A method for extracting pectin and protein from sunflower heads

Abstract

This invention relates to the field of natural product extraction technology, and discloses a method for extracting pectin and protein from sunflower discs, comprising the following steps: S1, hydrophobic matrix construction: fresh sunflower discs are pulverized and dispersed in N,N-dimethylcyclohexylamine, hydrophobic Schiff base ligands and β-cyclodextrin are added, and the mixture is homogeneous at 20℃-30℃ and 0.1MPa to obtain a hydrophobic mixed slurry; S2, phase change extraction and in-situ chelation: carbon dioxide gas is introduced into the hydrophobic mixed slurry, and the system pressure is maintained at 0.3MPa to 0.6MPa; S3, microemulsion fractionation; S4, product recovery. By directly using fresh sunflower discs as raw materials and utilizing the hydrophobic properties of N,N-dimethylcyclohexylamine to exchange cell water in situ at room temperature and pressure, an anhydrous hydrophobic matrix is ​​constructed, preserving the nitrogen solubility index and bioactivity of sunflower disc proteins, while maintaining the natural molecular structure of pectin.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically a method for extracting pectin and protein from sunflower heads. Background Technology

[0002] The sunflower disc, or receptacle, is the part remaining after threshing and harvesting sunflower seeds in the sunflower industry, accounting for over 30% of the total biomass of the sunflower plant. The sunflower disc is rich in low-methoxyl pectin and high-quality plant protein. Pectin possesses unique gelling, thickening, and stabilizing properties, making it widely used in the food and pharmaceutical fields, while sunflower disc protein is a plant protein source with significant development potential. Therefore, the synergistic extraction of pectin and protein from sunflower discs has significant economic and social value for achieving high-value utilization of large-scale agricultural and forestry waste, addressing resource waste, and extending the sunflower deep-processing industrial chain.

[0003] In existing technologies, to facilitate subsequent storage, transportation, and organic solvent extraction, fresh sunflower heads with extremely high water content typically require dehydration and drying. Industrially, prolonged hot air drying or sun-drying processes are commonly used to prepare them into dry powder. However, since the proteins and pectins in sunflower heads are heat-sensitive biomolecules, the heat effect can cause severe denaturation and aggregation of proteins, reducing their nitrogen solubility index and biological activity. Simultaneously, it can trigger thermal degradation and browning reactions of pectin molecular chains, destroying their natural molecular structure, thus leading to a significant decline in the quality of the finally extracted proteins and pectins. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting pectin and protein from sunflower heads, solving the problems of protein denaturation and pectin structural damage caused by drying pretreatment.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting pectin and protein from sunflower heads, comprising the following steps: S1. Construction of hydrophobic matrix: Fresh sunflower heads were crushed and dispersed in N,N-dimethylcyclohexylamine, hydrophobic Schiff base ligands and β-cyclodextrin were added, and the mixture was mixed evenly under the conditions of 20℃-30℃ and 0.1MPa to obtain hydrophobic mixed slurry. S2. Phase change extraction and in-situ chelation: Carbon dioxide gas is introduced into the hydrophobic mixed slurry, and the system pressure is controlled to be maintained at 0.3MPa to 0.6MPa, and the reaction is carried out by stirring. S3. Microemulsion fractionation: Under the condition of maintaining carbon dioxide pressure, hydrofluoroether is injected into the system and shear force is applied to form a microemulsion. Then, based on the pressure value set in step S2, periodic pressure pulsation is applied to the system to separate the mixture into an upper oil phase, a middle pectin-enriched phase, and a lower protein-containing liquid phase. S4. Product recovery: The middle layer pectin-enriched phase is separated and collected, and then washed and dried to obtain pectin; the lower layer protein-containing liquid phase is collected and treated to remove carbon dioxide, separating the upper hydrophobic solvent phase and the lower aqueous precipitate layer, and the lower aqueous precipitate layer is collected to obtain protein.

