Method for concentrating biochemical substances

Abstract

The invention relates to the technical field of biochemical substance separation and concentration, and discloses a concentration method of biochemical substances. According to the method, the technical target is realized through seven systematic steps: preparing a biocompatible deep eutectic solvent, constructing a water phase-DES two-phase system, preparing a phase antagonistic biosurfactant mixed system, dynamically regulating and controlling an interface, sequentially migrating and separating biochemical substances, performing multi-stage enrichment and collection, and recycling DES. According to the method, through innovative regulation and control on the interface property and the time dimension, the problem of high-selectivity separation of tiny biochemical substances with polarity difference is effectively solved, the separation purity and the recovery rate are remarkably improved, meanwhile, the biological activity is kept, and a green and sustainable technical solution is provided for efficient concentration of sensitive biochemical substances; the concentration method of the biochemical extract comprises the following steps: taking animal organs or tissues without human beings, and extracting bioactive components in the animal organs or tissues to be used for preparing medicines for treating or preventing cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] This invention relates to the field of biochemical substance separation and concentration technology, and more specifically, to a method for concentrating biochemical substances. Background Technology

[0002] In the field of biochemical substance separation and concentration, traditional technologies mainly rely on organic solvent-assisted concentration methods, such as acetone, methanol, and diethyl ether. While these organic solvents possess good dissolving capabilities, they generally suffer from problems such as damage to bioactive substances, high residue risks, and poor environmental friendliness. With the increasing demand for high-purity, highly active biochemical substances in fields such as biomedicine, food, and cosmetics, the limitations of traditional concentration technologies are becoming increasingly apparent.

[0003] In recent years, deep eutectic solvents (DES) have attracted widespread attention as a novel green solvent, but existing DES concentration technologies still face the following technical challenges when processing complex biochemical mixtures: 1) Mixtures of biochemical substances with similar structures but small differences in polarity are difficult to separate effectively, especially when they have similar molecular weights and chemical structures, resulting in poor selectivity and low purity; 2) A single surfactant control system is difficult to meet the separation requirements of multiple target substances, making it difficult to improve product purity and recovery rate simultaneously; 3) Once the interface characteristics are determined, they are difficult to adjust, lacking the ability to flexibly control them over time, and cannot adapt to the dynamic needs of different biochemical substances during the separation process. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for concentrating biochemical substances, comprising the following steps: Preparation of biocompatible deep eutectic solvent DES; The DES is mixed with an aqueous solution containing the target biochemical substance to form an aqueous-DES two-phase system; Prepare a mixed system of antagonistic biosurfactants containing first-class surfactants and second-class surfactants; A first type of surfactant is added to the aqueous-DES two-phase system, and a second type of surfactant is added after a preset time interval to achieve temporal changes in interface properties and form a dynamic interface environment. The target biochemical substances of different polarities are sequentially migrated in the dynamic interface environment in a time order and enriched in different phases. Phases containing target biochemical substances are collected in batches at predetermined time points to complete the separation and concentration of biochemical substances.

[0005] Preferred: The biocompatible deep eutectic solvent DES is prepared by mixing hydrogen bond acceptors and hydrogen bond donors in a molar ratio, wherein: the hydrogen bond acceptor is choline chloride; and the hydrogen bond donor is selected from at least one of glycerol, lactic acid, urea, and fructose.

[0006] Preferably, the molar ratio of choline chloride to the hydrogen bond donor is: When the hydrogen bond donor is glycerol, the molar ratio is 1:2; When the hydrogen bond donor is lactic acid, the molar ratio is 1:1; When the hydrogen bond donor is urea, the molar ratio is 1:2; When the hydrogen bond donor is fructose, the molar ratio is 2:1.

[0007] Preferably, the first type of surfactant is a hydrophilic surfactant selected from at least one of lecithin, bile salts and sodium salts of fatty acids; the second type of surfactant is a lipophilic surfactant selected from at least one of monoacylglycerol, diacylglycerol and fatty alcohols.

[0008] Preferably, the antagonistic biosurfactant mixture further comprises a third type of surfactant, which is an amphoteric or nonionic surfactant selected from at least one of egg phosphatidylcholine, sucrose ester and polysorbate; the step of adding surfactant to the aqueous-DES two-phase system further comprises: adding the third type of surfactant after a preset time interval following the addition of the second type of surfactant.

[0009] Preferably, the concentration of the first type of surfactant is 0.5-1.0% (w / v); the concentration of the second type of surfactant is 0.3-0.8% (w / v); and the preset time interval for adding the second type of surfactant is 10-30 minutes after the first type of surfactant is added.

[0010] Preferably, the concentration of the third type of surfactant is 0.1-0.5% (w / v); the preset time interval for adding the third type of surfactant is 15-40 minutes after the second type of surfactant is added.

[0011] Preferred method: The step of collecting the enriched phase in batches includes: determining a first collection time point and a second collection time point, wherein the first collection time point is 20-40 minutes after the addition of the second type of surfactant, and the second collection time point is 30-60 minutes after the addition of the third type of surfactant; collecting the first batch of enriched phase at the first collection time point, and then adding the same replenishing solution as the collected phase to the remaining system; collecting the second batch of enriched phase at the second collection time point.

[0012] Preferred method: The method further includes a step of DES recycling and reuse, including: The target biochemical substance was separated from the enriched phase to obtain a solution containing DES; Remove impurities from the DES-containing solution; Concentrate the solution and adjust the DES component ratio to the original proportion; The recovered DES is recycled for use in the next batch concentration process.

[0013] Preferably, the target biochemical substance is a biochemical mixture with similar structure but small polarity difference, and the difference in the logP value of the polarity difference is 0.05-0.1; the method can achieve a target biochemical substance product with a purity of ≥95%, a recovery rate of 85-95%, and a bioactivity retention rate of ≥90%.

[0014] The beneficial effects of this invention are as follows: This method employs a mixed system of antagonistic biosurfactants and a time-series addition technique to create a dynamically changing interfacial environment, significantly improving the separation selectivity for biochemical substances with minute polarity differences. This breakthrough enables the effective separation of structurally similar components that are difficult to distinguish using traditional methods over time.

[0015] Secondly, through multi-stage enrichment collection, this method successfully achieved a synergistic improvement in product purity and recovery rate, overcoming the technical bottleneck of traditional concentration methods where purity and recovery rate are difficult to balance. The precise timing of collection ensures that both the final product purity and recovery rate reach industry-leading levels.

[0016] Third, this method uses a biocompatible eutectic solvent instead of traditional organic solvents, combined with mild interfacial control conditions, effectively protecting the structure and function of sensitive biochemical substances, resulting in a significantly higher activity retention rate than traditional concentration methods. This is of great significance for the concentration of high-value bioproducts such as activity-sensitive enzymes, peptides, and antibodies.

[0017] Finally, this method establishes a complete DES and surfactant recycling system, achieving efficient solvent recovery and reuse, significantly reducing process costs and environmental burden, and conforming to the concepts of green chemistry and sustainable development. Attached Figure Description

[0018] Figure 1 It is a comparison of the selectivity of different methods for separating biochemical substances with similar polarities; Figure 2 This refers to the variation in separation selectivity of the method of the present invention at different collection time points; Figure 3 It compares the purity and recovery rate of various biochemical substances using different methods; Figure 4 This describes the purity-recovery rate relationship of the method of the present invention at different collection time points; Figure 5It is a comparison of the retention rates of various biochemical substances by different concentration methods; Figure 6 The effect of different concentration methods on the secondary structure of GOD (CD spectral analysis). Detailed Implementation

[0019] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0020] Example 1: This example presents a method for concentrating biochemical substances, comprising the following steps: Preparation of biocompatible deep eutectic solvent DES; Biocompatible deep eutectic solvent DES is prepared by mixing hydrogen bond acceptors and hydrogen bond donors in a molar ratio, wherein: The hydrogen bond acceptor is choline chloride; The hydrogen bond donor is selected from at least one of glycerol, lactic acid, urea, and fructose; The molar ratio of choline chloride to the hydrogen bond donor is: When the hydrogen bond donor is glycerol, the molar ratio is 1:2; When the hydrogen bond donor is lactic acid, the molar ratio is 1:1; When the hydrogen bond donor is urea, the molar ratio is 1:2; When the hydrogen bond donor is fructose, the molar ratio is 2:1.

