillary resistance within the material, allowing the last remaining stubborn bound water to escape rapidly. This combined drying method reduced the total drying time by 30%, and the porous structure of the product facilitated rapid wetting during application. The resulting sea buckthorn fruit polysaccharide coupled with flaxseed protein peptides exhibited a unique honeycomb-like arrangement in its microstructure, significantly increasing the specific surface area and providing excellent physical properties for its use as a functional raw material.

[0058] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a glycopeptide coupled with sea buckthorn fruit polysaccharide and flaxseed protein peptide, characterized in that, Includes the following steps: Step 1: Pre-treat the sea buckthorn fruit raw material to extract high-purity sea buckthorn fruit polysaccharide. After drying, the sea buckthorn fruit is crushed, defatted, and decolorized. A crude polysaccharide solution is obtained through hot water extraction combined with enzymatic hydrolysis. This solution is then purified by alcohol precipitation, dialysis, and freeze-drying. Step 2: Grade and separate flaxseed raw material to obtain highly active flaxseed protein peptides. The hulled flaxseed powder is extracted for flaxseed protein using an alkali-soluble acid precipitation method. Subsequently, a complex protease is used for targeted enzymatic hydrolysis, controlling the hydrolysis time and temperature within a specific range. The resulting hydrolysate is then subjected to enzyme inactivation, centrifugation, ultrafiltration, and spray drying to obtain flaxseed protein peptide powder. Step 3: Construct a controllable coupled reaction system. The purified sea buckthorn fruit polysaccharide and the flaxseed protein peptide powder are dissolved in a buffer solution at a predetermined molar ratio. The pH of the system was adjusted to a slightly alkaline range, and a specific catalytic agent was introduced to activate the polysaccharide reduction terminus, forming an intermediate with directional reaction capability. Step four involved implementing a low-temperature gradient coupling reaction. Under an inert gas environment, the intermediate was placed in a programmable temperature control device, initially maintained at a lower temperature range to promote molecular recognition and initial docking, and then gradually heated to a medium temperature range to complete covalent bonding. Throughout the reaction, changes in system viscosity and turbidity were monitored to determine the coupling progress. Step five involved purifying and conformationally stabilizing the coupling product. The reaction was immediately terminated after completion, and the pH was adjusted to neutral. Unreacted components were removed by ultrafiltration. The resulting concentrate was then freeze-dried after adding a conformation stabilizer to obtain the finished product of sea buckthorn fruit polysaccharide and flaxseed protein peptide coupled glycopeptide.

2. The method for preparing a sea buckthorn fruit polysaccharide coupled with flaxseed protein peptide according to claim 1, characterized in that: In step one, the pulverization process employs low-temperature airflow pulverization. During pulverization, the airflow pressure within the pulverization chamber is adjusted, and the feeding speed is controlled to ensure that the heat generated during material collision is immediately removed, maintaining the outlet temperature of the pulverization chamber below a predetermined range. The pulverized sea buckthorn fruit powder is then sieved to ensure uniform particle size distribution. In the degreasing process, the pulverized sea buckthorn fruit powder is placed in an extraction vessel, and a predetermined proportion of petroleum ether is added as a solvent. Under constant temperature conditions, a mechanical stirring device is activated for dissolution. After stirring, solid-liquid separation is achieved through pressure filtration, and the filter cake is collected. In the decolorization process, the filter cake is redispersed in an ethanol solution of a specific concentration and subjected to multi-stage cyclic decolorization under constant temperature conditions. The process continues until the ethanol washing solution becomes colorless and transparent. In the hot water extraction, deionized water is added according to a predetermined liquid-to-solid ratio, and the pH of the system is adjusted to an acidic range. The extraction time is maintained within a specific temperature range for a predetermined duration. The hot water extraction process utilizes a composite system of cellulase and pectinase for enzymatic hydrolysis assistance. The amount of the composite system added is based on a predetermined ratio of substrate mass, and the process is carried out at a constant stirring rate to ensure sufficient degradation of the cell wall and release of intracellular polysaccharides. In the dialysis step of the purification process, the polysaccharide solution is placed in a dialysis bag with a specific molecular weight cutoff and dialyzed in flowing deionized water. The conductivity of the dialysate is monitored online, and dialysis is stopped when the conductivity stabilizes and approaches the background value of deionized water.

3. The method for preparing a sea buckthorn fruit polysaccharide coupled with flaxseed protein peptide according to claim 1, characterized in that: In step two, the alkali extraction involves dispersing flaxseed powder in deionized water and controlling the liquid-to-solid ratio. The pH of the system is adjusted to a specific alkaline range by adding alkaline solution dropwise, and the mixture is continuously stirred in a constant temperature water bath to ensure that the protein molecules are fully dissolved in the aqueous phase. In the acid precipitation stage, the alkali-dissolved suspension is centrifuged and the supernatant is collected. The pH is adjusted using an acidic solution, and the isoelectric point of the protein is located using an online potential monitor. The final pH is controlled within a specific range near the isoelectric point of the protein to produce flocculation and precipitation. The precipitate is collected and washed with deionized water to remove impurities and salts. The targeted enzymatic hydrolysis involves resuspending the flaxseed protein obtained from acid precipitation in deionized water and adjusting the substrate mass fraction. A complex protease, composed of alkaline protease and flavor protease in a specific mass ratio, is added. During the enzymatic hydrolysis process, alkali solution is automatically added in real time via titration to maintain pH stability, and the degree of hydrolysis is monitored online. When the degree of hydrolysis reaches a predetermined target range, enzyme inactivation is performed by instantaneous heating to lock the molecular weight distribution of peptides. The ultrafiltration separation employs a multi-stage ultrafiltration system. The molecular weight cutoff of the ultrafiltration membrane is set to two different predetermined values. First, a large-pore ultrafiltration membrane is used to remove larger molecular weight peptides, and then a small-pore ultrafiltration membrane is used for concentration and desalting, retaining highly active target peptides within a specific molecular weight distribution range. The spray drying uses a pressure spray drying tower. By controlling the inlet air temperature, outlet air temperature, and atomizer rotation speed, the droplet size is ensured to be fine, resulting in soluble flaxseed protein peptide powder.