[0006] By adopting the above technical solution, in step S1, fresh sunflower discs are dispersed in N,N-dimethylcyclohexylamine. The hydrophobic properties of N,N-dimethylcyclohexylamine are used to replace cell water, constructing an anhydrous hydrophobic matrix, avoiding hot air drying pretreatment, thereby preserving the nitrogen solubility index and bioactivity of sunflower disc proteins and maintaining the natural molecular structure of pectin. In step S2, carbon dioxide gas is introduced to convert N,N-dimethylcyclohexylamine into a hydrophilic ionic liquid to dissolve pectin and protein, while hydrophobic Schiff base ligands are used to capture metal ions. In step S3, hydrofluoroether was injected to construct a microemulsion system and periodic pressure pulsation was applied. The low dielectric constant of hydrofluoroether was used to form a dielectric gradient at the microscopic interface, and the Gibbs-Thomson effect was generated by the curvature change caused by pressure fluctuation, which induced the macromolecular pectin to accumulate at the liquid-liquid interface to form a middle pectin-enriched phase, thus realizing the physical stratification and purification of pectin and protein. In step S4, carbon dioxide is removed from the lower protein-containing liquid phase, causing the solvent to reverse back to a hydrophobic state. The hydrophobic Schiff base ligands are transferred to the upper hydrophobic solvent phase in the form of metal ion-locking complexes, while the protein precipitates in the lower aqueous phase. This achieves physical isolation between the metal catalyst and the protein product to solve the problem of spoilage during storage. At the same time, it realizes automatic solvent separation and recovery without the need to add strong acid or strong base reagents.

[0007] Preferably, in step S1, the mass-to-volume ratio of fresh sunflower heads to N,N-dimethylcyclohexylamine is 1:4-1:8 g / mL; the amount of hydrophobic Schiff base ligand added is 0.1%-0.3% of the mass of N,N-dimethylcyclohexylamine; and the amount of β-cyclodextrin added is 2%-4% of the dry weight of fresh sunflower heads.

[0008] By employing the above technical solutions, and controlling the mass-to-volume ratio of fresh sunflower heads to N,N-dimethylcyclohexylamine, sufficient volumetric capacity of N,N-dimethylcyclohexylamine is ensured to wet the pulverized biomass particles and replace intercellular and internal water, thereby constructing a continuous anhydrous hydrophobic matrix. This avoids the impact of heat treatment processes on the protein nitrogen solubility index and pectin molecular structure. By controlling the addition ratio of hydrophobic Schiff base ligands to solvent, sufficient ligand molecules are ensured to capture free endogenous metal ions in situ in a carbon dioxide-induced ionic liquid environment. These metal ions are then transferred to the oil phase along with the hydrophobic ligands during subsequent carbon dioxide removal, achieving physical isolation between the metal catalyst and the protein precipitated in the aqueous phase. By controlling the addition ratio of β-cyclodextrin to dry weight, its dispersing effect promotes uniform mixing of the hydrophobic Schiff base ligands and biomass particles in the solvent, forming a homogeneous hydrophobic slurry. This provides a material basis for subsequent phase change extraction and microemulsion interface fractionation.

[0009] Preferably, in step S1, the hydrophobic Schiff base ligand is N-(2-hydroxybenzylmethyl)dodecane-1-amine.

[0010] By adopting the above technical solution, N-(2-hydroxybenzylmethyl)dodecane-1-amine is selected as a hydrophobic Schiff base ligand. Utilizing the unique chelating groups and long-chain hydrophobic tails in its molecular structure, endogenous metal ions are captured in situ through conformational adjustment in a carbon dioxide-induced ionic liquid environment. During the process of removing carbon dioxide and restoring the solvent to a hydrophobic state, the lipophilic properties of the long-chain hydrophobic tail drive the directional transfer of the metal ion complex to the upper hydrophobic solvent phase, allowing it to physically separate from the protein precipitated in the lower aqueous phase. This eliminates the catalytic oxidation of proteins by metal ions and solves the problem of easy spoilage of protein products during storage.

[0011] Preferably, in step S2, during phase change extraction and in-situ chelation, the temperature of the stirring reaction is 25℃-35℃, the stirring speed is 300rpm to 500rpm, and the pressure is maintained for 40min-60min.