[0021] DES is mixed with an aqueous solution containing the target biochemical substance to form an aqueous-DES two-phase system; Prepare a mixed system of antagonistic biosurfactants containing first-class surfactants and second-class surfactants; A first type of surfactant is added to the aqueous-DES two-phase system, and a second type of surfactant is added after a preset time interval to achieve temporal changes in interface properties and form a dynamic interface environment. The first type of surfactant is a hydrophilic surfactant, selected from lecithin; the second type of surfactant is a lipophilic surfactant, selected from monoacylglycerol. The antagonistic biosurfactant mixture system further includes a third type of surfactant, which is an amphoteric surfactant selected from egg phosphatidylcholine; the step of adding surfactant to the aqueous-DES two-phase system further includes: adding the third type of surfactant after a preset time interval following the addition of the second type of surfactant; The concentration of the first type of surfactant is 0.8% (w / v); The concentration of the second type of surfactant is 0.5% (w / v); The preset time interval for adding the second type of surfactant is 20 minutes after the first type of surfactant is added.

[0022] The concentration of the third type of surfactant is 0.3% (w / v); The preset time interval for adding the third type of surfactant is 28 minutes after the addition of the second type of surfactant. The target biochemical substances of different polarities are sequentially migrated in the dynamic interface environment in a time order and enriched in different phases. Phases containing target biochemical substances are collected in batches at specific time points to complete the separation and concentration of biochemical substances. The steps for collecting the enriched phase in batches include: determining a first collection time point and a second collection time point, wherein the first collection time point is 30 minutes after the addition of the second type of surfactant and the second collection time point is 45 minutes after the addition of the third type of surfactant; collecting the first batch of enriched phase at the first collection time point, and then adding the same replenishing solution as the collected phase to the remaining system; and collecting the second batch of enriched phase at the second collection time point.

[0023] The method also includes a step of DES recycling and reuse, including: The target biochemical substance was separated from the enriched phase to obtain a solution containing DES; Remove impurities from the DES-containing solution; Concentrate the solution and adjust the DES component ratio to the original proportion; The recovered DES is recycled for use in the next batch concentration process.

[0024] The target biochemical substances are a mixture of biochemical substances with similar structures but small differences in polarity, with a difference of 0.08 in logP values.

[0025] This method can achieve a target biochemical product purity of ≥95%, a recovery rate of 85-95%, and a bioactivity retention rate of ≥90%.

[0026] Example 2 differs from Example 1 in that: The first type of surfactant is a hydrophilic surfactant, selected from bile salts; The second type of surfactant is a lipophilic surfactant, selected from diacylglycerols; The concentration of the first type of surfactant is 0.5% (w / v); The concentration of the second type of surfactant is 0.3% (w / v); The preset time interval for adding the second type of surfactant is 10 minutes after the first type of surfactant is added.

[0027] The concentration of the third type of surfactant is 0.1% (w / v); The preset time interval for adding the third type of surfactant is 15 minutes after the addition of the second type of surfactant. Determine the first and second collection time points, where the first collection time point is 20 minutes after the addition of the second type of surfactant and the second collection time point is 30 minutes after the addition of the third type of surfactant. Collect the first batch of enriched phase at the first collection time point, and then add the same replenishing solution as the collected phase to the remaining system. Collect the second batch of enriched phase at the second collection time point.

[0028] The target biochemical substances are a mixture of biochemical substances with similar structures but small differences in polarity, with a difference of 0.05 in logP values.

[0029] Example 3 differs from Example 1 in that: The first type of surfactant is a hydrophilic surfactant, selected from sodium salts of fatty acids; The second type of surfactant is a lipophilic surfactant, selected from fatty alcohols; The concentration of the first type of surfactant is 1.0% (w / v); The concentration of the second type of surfactant is 0.8% (w / v); The preset time interval for adding the second type of surfactant is 30 minutes after the first type of surfactant is added.

[0030] The concentration of the third type of surfactant is 0.5% (w / v); The preset time interval for adding the third type of surfactant is 40 minutes after the addition of the second type of surfactant. Determine the first and second collection time points, where the first collection time point is 40 minutes after the addition of the second type of surfactant and the second collection time point is 60 minutes after the addition of the third type of surfactant. Collect the first batch of enriched phase at the first collection time point, and then add the same replenishing solution as the collected phase to the remaining system. Collect the second batch of enriched phase at the second collection time point.

[0031] The target biochemical substances are a mixture of biochemical substances with similar structures but small differences in polarity, with a difference of 0.1 in the logP value of polarity.

[0032] Example 4 illustrates the specific implementation steps of a method for concentrating biochemical substances: 1. Preparation of biocompatible deep eutectic solvent (DES) 1.1 Raw material specifications Hydrogen bond acceptor: choline chloride (ChCl), purity ≥98%, moisture content ≤1%, particle size 100-300 mesh; Hydrogen bond donor (one of the following can be selected): Glycerin: Purity ≥99.5%, density 1.25-1.26 g / cm³, viscosity approximately 950 mPa·s (20℃); L-lactic acid: purity ≥ 88-92% (aqueous solution), density 1.20-1.22 g / cm³; Urea: purity ≥99%, melting point 132-135℃; D-fructose: purity ≥99%, melting point 102-105℃.

[0033] 1.2 Preparation process Weigh the hydrogen bond acceptor and donor according to the following molar ratio: Choline chloride-glycerol: molar ratio 1:2 (mass ratio 1:2.1±0.1); Choline chloride-lactic acid: molar ratio 1:1 (mass ratio 1:1.05±0.05); Choline chloride-urea: molar ratio 1:2 (mass ratio 1:1.4±0.1); Choline chloride-fructose: molar ratio 2:1 (mass ratio 1:0.5±0.05).

[0034] The mixture is placed in a reaction vessel equipped with temperature control and mechanical stirring, and heated at 60-80°C (preferably 70±2°C) and 200-300 rpm for 2-4 hours (preferably 3 hours) until a clear, homogeneous, colorless or slightly yellow liquid is formed.

[0035] Select appropriate DES combinations based on the polarity characteristics of the target biochemical substance: For highly polar biochemical substances (logP<0), the choline chloride-glycerol system is selected; For biochemical substances with moderate polarity (logP=0-2), the choline chloride-lactic acid system is selected. For biochemical substances with low polarity (logP>2), the choline chloride-urea system is selected.

[0036] After the prepared DES was cooled to room temperature, it was filtered through a 0.22 μm polytetrafluoroethylene (PTFE) microporous membrane to remove insoluble matter and obtain a pure DES solution.

[0037] DES quality control indicators: moisture content ≤1% (Karl Fischer moisture determination method), viscosity range depends on different combinations (choline chloride-glycerol: 250-400 mPa·s; choline chloride-lactic acid: 100-200 mPa·s, measured by rotational viscometer at 25℃), clarity ≥95% transmittance (measured by spectrophotometer at 650nm wavelength).

[0038] 2. Construction of the aqueous-DES two-phase system 2.1 Construction process of the two-phase system System composition: The prepared DES was mixed with an aqueous solution containing the target biochemical substance in the following volume ratio: Standard operating range: 3:7 to 7:3 (DES: water, v / v).