4. The method for preparing a sea buckthorn fruit polysaccharide coupled with flaxseed protein peptide according to claim 1, characterized in that: In step three, the buffer solution uses a phosphate buffer system or a carbonate buffer system, and the ionic strength of the buffer solution is adjusted by adding sodium chloride to maintain the system conductivity within a preset threshold range. The predetermined molar ratio is calculated based on the average molecular weight of the refined sea buckthorn fruit polysaccharide and flaxseed protein peptide powder to ensure that there is a sufficient probability of collision between the polysaccharide molecular chain and the peptide segment. The specific catalytic aid is an organic small molecule compound with a vicinal diol structure, and its addition amount is added according to a preset ratio of the total volume of the reaction system. The mechanism of the catalytic aid is to utilize the vicinal diol structure to form a temporary cyclic transition state with the aldehyde or hemiacetal structure at the end of the polysaccharide chain, thereby reducing the activation energy of the subsequent reaction with the amino group of the peptide segment, achieving selective activation of the polysaccharide reduction end without destroying the peptide bond inside the peptide segment. The pH of the system is adjusted using a precision pH meter to ensure that the system is in a weakly alkaline environment to facilitate the ring-opening of the polysaccharide reduction end and the nucleophilic attack of the peptide amino group.

5. The method for preparing a glycopeptide coupled with sea buckthorn fruit polysaccharide and flaxseed protein peptide according to claim 1, characterized in that: In step four, the low-temperature gradient coupling reaction is carried out in a reaction vessel equipped with a jacket and a precision stirrer. The dissolved oxygen content in the system is reduced to below a predetermined value by repeatedly evacuating and filling with high-purity nitrogen or argon. During the reaction, a weak positive pressure environment is maintained in the vessel to prevent non-specific oxidative degradation. The programmed temperature control of the low-temperature gradient coupling reaction includes an initial recognition stage, a linear heating stage, and a medium-temperature covalent bonding stage. In the initial recognition stage, the programmed temperature control device sets the system temperature in a low temperature range and maintains it for a predetermined duration. The low thermal energy is used to promote the initial recognition and docking of molecules through non-covalent forces to form a physical complex. During the linear heating phase, the temperature control device steadily increases the system temperature at a preset temperature rise rate to maintain the microscopic order of the system and prevent violent molecular aggregation. During the intermediate-temperature covalent bonding phase, the temperature is maintained in a moderate temperature range that does not cause irreversible denaturation of the peptide's secondary or tertiary structure, promoting the condensation reaction between the activated polysaccharide reducing ends and the free amino groups of the peptide and removing water molecules to form a stable covalent bond. The viscosity of the system is monitored using an online rotational viscometer, and the turbidity is monitored using a light scattering sensor. By collecting data in real time and calculating the rate of change of viscosity over time, when the rate of change is continuously less than a preset small threshold for a predetermined period of time and the turbidity curve tends to flatten, the reaction is determined to have reached a coupling equilibrium state.

6. The method for preparing a glycopeptide coupled with sea buckthorn fruit polysaccharide and flaxseed protein peptide according to claim 1, characterized in that: In step five, the reaction is terminated by rapidly reducing the temperature inside the reactor by activating the circulating cooling water system, and an acidic solution is added to restore the pH of the system to the neutral range. The ultrafiltration membrane separation employs cross-flow ultrafiltration technology. The molecular weight cutoff of the ultrafiltration membrane is selected based on the theoretical molecular weight of the target coupled glycopeptide. Unreacted residual components and catalysts are removed by continuous addition of purified water at a predetermined operating pressure using an equal-volume filtration method. The conformation stabilizer is selected from natural polyols, and its addition amount is calculated according to a preset ratio of the product dry weight. The stabilizer molecules form multi-point hydrogen bonds with the hydroxyl or amide groups on the glycopeptide molecular chain, constructing a stable hydration film on the molecular surface to prevent conformational collapse during the drying process. The freeze-drying employs a step-by-step freeze-drying strategy, including a rapid pre-freezing stage, a cooling stage, and a freezing stage. In the drying and desorption stages, the temperature and vacuum of the heating plate are controlled in stages, and the thickness of the material layer is kept within a predetermined range to ensure that the internal moisture gradient of the product escapes uniformly. The preparation method also introduces online analysis technology based on near-infrared spectroscopy. An optical probe installed on the side wall of the reactor acquires the absorption spectrum of the system in a specific wavelength band in real time. The characteristic peak information related to specific chemical bonds is extracted using chemometric methods. When the intensity change rate of the characteristic peak tends to be constant, the reaction termination program is automatically triggered. In large-scale industrial production, external circulation heating technology is used in conjunction with a multi-segment slope control mode to control temperature fluctuations. Online acoustic vibration frequency monitoring technology is used to analyze the propagation and attenuation characteristics of sound waves in the reaction medium, thereby inverting the viscosity change of the system and determining the endpoint of the coupled reaction.

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