[0012] By employing the above technical solution, and by controlling the temperature of the stirring reaction, the process of generating a hydrophilic ionic liquid from N,N-dimethylcyclohexylamine with carbon dioxide and water is ensured to occur under mild conditions. This avoids thermal denaturation of sunflower disc protein and thermal degradation of pectin molecules caused by high temperatures, preserving the nitrogen solubility index and bioactivity of the protein and maintaining the natural molecular structure of pectin. By controlling the stirring speed, mass transfer and mixing within the system are promoted, accelerating the generation of the hydrophilic ionic liquid to dissolve pectin and protein. At the same time, the contact probability between hydrophobic Schiff base ligands and endogenous metal ions is increased, improving in-situ chelation efficiency. By controlling the duration of pressure maintenance, the phase change extraction and metal ion capture processes are ensured to reach equilibrium, allowing the ligands to fully complete conformational inversion to lock in metal ions. This provides a guarantee for subsequent physical isolation between the metal catalyst and the protein product and for solving the problem of spoilage during storage.

[0013] Preferably, in step S3, the hydrofluoroether is methoxynonafluorobutane, and the injection volume is 10%-15% of the total liquid volume of the system. During the shearing, the shearing speed is 2000rpm-3000rpm, and the shearing time is 10min-15min.

[0014] By employing the above technical solution, selecting methoxynonfluorobutane as the hydrofluoroether and controlling its injection volume, the aim is to construct a microemulsion system based on hydrofluoroether and ionic liquid. The low dielectric constant of the hydrofluoroether creates a dielectric gradient at the microscopic interface. By controlling the shear rate and duration, the hydrofluoroether is dispersed in the system to form a microemulsion interface. Combined with subsequent periodic pressure pulsation technology, the curvature of the microscopic interface changes, inducing the Gibbs-Thomson effect and directionally inducing the enrichment of macromolecular pectin at the liquid-liquid interface. This solves the problem of efficient separation of macromolecular polysaccharides and small molecule proteins in complex biomass systems using a single solvent and improves the purity of the pectin product.

[0015] Preferably, in step S3, the periodic pressure pulsation is based on the pressure value set in step S2, and the pressure is controlled to fluctuate within the range of ±0.05MPa of the base pressure, with a fluctuation frequency of 0.05Hz-0.2Hz, and the pulsation lasts for 10-20 minutes.

[0016] By adopting the above technical solution, periodic pressure pulsations are applied based on the pressure value set in step S2, driving periodic curvature changes in the microscopic interface of the microemulsion system constructed based on hydrofluoroether and ionic liquid. This utilizes the chemical potential driving force generated by the Gibbs-Thomson effect to directionally induce the migration and enrichment of macromolecular pectin to the liquid-liquid interface. Physical stratification of macromolecular polysaccharides and small molecule proteins is achieved in a single solvent system, solving the problem of difficult efficient separation of components with different molecular weights in complex biomass systems and improving the purity of pectin products.

[0017] Preferably, in step S3, after the pressure pulsation ends, the microemulsion fractionation further includes a pressurized centrifugation step, with a centrifugation speed of 3000rpm-5000rpm and a centrifugation time of 5min-10min.

[0018] By adopting the above technical solution, a pressurized centrifugation step is performed after the pressure pulsation ends. The centrifugal force field enhances the density gradient and interfacial tension differences between the phases induced by the Gibbs-Thomson effect, forcing the middle pectin-rich phase to form a clearly defined macroscopic stratification with the lower protein-containing liquid phase and the upper oil phase. By maintaining the system pressure during centrifugation, the bubble disturbance and solvent phase change caused by the escape of carbon dioxide from the ionic liquid due to pressure release are prevented, thus maintaining the stability of the microemulsion fractionation system. This physically achieves efficient separation of macromolecular pectin and small molecule proteins, solving the problem of difficult component separation in complex biomass systems and further improving the purity of the pectin product.

[0019] Preferably, in step S4, the processing steps after separating and collecting the middle layer pectin-enriched phase include: adding a 75% ethanol solution with a volume of 2 to 3 times the volume of the middle layer pectin-enriched phase to the middle layer pectin-enriched phase for demulsification, filtering and collecting the solid, and vacuum drying at 40°C-50°C.

[0020] By adopting the above technical solution, a 75% ethanol solution is added to the middle pectin-enriched phase. The ethanol changes the dielectric environment and polarity of the system, disrupts the stability of the microemulsion interface, and causes demulsification, which promotes the aggregation of pectin molecules and their precipitation from the liquid phase, thus achieving solid-liquid separation of pectin. Subsequently, the collected solid is vacuum dried. By controlling the drying temperature and removing residual solvent under negative pressure, the thermal degradation and browning reaction of pectin sugar chains under high temperature are avoided, thereby maintaining the natural molecular structure of pectin.