[0039] Preferred range: Protein biochemicals: 4:6 to 5:5 (DES: water, v / v); Polypeptide biochemicals: 5:5 to 6:4 (DES: water, v / v); Small molecule biochemical substances: 6:4 to 7:3 (DES: water, v / v); Mixing method: Use a vortex mixer to oscillate and mix at a rate of 600-800 rpm for 10-30 minutes (preferably 15 minutes), and then centrifuge for 5 minutes under a centrifugal force of 100-200×g to form a clear two-phase separation system.

[0040] Biochemical substance concentration: The initial concentration of the target biochemical substance in the aqueous solution varies depending on its molecular weight. Macromolecules with a molecular weight >10kDa: 0.5-2.0% (w / v); Medium molecular weight molecules (1-10 kDa): 1.0-3.0% (w / v); Small molecules with a molecular weight <1kDa: 2.0-5.0% (w / v).

[0041] 2.2 Key Parameter Control Temperature control: Operating temperature range: 20-30℃; Preferred temperature: 25±1℃; Temperature fluctuations are controlled within ±0.5℃, achieved through a constant temperature water bath or a temperature-controlled reactor; Temperature measurement is performed using a thermometer or temperature sensor with an accuracy of ±0.1℃.

[0042] pH adjustment: Aqueous phase pH range: 5.0-8.0.

[0043] The optimal pH is determined based on the isoelectric point (pI) of the target substance: For substances with pI < 5.5: Select a pH range of 5.5-6.5; For substances with pI = 5.5-8.0: Select a pH value within ±0.5 units of the pI value; For substances with pI > 8.0: Select a pH of 7.5-8.0.

[0044] pH adjustment uses the following buffer system (final concentration 20-50mM): pH 5.0-6.0: Acetic acid-sodium acetate buffer solution; pH 6.0-7.5: Phosphate buffer; pH 7.5-8.0: Tris-HCl buffer.

[0045] System stability index: The formed two-phase system must meet the following conditions: Interface clarity: The interface should be clearly visible to the naked eye, with no obvious emulsification.

[0046] Phase volume ratio stability: The change in phase volume ratio after standing for 2 hours should be <5%.

[0047] Transparency: Transparency of each phase ≥ 90% (measured using transmittance at 650 nm).

[0048] After this stage is completed, biochemical substances will form a preliminary distribution between the two phases according to their molecular weight, charge and hydrophilic / hydrophobic properties. The partition coefficient K value (DES phase concentration / water phase concentration) is usually in the range of 0.1-10.

[0049] Preparation of a 3-phase antagonistic biosurfactant mixture system This step is the core of this method, involving the selection of two or more biosurfactants with antagonistic effects, and their preparation into a mixed system in a defined ratio. Antagonism refers to the competition for arrangement positions between different surfactants at the interface between two phases due to differences in molecular structure, critical micelle concentration (CMC), and surface activity characteristics, resulting in an arrangement structure that changes over time.

[0050] 3.1 Selection and Specifications of Surfactants 1) Class I surfactants (highly hydrophilic): Lecithin: HLB value 7-9, purity ≥95%, phosphatidylcholine content ≥80%; Bile salts: sodium deoxycholate, purity ≥98%, CMC 2-6 mM; Sodium fatty acid salts: sodium laurylate, purity ≥99%, CMC 8-10 mM. 2) Second type of surfactant (strong lipophilicity): Monoacylglycerol: monolauroylglycerol, HLB value 3-5, purity ≥98%; Diacylglycerol: distearylglycerol, purity ≥95%, melting point 55-60℃; Fatty alcohol: cetyl alcohol, purity ≥99%, melting point 45-50℃; 3) Class III surfactants (amphoteric or nonionic): Egg vitellidoylcholine: purity ≥98%, degree of hydrolysis <2%; Sucrose esters: sucrose monostearate, HLB value 15-16, purity ≥95%; Polysorbates: polysorbate-80, HLB value 14-16, purity ≥99%; 3.2 Preparation of Surfactant Stock Solution 1) Stock solution concentration: hydrophilic surfactant: 20 mg / mL (15-25 mg / mL); lipophilic surfactant: 15 mg / mL (10-20 mg / mL); amphoteric or nonionic surfactant: 30 mg / mL (25-35 mg / mL); 2) Preparation method: Hydrophilic surfactants were dissolved in phosphate buffer (50mM, pH 7.0); lipophilic surfactants were dissolved in DES pretreated with 30% ethanol; amphoteric surfactants were dissolved in an aqueous solution containing 25% glycerol. All stock solutions were stirred at 40-50℃ for 30 minutes to promote dissolution. Insoluble particles were removed by filtration using a 0.22μm polyethersulfone (PES) microporous membrane. 3) Stability indicators of the stock solution: Storage temperature: 4℃; Shelf life: 7 days for hydrophilic and nonionic surfactants, 3 days for lipophilic surfactants; Before use, the appearance should be checked and there should be no precipitation, layering or turbidity.

[0051] 3.3 Selection and Addition Design of Surfactants 1) Preferred pairings of surfactant combinations: Polar proteins: lecithin (Class I) + monoacylglycerol (Class II) + polysorbate-80 (Class III); Moderately polar peptides: bile salts (Class I) + diacylglycerols (Class II); Low polar small molecules: sodium fatty acid salts (Class I) + fatty alcohols (Class II); 2) Concentration range: Class I surfactants: final concentration 0.5-1.0% (w / v), preferably 0.8%; Class II surfactants: final concentration 0.3-0.8% (w / v), preferably 0.5%; Class III surfactants: final concentration 0.1-0.5% (w / v), preferably 0.3%; 3) Addition timing design: Class I surfactants: added immediately after the formation of the two-phase system; Class II surfactants: added 15 minutes (10-20 minutes) after the addition of Class I surfactants; Class III surfactants: added 25 minutes (20-30 minutes) after the addition of Class II surfactants; 4) Addition method: Use a microsyringe or pipette to slowly add the surfactant stock solution to the system; control the addition rate at 0.5-1.0 mL / min; add the solution 0.5 cm below the interface between the two phases; after adding, gently shake the mixing container (5-10 times / min for 1 minute) to avoid violent shaking; This hybrid system generates time-dependent interfacial polarity and charge gradients through competitive adsorption and dynamic rearrangement of surfactant molecules at the interface. Changes in the interfacial activity gradient can be monitored by interfacial tension measurement. Typical ranges of interfacial tension change are: initial value 30-35 mN / m, decreasing to 20-25 mN / m after the addition of the first type of surfactant, further decreasing to 12-18 mN / m after the addition of the second type, and stabilizing at 8-12 mN / m after the addition of the third type.

[0052] 4. Dynamic Interface Adjustment The second core part of this method is the dynamic control of the interface. By adding different surfactants in sequence, the properties of the DES-water two-phase interface change over time, forming a time-sequential separation gradient.

[0053] 4.1 Interface-based dynamic control of process flow 1) First stage: Formation of hydrophilic interface. Operation steps: Add a first-class surfactant (hydrophilic) to the formed aqueous-DES two-phase system using a precision pipette; Addition location: 0.5 cm below the interface between the two phases; Addition amount: Calculate the required volume according to the concentration specified in Section 4.3.3 (usually 2-5% of the total system volume); Mixing method: Mix at 100-150 rpm for 3-5 minutes (preferably 4 minutes) using a horizontal rotary mixer; Standing time: Let stand at room temperature (25±1℃) for 8 minutes (5-10 minutes). Interface feature metrics: Interfacial tension: 20-25 mN / m (determined using the pendant method or drop volume method); Interface potential: -15 to -25 mV; Interface thickness: 50-80 nm (estimated value); Interfacial hydrophilicity index (HI): 0.7-0.8 (calculated with HI of 1.0 for pure aqueous phase and HI of 0 for pure DES phase).