[0021] Preferably, in step S4, the carbon dioxide removal process includes the following steps: purging the lower protein-containing liquid phase with a nitrogen gas flow, or heating it to 50°C to 60°C until the system becomes turbid and separates into an upper hydrophobic solvent phase and a lower aqueous precipitate layer.

[0022] By employing the above technical solution, the lower protein-containing liquid phase is treated using physical methods such as purging with nitrogen gas or heating. This removes carbon dioxide from the system and adjusts the polarity and phase state of N,N-dimethylcyclohexylamine, reversing it from a hydrophilic ionic liquid to a hydrophobic solvent, causing the system to become turbid and automatically separate into layers. During this process, the hydrophobic Schiff base ligand dissolves and transfers to the upper hydrophobic solvent phase as a complex that locks in metal ions, while the protein exists as a precipitate in the lower aqueous precipitate layer. This achieves physical isolation between the metal catalyst and the protein product, solving the problem of easy spoilage of the protein product during storage. At the same time, the separated hydrophobic solvent can be recycled without the need for adding strong acid or strong base reagents or high-energy-consuming distillation, reducing the loss of organic solvents and energy costs in industrial production.

[0023] Preferably, in step S4, after the carbon dioxide removal treatment, a solid-liquid separation step is also included: the layered system is centrifuged to remove the upper hydrophobic solvent phase containing hydrophobic Schiff base ligands, and the lower aqueous precipitate layer containing protein precipitates is retained.

[0024] By adopting the above technical solution, in the solid-liquid separation step, the layered system after carbon dioxide removal is physically separated by centrifugation. The upper hydrophobic solvent phase containing hydrophobic Schiff base ligands is removed. Utilizing the solubility characteristics of hydrophobic Schiff base ligands in hydrophobic solvents, the complex that locks in endogenous metal ions is removed from the system along with the upper hydrophobic solvent phase. At the same time, the lower aqueous precipitate layer containing protein precipitate is retained. This achieves physical isolation between the metal catalyst and the protein product, breaks the catalytic chain of oxidation of defatting residues, solves the problem of easy spoilage of protein products during storage, and realizes automatic layer separation of hydrophobic solvent and product.

[0025] This invention provides a method for extracting pectin and protein from sunflower heads. It has the following beneficial effects: 1. This invention uses fresh sunflower discs as raw materials and utilizes the hydrophobic properties of N,N-dimethylcyclohexylamine to replace cell water in situ under normal temperature and pressure, thereby constructing an anhydrous hydrophobic matrix. This preserves the nitrogen solubility index and biological activity of sunflower disc proteins while maintaining the natural molecular structure of pectin.

[0026] 2. This invention introduces a hydrophobic Schiff base ligand with phase transfer properties. By utilizing its conformational inversion properties in a carbon dioxide-induced ionic liquid environment, it can efficiently capture endogenous metal ions in the system in situ. This locks in the automatic transfer of metal ions to the upper oil phase, while the protein precipitates in the lower aqueous phase. This achieves physical isolation between the metal catalyst and the protein product, solving the problem of easy spoilage of the protein product during storage.

[0027] 3. This invention constructs a microemulsion system based on hydrofluoroether and ionic liquid, and supplements it with periodic pressure pulsation technology. The low dielectric constant of hydrofluoroether is used to form a dielectric gradient at the microscopic interface, and the curvature change caused by pressure fluctuation generates the Gibbs-Thomson effect, which directionally induces the enrichment of macromolecular pectin at the liquid-liquid interface. This solves the problem that it is difficult to efficiently separate macromolecular polysaccharides and small molecule proteins in complex biomass systems using a single solvent, and also improves the purity of pectin products.

[0028] 4. This invention uses N,N-dimethylcyclohexylamine, a carbon dioxide-responsive switchable solvent, as the extraction medium. The polarity and phase of the solvent can be adjusted simply by introducing or purging gas, without the need to add strong acid or strong base reagents or perform energy-intensive distillation recovery. After the product precipitates, the hydrophobic solvent and the aqueous phase automatically separate into layers, and most of the solvent can be directly recycled. This not only significantly reduces the loss of organic solvents and environmental pollution, but also saves energy costs in industrial production. Attached Figure Description

[0029] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

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

[0031] Example 1 This embodiment provides S1, hydrophobic matrix construction: fresh sunflower discs are crushed and dispersed in N,N-dimethylcyclohexylamine, hydrophobic Schiff base ligands and β-cyclodextrin are added, and the mixture is uniformly mixed at 20°C and 0.1 MPa to obtain a hydrophobic mixed slurry; The mass-to-volume ratio of fresh sunflower heads to N,N-dimethylcyclohexylamine was 1:4 g / mL; the amount of hydrophobic Schiff base ligand added was 0.1% of the mass of N,N-dimethylcyclohexylamine; and the amount of β-cyclodextrin added was 2% of the dry weight of fresh sunflower heads. The hydrophobic Schiff base ligand is N-(2-hydroxybenzylmethyl)dodecane-1-amine.

[0032] S2. Phase change extraction and in-situ chelation: Carbon dioxide gas is introduced into the hydrophobic mixed slurry, and the system pressure is controlled at 0.3 MPa for stirring reaction. The stirring reaction was carried out at a temperature of 25°C, a stirring speed of 300 rpm, and a pressure maintained for 40 minutes.

[0033] S3. Microemulsion fractionation: Under the condition of maintaining carbon dioxide pressure, hydrofluoroether is injected into the system and shear force is applied to form a microemulsion. Then, based on the pressure value set in step S2, periodic pressure pulsation is applied to the system to separate the mixture into an upper oil phase, a middle pectin-enriched phase, and a lower protein-containing liquid phase. The hydrofluoroether is methoxynonafluorobutane, and the injection volume is 10% of the total liquid volume of the system; during shearing, the shearing rate is 2000 rpm and lasts for 10 min; The periodic pressure pulsation is based on a reference pressure of 0.3 MPa, with the pressure controlled to fluctuate within the range of ±0.05 MPa of the reference pressure, the fluctuation frequency is 0.05 Hz, and the pulsation lasts for 10 minutes. After the pressure pulsation ends, the microemulsion fractionation process also includes a pressurized centrifugation step at 3000 rpm for 5 minutes.

[0034] S4. Product recovery: Separate and collect the middle layer of pectin-enriched phase, wash and dry to obtain pectin; collect the lower layer of protein-containing liquid phase, remove carbon dioxide from it, separate the upper layer of hydrophobic solvent phase and the lower layer of aqueous precipitate, and collect the lower layer of aqueous precipitate to obtain protein.

[0035] The processing steps after separating and collecting the middle layer pectin-enriched phase include: adding a 75% ethanol solution with a volume twice that of the middle layer pectin-enriched phase to break the emulsion, filtering and collecting the solid, and vacuum drying at 40°C. The carbon dioxide removal process includes the following steps: heating the lower protein-containing liquid phase to 50°C until the system becomes turbid and separates into an upper hydrophobic solvent phase and a lower aqueous precipitate layer; After carbon dioxide removal, the process also includes a solid-liquid separation step: the layered system is centrifuged to remove the upper hydrophobic solvent phase containing hydrophobic Schiff base ligands, while retaining the lower aqueous precipitate layer containing protein precipitates. The hydrophobic Schiff base ligands are dissolved in the upper hydrophobic solvent phase as a complex that locks in metal ions, while the protein exists in the lower aqueous precipitate layer as a precipitate.

[0036] Example 2 This embodiment provides a method for extracting pectin and protein from sunflower heads, including the following steps: S1. Construction of hydrophobic matrix: Fresh sunflower heads were crushed and dispersed in N,N-dimethylcyclohexylamine, hydrophobic Schiff base ligands and β-cyclodextrin were added, and the mixture was mixed evenly at 25℃ and 0.1MPa to obtain hydrophobic mixed slurry. The mass-to-volume ratio of fresh sunflower heads to N,N-dimethylcyclohexylamine was 1:6 g / mL; the amount of hydrophobic Schiff base ligand added was 0.2% of the mass of N,N-dimethylcyclohexylamine; and the amount of β-cyclodextrin added was 3% of the dry weight of fresh sunflower heads. The hydrophobic Schiff base ligand is N-(2-hydroxybenzylmethyl)dodecane-1-amine.

[0037] S2. Phase change extraction and in-situ chelation: Carbon dioxide gas is introduced into the hydrophobic mixed slurry, and the system pressure is controlled at 0.45 MPa for stirring reaction. The stirring reaction was carried out at a temperature of 30°C, a stirring speed of 400 rpm, and a pressure maintained for 50 minutes.