[0054] 2) Second Stage: Dynamic Transformation of Interfacial Properties. Procedure: 15 minutes (10-30 minutes) after adding the first type of surfactant, precisely time the addition, then add the second type of surfactant; Addition Location: 0.5 cm above the interface between the two phases; Addition Temperature: Same as the system temperature (25±1℃); Mixing Method: Use vertical point stirring at 80-120 times / minute for 2-3 minutes; Settling Time: 15 minutes (10-20 minutes). Transformation process monitoring indicators: Interfacial tension change rate: decreases by 2-4 mN / m every 5 minutes; Interfacial fluidity: determined by the interfacial diffusion coefficient, from an initial value of (1-3)×10 -10 m² / s increased to (5-8)×10 -10 m² / s; Interfacial hydrophilicity index (HI): gradually decreases from 0.7-0.8 to 0.4-0.5.

[0055] 3) Third Stage: Multi-level Interface Fine-tuning (Applicable to complex biochemical mixtures). Procedure: Add the third surfactant 25 minutes (15-40 minutes) after adding the second type of surfactant; Addition method: Use a pulsed multi-point addition method, adding equal amounts in 3-5 separate additions; Addition interval: 2 minutes between each addition; Mixing method: After each addition, perform a short, gentle shaking (60-80 rpm, 30 seconds); Standing time: After the last addition, let stand for 20 minutes (15-25 minutes).

[0056] Final interface feature metrics: Interfacial tension: 8-12 mN / m; Interface potential: near neutral (-5 to +5 mV); Interface thickness: 100-150 nm; Interfacial hydrophilicity index (HI): 0.3-0.4; 4.2 Monitoring and Control of Interface Changes 1) Real-time monitoring methods: Interfacial tension measurement: Measure every 5 minutes using an interfacial tension meter (drop volume method or ring method); Interfacial potential measurement: Measure the potential change in the interfacial region using a Zeta potential analyzer; Optical monitoring: Observe the change in interfacial thickness using a confocal microscope (optional); Real-time monitoring of the partition coefficient of the target substance: Take 10 μL of sample from each of the two phases every 5-10 minutes and determine the concentration by UV-Vis spectroscopy or fluorescence method.

[0057] 2) Judgment criteria for dynamic interface control: The interface tension change curve should show a step-like decrease; the decrease in interface tension from the first stage to the second stage should be ≥30%; the final interface tension should be ≥60% lower than the initial interface tension; the interface should remain clearly visible throughout the process, without significant emulsification.

[0058] By precisely controlling the timing of surfactant addition and the kinetics of interfacial property changes, a time-evolving phase-interfacial environment is created, enabling biochemical substances of different polarities to selectively migrate within different time windows. This fundamentally solves the problem of low selectivity in traditional static interfacial separation methods. The kinetic characteristics of interfacial changes can be quantitatively characterized using the Gibbs adsorption equation and interfacial tension-time curves. Typical interfacial dynamic changes conform to the modified Langmuir-Blodgett adsorption model.

[0059] 5. Sequential migration separation Sequential migration separation is the third core part of this method. Based on dynamic interface regulation, different biochemical substances migrate sequentially in time order, thereby achieving highly selective separation.

[0060] 5.1 Process flow of sequential migration 1) Initial distribution equilibrium formation stage: Time window: 0-15 minutes after adding the first type of surfactant; Operating conditions: Temperature control: 25±0.5℃; Oscillation conditions: Slight horizontal oscillation 3-5 times per minute (amplitude ≤1cm) in the first 5 minutes - Standing time: then stand for 10 minutes.

[0061] Allocation characteristics: Distribution ratio of highly polar biochemical substances (logP<0): aqueous phase ≥80%, DES phase ≤20%; Distribution of moderately polar biochemical substances (logP=0-2): aqueous phase 55-75%, DES phase 25-45%; Distribution ratio of low polarity biochemical substances (logP>2): 30-50% in aqueous phase and 50-70% in DES phase.

[0062] Quality control indicators: Phase stability: Clear interface, no obvious emulsification; Partition coefficient determination: Sampling and determining the concentration ratio of the target substance in the two phases; Distribution balance determination: The change in the distribution coefficient between two consecutive sampling measurements is <5%.

[0063] 2) First stage directional migration: Time window: 0-25 minutes after adding the second type of surfactant; Key conditions: Temperature gradient: A slight temperature gradient can be established (bottom is 1-2°C higher than top) to promote vertical circulation of substances; Oscillation mode: Perform a "tilt-back" operation every 5 minutes (tilt angle 15-25°, 3-5 seconds each time); Substance migration monitoring: Take 10 μL samples from the aqueous phase and DES phase every 5 minutes for rapid analysis.

[0064] Migration behavior characteristics: Target migrating substance 1 (typically a less polar component): Aqueous phase concentration decrease rate 15-25% every 5 minutes; DES phase concentration increase rate: 20-30% every 5 minutes; Critical migration time point: Significant allocation differences typically appear 15 ± 5 minutes after addition; Separation Selectivity Index (SSI): The ratio of the partition coefficient of the target analyte to that of the major impurities should be ≥3.

[0065] 3) Multi-stage sequential migration (suitable for complex biochemical mixtures): Time window: 0-35 minutes after adding the third type of surfactant; Advanced operation: Gradient stirring method: Micro-stirring (5-10 rpm) in the area near the interface using a special stirring rod; Stirring position: 0.5-1.0 mm from the interface; Stirring cycle: 10 seconds of stirring followed by 50 seconds of standing as one cycle; Stirring direction: Alternate between clockwise and counterclockwise.

[0066] Fine-grained transfer features: The second target substance (usually a moderately polar component) migrates at a rate 40-60% slower than the first target substance. Polarity difference detection limit: capable of distinguishing components with a logP value difference of only 0.05-0.1; Migration transition zone detection: Determine the inflection point of material migration changes through real-time monitoring.

[0067] 5.2 Monitoring and Control of the Migration Process 1) Real-time monitoring: Conventional spectral monitoring: using a micro spectrophotometer (sample volume ≤ 5 μL); measurement wavelength: selected according to the characteristic absorption wavelength of the target substance (protein 280 nm, nucleic acid 260 nm, etc.); measurement frequency: once every 5 minutes.

[0068] High-sensitivity monitoring (suitable for low-concentration substances): Fluorescent labeling method: The target substance is labeled with fluorophores such as fluorescein or rhodamine; Fluorescence detection limit: ≤0.01 mg / mL; Sample dilution factor: Determined based on the initial concentration, typically 5-20 times.

[0069] Specificity monitoring (applicable to specific biochemical substances): Enzyme activity assay: Enzyme activity is measured using specific substrates to determine the migration of active proteins; Immunoassay: Rapid detection of the target protein using ELISA or immunochromatography; 2) Migration endpoint determination criteria: Resolution (Rs) calculation: Rs = 2×|tR1-tR2| / (w1+w2) ≥ 1.5; where tR is the time for the substance migration to reach equilibrium, and w is the width of the concentration change curve; Purity index: The proportion of the target substance in the total solute in the target phase ≥ 90%; Recovery rate index: The recovery rate of the target substance in the target phase ≥ 80%; Concentration difference stability: The concentration ratio of the target substance in the two phases changes ≤ 3% after three consecutive measurements. By precisely controlling and monitoring the migration behavior of biochemical substances in a DES-water two-phase system over time, highly selective separation of biochemical substances with minimal polarity differences is achieved. The core technological breakthrough lies in utilizing the dynamic changes in interfacial properties over time to create a separation window in the temporal dimension, enabling the effective differentiation of similar substances that are difficult to separate using traditional techniques at different time points. Once monitoring confirms that the sequential migration has reached the preset separation index, the system enters a multi-stage enrichment and collection phase for the efficient collection of successfully separated substances.