[0038] S3. Microemulsion fractionation: Under the condition of maintaining carbon dioxide pressure, hydrofluoroether is injected into the system and shear force is applied to form a microemulsion. Then, based on the pressure value set in step S2, periodic pressure pulsation is applied to the system to separate the mixture into an upper oil phase, a middle pectin-enriched phase, and a lower protein-containing liquid phase. The hydrofluoroether is methoxynonafluorobutane, and the injection volume is 12.5% ​​of the total liquid volume of the system; during shearing, the shearing rate is 2500 rpm and lasts for 12.5 min; The periodic pressure pulsation is based on a reference pressure of 0.45 MPa, with the pressure controlled to fluctuate within the range of ±0.05 MPa of the reference pressure, the fluctuation frequency is 0.12 Hz, and the pulsation lasts for 15 minutes. After the pressure pulsation ends, the microemulsion fractionation process also includes a pressurized centrifugation step at 4000 rpm for 7.5 min.

[0039] S4. Product recovery: Separate and collect the middle layer of pectin-enriched phase, wash and dry to obtain pectin; collect the lower layer of protein-containing liquid phase, remove carbon dioxide from it, separate the upper layer of hydrophobic solvent phase and the lower layer of aqueous precipitate, and collect the lower layer of aqueous precipitate to obtain protein.

[0040] The processing steps after separating and collecting the middle layer pectin-enriched phase include: adding a 75% ethanol solution with a volume of 2.5 times that of the middle layer pectin-enriched phase to the middle layer pectin-enriched phase for demulsification, filtering and collecting the solid, and vacuum drying at 45°C. The carbon dioxide removal process includes the following steps: heating the lower protein-containing liquid phase to 55°C until the system becomes turbid and separates into an upper hydrophobic solvent phase and a lower aqueous precipitate layer; After carbon dioxide removal, the process also includes a solid-liquid separation step: the layered system is centrifuged to remove the upper hydrophobic solvent phase containing hydrophobic Schiff base ligands, while retaining the lower aqueous precipitate layer containing protein precipitates. The hydrophobic Schiff base ligands are dissolved in the upper hydrophobic solvent phase as a complex that locks in metal ions, while the protein exists in the lower aqueous precipitate layer as a precipitate.

[0041] Example 3 This embodiment provides a method for extracting pectin and protein from sunflower heads, including the following steps: S1. Construction of hydrophobic matrix: Fresh sunflower heads were crushed and dispersed in N,N-dimethylcyclohexylamine, hydrophobic Schiff base ligands and β-cyclodextrin were added, and the mixture was mixed evenly at 30℃ and 0.1MPa to obtain hydrophobic mixed slurry. The mass-to-volume ratio of fresh sunflower heads to N,N-dimethylcyclohexylamine was 1:8 g / mL; the amount of hydrophobic Schiff base ligand added was 0.3% of the mass of N,N-dimethylcyclohexylamine; and the amount of β-cyclodextrin added was 4% of the dry weight of fresh sunflower heads. The hydrophobic Schiff base ligand is N-(2-hydroxybenzylmethyl)dodecane-1-amine.

[0042] S2. Phase change extraction and in-situ chelation: Carbon dioxide gas is introduced into the hydrophobic mixed slurry, and the system pressure is controlled at 0.6 MPa for stirring reaction. The stirring reaction was carried out at a temperature of 35°C, a stirring speed of 500 rpm, and a pressure maintained for 60 minutes.

[0043] S3. Microemulsion fractionation: Under the condition of maintaining carbon dioxide pressure, hydrofluoroether is injected into the system and shear force is applied to form a microemulsion. Then, based on the pressure value set in step S2, periodic pressure pulsation is applied to the system to separate the mixture into an upper oil phase, a middle pectin-enriched phase, and a lower protein-containing liquid phase. The hydrofluoroether is methoxynonafluorobutane, and the injection volume is 15% of the total liquid volume of the system; during shearing, the shearing rate is 3000 rpm and lasts for 15 min; The periodic pressure pulsation is based on a reference pressure of 0.6 MPa, with the pressure controlled to fluctuate within the range of ±0.05 MPa of the reference pressure, the fluctuation frequency is 0.2 Hz, and the pulsation lasts for 20 minutes. After the pressure pulsation ends, the microemulsion fractionation process also includes a pressurized centrifugation step at a speed of 5000 rpm for 10 minutes.

[0044] S4. Product recovery: Separate and collect the middle layer of pectin-enriched phase, wash and dry to obtain pectin; collect the lower layer of protein-containing liquid phase, remove carbon dioxide from it, separate the upper layer of hydrophobic solvent phase and the lower layer of aqueous precipitate, and collect the lower layer of aqueous precipitate to obtain protein.

[0045] The processing steps after separating and collecting the middle layer pectin-enriched phase include: adding a 75% ethanol solution with a volume of 3 times that of the middle layer pectin-enriched phase to break the emulsion, filtering and collecting the solid, and vacuum drying at 50°C. The carbon dioxide removal process includes the following steps: heating the lower protein-containing liquid phase to 60°C until the system becomes turbid and separates into an upper hydrophobic solvent phase and a lower aqueous precipitate layer; After carbon dioxide removal, the process also includes a solid-liquid separation step: the layered system is centrifuged to remove the upper hydrophobic solvent phase containing hydrophobic Schiff base ligands, while retaining the lower aqueous precipitate layer containing protein precipitates. The hydrophobic Schiff base ligands are dissolved in the upper hydrophobic solvent phase as a complex that locks in metal ions, while the protein exists in the lower aqueous precipitate layer as a precipitate.

[0046] Comparative Example 1 The only difference from Example 2 is that the raw material used is not fresh sunflower discs, but sunflower disc powder that has been dried by hot air at 60°C for 24 hours and then pulverized. When dispersing N,N-dimethylcyclohexylamine, an additional amount of water equal to that of fresh sunflower discs is added to simulate a water-containing environment.

[0047] Comparative Example 2 The only difference from Example 2 is that hydrophobic Schiff base ligands are not added in step S1.

[0048] Comparative Example 3 The only difference from Example 2 is that in step S1, N-(2-hydroxybenzylmethyl)dodecane-1-amine is replaced with an equal mass of disodium ethylenediaminetetraacetate.

[0049] Comparative Example 4 The only difference from Example 2 is that in step S3, methoxynonafluorobutane is not injected, but only periodic pressure pulsations are applied.

[0050] Comparative Example 5 The only difference from Example 2 is that in step S3, after injecting hydrofluoroether and forming a microemulsion, the system is kept at a constant pressure of 0.45 MPa for 15 minutes without applying periodic pressure pulsations.

[0051] Experiment 1: Assessment of Protein Thermosensitive Damage and Experiment on Pectin Grading Purity First, 1.0 g of each protein sample prepared in Examples 1-3 and Comparative Examples 1-5 was dispersed in 100 mL of distilled water (pH 7.0). After stirring at 25 °C for 1 hour, the sample was centrifuged at 4000 rpm for 20 minutes. The nitrogen content of the supernatant and the original sample was determined by the Kjeldahl method, and the NSI value was calculated (NSI = nitrogen content of supernatant / nitrogen content of original sample × 100%). This index directly reflects whether the protein has undergone thermal denaturation. At the same time, the pectin samples prepared in each group were taken, and the galacturonic acid content was determined by the carbazole colorimetric method to characterize the purity and fractionation effect of the pectin.

[0052] Experiment 2: Residual Metal Ions and Oxidative Stability During Storage The residual iron ion content in each group of protein powders was determined by inductively coupled plasma mass spectrometry (ICP-MS). Subsequently, the protein samples were placed in a constant temperature and humidity chamber at 45°C and 75% relative humidity for accelerated storage. Samples were taken after 14 days to determine the carbonyl content of the proteins. Carbonyl content is a core indicator for evaluating the degree of protein oxidation; a higher value indicates more severe oxidation.

[0053] Table 1. Experimental data on protein thermosensitive damage assessment and pectin grading purity.

[0054] Table 2 Experimental data on metal ion residue and oxidative stability during storage.