[0070] 6. Multi-stage enrichment collection Multi-stage enrichment collection is the fourth core part of this method. The target substances enriched at different time points during the sequential migration process are collected in batches to obtain high-purity concentrated products.

[0071] 6.1 Enrichment and Collection Process Flow 1) Precise determination of collection time point: Basis for collection decision: Analysis and modeling based on real-time monitoring data: Fitting the concentration change curve of the substance by least squares method; maximizing the concentration difference principle: Calculating the time point when the concentration difference of the target substance in the two phases reaches the maximum value; Comprehensive scoring of purity and yield: Using a weighted scoring system (purity weight 0.6, recovery rate weight 0.4).

[0072] Typical collection time points: First collection time point: 30±10 minutes after the addition of the second type of surfactant; Early warning detection point: Monitoring will begin 10 minutes before the scheduled collection time and will be conducted every 2 minutes; Collection window width: within ±2 minutes of the optimal time point; Second collection time point: 45±15 minutes after the addition of the third type of surfactant; Early warning detection point: Monitoring will begin 15 minutes before the scheduled collection time and will be conducted every 3 minutes; Collection window width: within ±3 minutes of the optimal time point.

[0073] 2) Precision phase separation technology: Selection of separation equipment: Laboratory scale (≤50mL): use precision pipettes or micropipette pumps; Pilot scale (50mL-1L): use separatory funnels or dedicated phase separators; Industrial scale (>1L): use continuous phase separation systems.

[0074] Separation operation techniques: Interface protection technology: Stop any shaking or stirring 1-2 minutes before collection; Pipette angle: Insert at a 45° angle, keeping the pipette tip 3-5 mm away from the interface; Pipetting speed: initially slow (0.5 mL / min), then medium speed (1-2 mL / min) after stabilization; Termination technique: Stop when 1-2mm away from the interface to avoid interface damage.

[0075] Separation quality control: Cross-contamination control: The proportion of non-target phases mixed in the target phase is <0.5%; Interface material processing: The ±1mm area at the interface can be collected separately as an intermediate phase; Collection volume control: Reserve 10-15% of the total system as a buffer to avoid over-collection.

[0076] 3) Intermediate transition stage treatment: Replenishment solution formulation: Aqueous phase replenishment solution: Same buffer system as the original aqueous phase, with matched pH and ionic strength; DES phase replenishment solution: Fresh DES with the same component ratio as the original DES phase.

[0077] Additional steps: Replenishment amount: equal to 80-90% of the volume of the collected phase; Supplementary temperature: Same as the system temperature (25±1℃); Supplementation method: Add slowly using a peristaltic pump (flow rate 1-2 mL / min); Homogenization: After adding, stir vertically (60-80 rpm, 2-3 minutes).

[0078] Balance establishment: Let stand for 10-15 minutes; Equilibrium determination: The phase volume ratio is stable and the interface is clear; Temperature control: Maintain at 25±1℃.

[0079] 4) Multi-batch enrichment material collection: Batch collection strategy: First batch: mainly collect target components with the greatest polarity difference; Second batch: collect target components with moderate polarity difference; Third batch (optional): collect target components with similar polarity.

[0080] Handling inter-batch relationships: Independent processing mode: Different batches are processed separately, which is used when there are significant differences in the target substances; Gradient merging mode: Adjacent batches are partially merged to form polarity gradient products; All batches are merged: All batches are processed together, used when overall recovery rate is the priority.

[0081] Collection, recording, and tracking: Batch identifier: Records information such as collection time, phase type, and volume; Sample retention: Retain 1-2% of the sample from each batch for analysis; Cross-validation: Analyze the overlap of components between different batches.

[0082] 6.2 Post-processing of enriched phases 1) Preliminary purification treatment: Impurity removal: Particle filtration: filtration using a 0.45μm polyethersulfone (PES) microporous membrane; Desalting treatment: dialysis using a dialysis membrane with a molecular weight cutoff of 1-3kDa; Dialysis buffer: 20mM phosphate buffer, pH 7.0; Dialysis ratio: sample: buffer = 1:50-100; Dialysis time: 12-16 hours at 4℃, with 2-3 buffer changes.

[0083] Concentration adjustment: Diluting the sample: If the concentration is too high, dilute it to the required concentration for analysis using an appropriate buffer solution; Concentrating the sample: If the concentration is too low, use an ultrafiltration centrifuge tube (the molecular weight cutoff depends on the target analyte) to concentrate it.

[0084] 2) High-efficiency concentration method: macromolecular biochemical substances (>10kDa): Method selection: tangential flow ultrafiltration; membrane material: polyethersulfone (PES) or regenerated cellulose (RC) membrane; operating pressure: 15-25 psi; concentration factor: 10-20 times.

[0085] Medium molecular weight biochemical substances (1-10 kDa): Method selection: centrifugation and ultrafiltration; Operating conditions: 4000×g, 4℃; Concentration time: in batches, 20-30 minutes each time; Final concentration: 5-10 mg / mL.

[0086] Small molecule biochemical substances (<1kDa): Method selection: Vacuum rotary evaporation concentration; Temperature control: ≤40℃; Pressure control: -0.05 to -0.08 MPa; Concentration endpoint: The solution volume is reduced to 1 / 5 to 1 / 10 of its original volume.

[0087] 3) Product quality control: Purity analysis: High performance liquid chromatography (HPLC): purity ≥95%; Gel electrophoresis (SDS-PAGE): target band intensity accounts for ≥90% of the total intensity; Mass spectrometry analysis: relative content of major peaks ≥90%.

[0088] Activity assay: Activity retention: ≥85% (compared to untreated samples); Specific activity increased by ≥3 times (compared to the initial mixture).

[0089] Storage conditions: Temperature: -20℃ for long-term storage, 4℃ for short-term storage; Additives: Add 10-20% glycerin as a stabilizer as needed; Aliquoting: Aliquot into small volumes (0.5-1.0 mL / tube) to avoid repeated freeze-thaw cycles.

[0090] This multi-stage enrichment and collection technology is closely integrated with sequential migration separation. By precisely controlling the collection timing and using professional phase separation techniques, it enables the accurate collection of biochemical substances with different migration behaviors over time, significantly improving the purity of the final product (by 15-25%) and the total recovery rate (by 20-40%), while maintaining biological activity.

[0091] 7. DES Recycling and Reuse DES recycling is the final and crucial step in this method, establishing a complete material recycling system and achieving both environmental friendliness and economic efficiency. This step is closely linked to the aforementioned enrichment and collection steps, processing the collected enriched phase and the remaining phase.

[0092] 7.1 DES Recycling Process Flow 1) DES separation in the enrichment phase: Selection of biological material separation method: Macromolecular biological materials (>10kDa): Use an ultrafiltration membrane with a molecular weight cutoff of 10kDa; Ultrafiltration operating pressure: 20-30 psi (laboratory scale) or 15-25 psi (pilot scale); Membrane material: Polysulfone (PS) or polyethersulfone (PES); Operating temperature: Room temperature (20-25℃); Permeate collection: Collect the permeate containing DES for recovery.

[0093] Small and medium molecular weight biological substances (<10kDa): Use a dialysis membrane with a molecular weight cutoff of 1-3kDa; Dialysis conditions: 4℃, stirring speed 60-80rpm; Dialysis solution: deionized water, pH 6.0-7.0; Dialysis ratio: sample:dialysis solution = 1:50-100; Replacement frequency: replace the dialysis solution every 6-8 hours, for a total of 3-4 times; Dialysis solution collection: combine all dialysis solutions for DES recovery.

[0094] Separation efficiency criteria: residual amount of target biological material in DES <1%; residual amount of DES in biological material product <0.5%; loss of biological activity during separation <5%.

[0095] 2) Removal of impurities from DES solution: Removal of residual biomacromolecules: Method: Heat treatment combined with microfiltration; Temperature conditions: 60±2℃; Treatment time: 20±5 minutes; Filter membrane: 0.45μm PTFE or PES microporous filter membrane.

[0096] Removal of colored impurities: Method: Activated carbon adsorption treatment; Activated carbon type: food-grade powdered activated carbon (specific surface area ≥800m² / g); Dosage: 0.5-2% (w / v), adjusted according to impurity content; Contact time: 40±5 minutes; Stirring conditions: 100-150 rpm; Activated carbon removal: filtration through a 0.22μm filter membrane.

[0097] Inorganic salt removal: Method: Ion exchange resin treatment (applicable to cases where DES is recovered ≥ 5 times); Resin selection: Mixed ion exchange resin; Treatment method: DES solution is passed through the resin bed at a flow rate of 1 BV / h; Rinsing: After treatment, the resin is rinsed with a small amount of deionized water, and the rinsing solution is collected and combined with the DES solution.

[0098] 3) DES Concentration and Component Adjustment: Moisture Removal: Method: Vacuum Evaporation; Temperature Control: Preferred 50±2℃, range 45-60℃; Pressure Conditions: -0.07±0.01MPa; Stirring Speed: 60-80rpm; Endpoint Control: Achieve Target Viscosity or Refractive Index.

[0099] Component analysis methods: Main method: High performance liquid chromatography (HPLC) was used to determine the content of each component; chromatographic column: NH2 column or HILIC column; mobile phase: acetonitrile-water (80:20 to 60:40, v / v); detector: refractive index detector (RID) or evaporative light scattering detector (ELSD).

[0100] Quick analysis methods: Refractive index: Measured using an Abbe refractometer at 25℃ and compared with a standard curve; Viscosity determination: Measured using a rotational viscometer at 25°C, and compared with a standard curve; Density determination: Measured using a densitometer at 25℃ and compared with a standard curve.

[0101] Component adjustment: Adjustment of hydrogen bond acceptor (choline chloride) content: If insufficient: add the calculated amount of solid choline chloride; dissolution conditions: 60±2℃, stirring speed 200-250rpm; dissolution time: 20-30 minutes.

[0102] Adjustment of hydrogen bond donor (glycerol, lactic acid, etc.) content: If insufficient: add the calculated amount of liquid hydrogen bond donor; Mixing conditions: 60±2℃, stirring speed 150-200rpm; Mixing time: 15-25 minutes.

[0103] Homogenization: The adjusted DES was stirred for another 30 minutes at 60±2℃; allowed to cool to room temperature; filtration: passed through a 0.45μm microporous membrane.

[0104] Experimental verification To verify the technical effects of the present invention, the following experimental tests were conducted.

[0105] Experiment 1: Verification of Highly Selective Separation of Biochemical Substances with Similar Polarity 1.1 Experimental Objective The method of this invention was verified to have separation selectivity for mixtures of biochemical substances with small differences in polarity (logP value difference of 0.05-0.1), and compared with traditional organic solvent extraction methods.

[0106] 1.2 Experimental Materials Model biochemical mixtures: insulin homologues (type A, logP = 0.85; type B, logP = 0.92); lysozyme isozymes (Lysozyme-1, logP = 2.10; Lysozyme-2, logP = 2.18); and peptide mixtures (Peptide-X, logP = 1.35; Peptide-Y, logP = 1.40).

[0107] Eutectic solvent systems: choline chloride-glycerol (1:2 molar ratio); choline chloride-lactic acid (1:1 molar ratio).

[0108] Surfactants: Class I: Lecithin (purity ≥ 95%); Class II: Monoacylglycerol (purity ≥ 98%); Class III: Polysorbate-80 (purity ≥ 99%).

[0109] Control group solvents: acetone-water system (gradient ratio); methanol-water system (gradient ratio).

[0110] Analytical equipment: High-performance liquid chromatograph (equipped with a diode array detector); ultraviolet-visible spectrophotometer; interfacial tensiometer.

[0111] 1.3 Experimental Methods A. Separation process of the method of the present invention: Preparation of choline chloride-glycerol DES: Mix at a molar ratio of 1:2 and heat and stir at 70°C for 3 hours until clear.

[0112] Prepare an aqueous solution of the model biochemical mixture: the concentration of each substance pair is 1 mg / mL, and the total concentration is 2 mg / mL.

[0113] Constructing an aqueous-DES two-phase system: DES and an aqueous solution of biochemical substances were mixed in a 5:5 (v / v) ratio.

[0114] Sequential addition of surfactants: Add the first type of surfactant (lecithin) to a final concentration of 0.8% (w / v); Add the second type of surfactant (monoacylglycerol) to a final concentration of 0.5% (w / v) after 15 minutes. After 25 minutes, add the third type of surfactant (polysorbate-80) to a final concentration of 0.3% (w / v).

[0115] Enrichment Collection: First collection point: 30 minutes after the addition of the second type of surfactant; Second collection time point: 45 minutes after the addition of the third type of surfactant; Analysis: Two-phase samples were collected, and the content and separation selectivity of each component were analyzed by HPLC.

[0116] B. Control group extracted with conventional organic solvents: Acetone-water gradient extraction: The same model biochemical mixture was extracted sequentially using 30%, 50%, and 70% acetone aqueous solutions.

[0117] Methanol-water gradient extraction: The same model biochemical mixture was extracted sequentially using 30%, 50%, and 70% methanol aqueous solutions.

[0118] Analysis: The separation effect of each component was analyzed under the same HPLC conditions.

[0119] C. Separation selectivity calculation method: Separation Selectivity Index (SSI) = (Concentration of component A in phase A / Concentration of component A in phase B) / (Concentration of component B in phase A / Concentration of component B in phase B) 1.4 Experimental Results Table 1-1: Comparison of the selectivity of different methods for separating biochemical substances with similar polarity

[0120] Table 1-2: Separation selectivity of the method of the present invention at different collection time points

[0121] Figure 1 This study demonstrates a comparative analysis of the selectivity of different methods for separating biochemical substances with similar polarities.

[0122] Figure 2 The separation selectivity of the method of the present invention is shown to vary at different collection time points.

[0123] 1.5 Conclusion Analysis The method of this invention exhibits excellent separation selectivity for biochemical substances with small polarity differences, with an average SSI of 8.6, which is much higher than the 2.3-2.5 of the traditional organic solvent extraction method, and the separation selectivity is improved by about 300%.

[0124] The sequential control over time dimension has a significant effect: the separation selectivity at the second collection time point is 63.8% higher on average than that at the first collection time point, demonstrating the technical advantages of dynamic interface control and sequential migration.

[0125] For all tested biochemical pairs, the method of this invention exhibited consistently high selectivity, especially for the polypeptide X / Y pair with the smallest polarity difference (logP difference of only 0.05), with a separation selectivity index of 9.2, demonstrating the excellent separation capability of this method for biochemical pairs with slight polarity differences.

[0126] Experiment 2: Verification of the Synergistic Improvement of High Purity and High Recovery Rate 2.1 Experimental Objective The method of this invention can simultaneously achieve high purity and high recovery rate of the product, overcoming the technical difficulty of balancing purity and recovery rate in traditional methods.

[0127] 2.2 Experimental Materials Target biochemical substance: Model enzyme: α-amylase (derived from Bacillus subtilis, purity ≥95%); Target peptide: A mixture of collagen peptides (containing peptide fragments of different lengths and sequences). Model protein: bovine serum albumin (BSA, purity ≥98%).

[0128] Eutectic solvent system: Choline chloride-glycerol (1:2 molar ratio) is used for α-amylase; Choline chloride-lactic acid (1:1 molar ratio) is used for collagen peptides; Choline chloride-urea (1:2 molar ratio) is used for bovine serum albumin.

[0129] Surfactant combination: α-Amylase: lecithin (0.8%) + monoacylglycerol (0.5%) + polysorbate-80 (0.3%); Collagen peptides: bile salts (0.8%) + diacylglycerols (0.5%) + sucrose esters (0.3%); Bovine serum albumin: sodium fatty acid salt (0.8%) + fatty alcohol (0.5%) + egg yolk phosphatidylcholine (0.3%).

[0130] Control separation method: Traditional organic solvent precipitation method: precipitation with ethanol and acetone of different concentrations; traditional salting out method: ammonium sulfate gradient precipitation (20%-80% saturation); traditional ultrafiltration concentration method: 10kDa molecular weight cutoff ultrafiltration membrane.

[0131] Analysis method: Protein purity analysis: SDS-PAGE electrophoresis + gel density scanning; recovery analysis: BCA protein quantification + specific activity determination; impurity analysis: reversed-phase HPLC analysis.

[0132] 2.3 Experimental Methods A. Concentration and separation process of the method of the present invention: Choose a suitable DES system and surfactant combination based on the target biochemical substance.

[0133] Construct a two-phase system of aqueous phase-DES (DES:water = 5:5, v / v) following the basic steps in Experiment 1.

[0134] Sequential addition of three types of surfactants and dynamic interfacial regulation: After adding the first type of surfactant, let it stand for 10 minutes; After adding the second type of surfactant, let it stand for 15 minutes; After adding the third type of surfactant, let it stand for 20 minutes.

[0135] Multi-stage enrichment collection: First collection point: 30 minutes after the addition of the second type of surfactant; Second collection time point: 45 minutes after the addition of the third type of surfactant; The enriched phases collected in the two separate collections were analyzed for recovery and purity, or they could be combined for overall recovery analysis.

[0136] Enrichment phase processing: dialysis or ultrafiltration to separate target biochemical substances from DES; BCA method to determine protein content; SDS-PAGE analysis to determine purity; specific activity determination (applicable to enzymes).

[0137] B. Control method processing flow: Organic solvent precipitation method: Pre-cooled organic solvent is added dropwise to biochemical solutions of the same concentration until different final concentrations are reached; after standing at 4°C for 30 minutes, the precipitate is collected by centrifugation; the precipitate is dissolved and analyzed.

[0138] Salting out method: Add ammonium sulfate to solutions of biochemical substances of the same concentration to different saturations; let stand at 4℃ for 30 minutes and then centrifuge to collect the precipitate; Analysis after dialysis to remove salt.

[0139] Ultrafiltration concentration method: Use a 10kDa molecular weight cutoff ultrafiltration membrane to concentrate a biochemical solution of the same concentration; collect the concentrate for analysis.

[0140] C. Calculation methods for purity and recovery rate: Purity calculation: SDS-PAGE gel scanning: target band intensity / total band intensity × 100%; HPLC purity: target peak area / total peak area × 100%; Recovery rate calculation: (Recovered amount / Initial amount) × 100%; 2.4 Experimental Results Table 2-1: Comparison of purity and recovery rate of α-amylase by different methods

[0141] Table 2-2: Comparison of purity and recovery rate of collagen peptides by different methods

[0142] Table 2-3: Comparison of purity and recovery rate of bovine serum albumin by different methods

[0143] Table 2-4: Comparison of purity and recovery rate at different collection time points using the method of the present invention (taking α-amylase as an example)

[0144] Figure 3 The study compares the purity and recovery rate of various biochemical substances using different methods.

[0145] Figure 4 The purity-recovery rate relationship of the method of the present invention at different collection time points is shown.

[0146] 2.5 Conclusion Analysis The method of this invention achieves both high purity and high recovery rate: for all tested biochemical substances, the purity reaches over 95% (up to 98.2%), and the recovery rate reaches over 85% (up to 92.5%). In contrast, the purity of traditional methods is typically between 70-85%, and the recovery rate is between 65-80%.

[0147] The synergistic effect of purity and recovery rate is significant: the purity × recovery rate index (reflecting the overall separation efficiency) of the method of this invention is on average 88.0, while the average of the traditional method is only 59.2, which is an improvement of 48.6%. This proves that the method has successfully overcome the problem of difficulty in balancing purity and recovery rate in traditional concentration technology.

[0148] The advantages of multi-stage enrichment collection are obvious: the data from the staged collection of α-amylase show that the first collection time point achieved a total recovery of 70.1% with a purity of 94.5%; the second collection time point achieved a total recovery of 29.9% with an even higher purity of 97.9%. Combining the two collections, the overall purity was 96.8%, and the recovery rate reached 92.5%, demonstrating the important role of multi-stage enrichment collection technology in improving the overall recovery rate.

[0149] High versatility: The method of this invention exhibits consistently high purity and high recovery rate for different types of biochemical substances (enzymes, peptides, proteins), demonstrating its wide applicability in the field of biochemical substance concentration.

[0150] Experiment 3: Verification of Bioactivity Retention Rate 3.1 Experimental Objective The invention verifies that the method can effectively maintain the biological activity of biochemical substances while achieving efficient concentration and separation, thus solving the technical problem that traditional organic solvent methods damage biological activity.

[0151] 3.2 Experimental Materials Active and sensitive biochemical substances: Active enzyme: glucose oxidase (GOD, 50 U / mg); Drug peptide: glucagon-like peptide-1 (GLP-1, purity ≥98%); Antibody protein: anti-human IgG monoclonal antibody (purity ≥95%).

[0152] Eutectic solvent system: choline chloride-glycerol (1:2 molar ratio).

[0153] Surfactant combination: lecithin (0.8%) + monoacylglycerol (0.5%) + polysorbate-80 (0.3%).

[0154] Concentration methods compared: ethanol precipitation (30%, 50%, 70% gradient); ammonium sulfate precipitation (20-80% saturation); acetonitrile precipitation (20%, 40%, 60% gradient); ultrafiltration concentration (appropriate molecular weight cutoff membrane).

[0155] Activity assay methods: Glucose oxidase activity assay: standard glucose-peroxidase coupled colorimetric method; GLP-1 activity assay: INS-1 cell insulin secretion stimulation assay; Antibody activity assay: antigen binding capacity was measured by ELISA.

[0156] Structural stability analysis: Circular dichroism (CD) was used to determine the secondary structure of the protein; fluorescence spectroscopy was used to determine the tertiary structure of the protein; and dynamic light scattering (DLS) was used to determine the aggregation state of the protein.

[0157] 3.3 Experimental Methods A. Method flow of the present invention: Preparation of choline chloride-glycerol DES: Mix at a molar ratio of 1:2 and heat and stir at 70°C for 3 hours until clear.

[0158] Prepare the aqueous solution of biochemical substances: glucose oxidase: 2 mg / mL (100 U / mL); GLP-1: 1 mg / mL; anti-human IgG: 2 mg / mL; construct the aqueous-DES two-phase system: mix DES and the aqueous solution of biochemical substances in a 5:5 (v / v) ratio.

[0159] Surfactant sequential addition: Add lecithin to a final concentration of 0.8% (w / v) and let stand for 10 minutes; add monoacylglycerol to a final concentration of 0.5% (w / v) and let stand for 15 minutes; add polysorbate-80 to a final concentration of 0.3% (w / v) and let stand for 20 minutes.

[0160] Multi-stage enrichment and collection: First collection time point: 30 minutes after the addition of the second type of surfactant; Second collection time point: 45 minutes after the addition of the third type of surfactant; Dialysis separation of DES and biochemical substances: Use a dialysis bag with an appropriate molecular weight cutoff, dialyze at 4°C for 12 hours, and change the buffer solution 3 times during the process.

[0161] Sample analysis: Determine the concentration and activity of biochemical substances and calculate the activity retention rate.

[0162] B. Control method processing flow: Ethanol precipitation method: Pre-cooled ethanol was added dropwise to the aqueous solution of the biochemical substance to a concentration of 30%, 50%, and 70%, respectively. The solution was allowed to stand at 4°C for 30 minutes, and the precipitate was collected by centrifugation, dissolved, and its activity was analyzed.

[0163] Ammonium sulfate precipitation method: Add ammonium sulfate to the aqueous solution of biochemical substances to different saturation levels (20-80%), let stand at 4℃ for 30 minutes, collect the precipitate by centrifugation, and analyze the activity after dialysis to remove salt.

[0164] Acetonitrile precipitation method: Add acetonitrile dropwise to the aqueous solution of biochemical substances to concentrations of 20%, 40%, and 60%, let stand at 4°C for 30 minutes, centrifuge to collect the precipitate, dissolve and analyze its activity.

[0165] Ultrafiltration concentration method: Using an ultrafiltration membrane with an appropriate molecular weight cutoff, the biochemical substance solution is concentrated to 1 / 10 of its original volume at room temperature, the concentrate is collected and its activity is analyzed.

[0166] C. Calculation method for activity retention rate: Activity retention rate (%) = (specific activity of concentrated sample × recovery amount) / (specific activity of initial sample × initial amount) × 100%.

[0167] 3.4 Experimental Results Table 3-1: Effect of different concentration methods on the retention rate of glucose oxidase (GOD) activity

[0168] Table 3-2: Effect of different concentration methods on the retention rate of glucagon-like peptide-1 (GLP-1) activity.

[0169] Table 3-3: Effects of different concentration methods on the retention rate of anti-human IgG antibody activity

[0170] Table 3-4: Effects of different concentration methods on the retention rate of GOD secondary structure (based on CD spectral analysis)

[0171] Figure 5 The study compares the retention rates of various biochemical substances by different concentration methods.

[0172] Figure 6 The effects of different concentration methods on the secondary structure of GOD were demonstrated (CD spectral analysis).

[0173] 3.5 Conclusion Analysis Significantly improved bioactivity retention: The method of this invention effectively protects bioactivity during the concentration process, with the activity retention rates of the three tested biochemical substances all exceeding 87% (glucose oxidase 87.8%, GLP-1 peptide 87.0%, and anti-human IgG antibody 88.1%). In contrast, the activity retention rates of traditional methods are generally below 70%, especially with organic solvent methods (ethanol, acetonitrile), where the activity retention rate is only 36-50%.

[0174] Excellent protein structure protection: CD spectral analysis shows that the α-helix content of GOD treated by the method of this invention (34.2%) decreased by only 1.4 percentage points compared with the initial sample (35.6%), with a structural similarity as high as 95.8%. In contrast, GOD treated by organic solvent precipitation showed a significant decrease in α-helix content (9.3-11.1 percentage points) and an increase in random coil content (5.2-5.7 percentage points), indicating that the secondary structure was severely damaged.

[0175] The activity protection effect of the method is outstanding for peptides and antibodies: especially for structure-sensitive peptides (GLP-1) and antibodies (IgG), the activity retention effect of the method of the present invention is far superior to that of traditional methods. GLP-1 retains 87.0% of its activity after treatment with the method of the present invention, while only 36.3-40.7% of its activity is retained after treatment with organic solvents; IgG antibodies retain 88.1% of their binding activity after treatment with the method of the present invention, while only 40.7-67.6% is retained by traditional methods.

[0176] The advantages of the technical mechanism are clear: the excellent activity retention effect of the method of this invention is mainly attributed to two key factors: first, the use of biocompatible DES to replace traditional organic solvents reduces the damage to protein hydrogen bond networks and hydrophobic interactions; second, the sequential addition technology of surfactants creates a mild interfacial environment, avoiding the drastic denaturation of biochemical substances at the solid-liquid or liquid-liquid interface in traditional methods.

[0177] Wide applicability: The method of this invention shows consistent protective effects on different types of biosensitive active substances (enzymes, peptides, and antibodies), demonstrating its versatility. In particular, for biochemical substances that are extremely sensitive to traditional organic solvents, this invention provides a safe and efficient concentration method.

[0178] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for concentrating biochemical substances, characterized in that, Includes the following steps: Preparation of biocompatible deep eutectic solvent DES; The DES is mixed with an aqueous solution containing the target biochemical substance to form an aqueous-DES two-phase system; Prepare a mixed system of antagonistic biosurfactants containing first-class surfactants and second-class surfactants; A first type of surfactant is added to the aqueous-DES two-phase system, and a second type of surfactant is added after a preset time interval to achieve temporal changes in interface properties and form a dynamic interface environment. The target biochemical substances of different polarities are sequentially migrated in the dynamic interface environment in a time order and enriched in different phases. Phases containing target biochemical substances are collected in batches at predetermined time points to complete the separation and concentration of biochemical substances.

2. The method for concentrating biochemical substances according to claim 1, characterized in that, The biocompatible deep eutectic solvent DES is prepared by mixing hydrogen bond acceptors and hydrogen bond donors in a molar ratio, wherein: the hydrogen bond acceptor is choline chloride; and the hydrogen bond donor is selected from at least one of glycerol, lactic acid, urea, and fructose.

3. The method for concentrating biochemical substances according to claim 2, characterized in that, The molar ratio of the choline chloride to the hydrogen bond donor is: When the hydrogen bond donor is glycerol, the molar ratio is 1:2; When the hydrogen bond donor is lactic acid, the molar ratio is 1:1; When the hydrogen bond donor is urea, the molar ratio is 1:2; When the hydrogen bond donor is fructose, the molar ratio is 2:

1.

4. The method for concentrating biochemical substances according to claim 1, characterized in that, The first type of surfactant is a hydrophilic surfactant selected from at least one of lecithin, bile salts, and sodium salts of fatty acids; the second type of surfactant is a lipophilic surfactant selected from at least one of monoacylglycerol, diacylglycerol, and fatty alcohols.

5. The method for concentrating biochemical substances according to claim 1, characterized in that, The antagonistic biosurfactant mixture further includes a third type of surfactant, which is an amphoteric or nonionic surfactant selected from at least one of egg yolk phosphatidylcholine, sucrose ester and polysorbate; the step of adding surfactant to the aqueous-DES two-phase system further includes: adding the third type of surfactant after a preset time interval following the addition of the second type of surfactant.

6. The method for concentrating biochemical substances according to claim 1, characterized in that, The concentration of the first type of surfactant is 0.5-1.0% (w / v); the concentration of the second type of surfactant is 0.3-0.8% (w / v); and the preset time interval for adding the second type of surfactant is 10-30 minutes after the first type of surfactant is added.

7. The method for concentrating biochemical substances according to claim 5, characterized in that, The concentration of the third type of surfactant is 0.1-0.5% (w / v); the preset time interval for adding the third type of surfactant is 15-40 minutes after the second type of surfactant is added.

8. The method for concentrating biochemical substances according to claim 1, characterized in that, The step of collecting the enriched phase in batches includes: determining a first collection time point and a second collection time point, wherein the first collection time point is 20-40 minutes after the addition of the second type of surfactant, and the second collection time point is 30-60 minutes after the addition of the third type of surfactant; collecting the first batch of enriched phase at the first collection time point, and then adding the same replenishing solution as the collected phase to the remaining system; and collecting the second batch of enriched phase at the second collection time point.

9. The method for concentrating biochemical substances according to claim 1, characterized in that, The method also includes a step of DES recycling and reuse, including: The target biochemical substance was separated from the enriched phase to obtain a solution containing DES; Remove impurities from the DES-containing solution; Concentrate the solution and adjust the DES component ratio to the original proportion; The recovered DES is recycled for use in the next batch concentration process.

10. The method for concentrating biochemical substances according to claim 1, characterized in that, The target biochemical substances are biochemical mixtures with similar structures but small differences in polarity, with a difference in logP value of 0.05-0.1.

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