[0055] Based on the experimental data from the above embodiments and comparative examples, the following conclusions are drawn: Combining Examples 1-3 and Comparative Examples 1-5 with Table 1, it can be seen that the protein nitrogen solubility index (NSI) of Examples 1-3 remained above 91%, and the pectin galacturonic acid content exceeded 86%, achieving the dual goals of high activity retention and high purity separation. In contrast, Comparative Example 1, due to the use of traditional hot air drying pretreatment, resulted in severe protein denaturation, with the NSI value plummeting to 42.3%, demonstrating that the wet displacement process of directly constructing a hydrophobic matrix using fresh sunflower heads in this invention can effectively avoid heat-sensitive damage; Comparative Example 4, lacking the crucial hydrofluoroether medium, could not construct a microemulsion dielectric difference environment, resulting in a significant drop in pectin purity to 62.4%; Comparative Example 5, although constructing a microemulsion, lacked periodic pressure pulsation, making it impossible to utilize the driving force generated by curvature changes for fractionation, resulting in a purity of only 68.7%. This indicates that the present invention successfully constructed a separation field based on the difference in dielectric constant by combining a hydrofluoroether frequency-modulated microemulsion system with pressure pulsation induction, effectively solving the technical problem that it is difficult to efficiently separate macromolecular pectin and small molecule proteins in complex biomass systems using a single solvent.

[0056] As can be seen from Examples 1-3 and Comparative Examples 1-5, and Table 2, the present invention demonstrates significant advantages in blocking catalytic oxidation and removing residual metals. The residual iron ion content in Examples 1-3 was controlled below 2.1 mg / kg, and the protein carbonyl content after 14 days of storage was below 3.5 nmol / mgpro, exhibiting excellent antioxidant stability. In contrast, Comparative Example 2, lacking a hydrophobic Schiff base ligand, had a large amount of residual endogenous metal ions, leading to severe catalytic oxidation of the protein. Although Comparative Example 3 used the conventional chelating agent EDTA, the complex formed by EDTA could not be removed with the oil phase transfer and remained in the protein phase, continuing catalytic oxidation; its carbonyl content was more than four times that of the examples. This indicates that the hydrophobic Schiff base ligand of the present invention exerts a chelating effect, fundamentally breaking the catalytic chain of oxidation of defatting residues.

[0057] As can be seen from Examples 1-3 and Comparative Examples 1-5, and Tables 1-2, the present invention demonstrates superior overall performance in terms of product structural integrity, purity, and stability. Comparative Example 1 suffered from activity loss due to improper drying methods; Comparative Examples 4 and 5 resulted in product contamination due to the lack of a fractionation mechanism; and Comparative Examples 2 and 3 suffered from product degradation due to the lack of an effective metal removal mechanism. In summary, the present invention, through the deep integration of the phase transition characteristics of N,N-dimethylcyclohexylamine, in-situ allosteric capture of hydrophobic ligands, and dielectric frequency modulation fractionation technology of microemulsions, solves the common technical bottlenecks in sunflower disc extraction, such as the easy denaturation of heat-sensitive proteins, low purity of pectin separation, and easy oxidation after defatting, achieving efficient, low-temperature, and stable preparation of high-value products.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting pectin and protein from sunflower heads, characterized in that, Includes the following steps: S1. Construction of hydrophobic matrix: Fresh sunflower heads were crushed and dispersed in N,N-dimethylcyclohexylamine, hydrophobic Schiff base ligands and β-cyclodextrin were added, and the mixture was mixed evenly under the conditions of 20℃-30℃ and 0.1MPa to obtain hydrophobic mixed slurry. S2. Phase change extraction and in-situ chelation: Carbon dioxide gas is introduced into the hydrophobic mixed slurry, and the system pressure is controlled to be maintained at 0.3MPa to 0.6MPa, and the reaction is carried out by stirring. S3. Microemulsion fractionation: Under the condition of maintaining carbon dioxide pressure, hydrofluoroether is injected into the system and shear force is applied to form a microemulsion. Then, based on the pressure value set in step S2, periodic pressure pulsation is applied to the system to separate the mixture into an upper oil phase, a middle pectin-enriched phase, and a lower protein-containing liquid phase. S4. Product recovery: The middle layer pectin-enriched phase is separated and collected, and then washed and dried to obtain pectin. The lower protein-containing liquid phase is collected and subjected to carbon dioxide removal treatment to separate the upper hydrophobic solvent phase and the lower aqueous precipitate layer. The lower aqueous precipitate layer is collected to obtain the protein.

2. The method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of fresh sunflower heads to N,N-dimethylcyclohexylamine is 1:4-1:8 g / mL; the amount of hydrophobic Schiff base ligand added is 0.1%-0.3% of the mass of N,N-dimethylcyclohexylamine; and the amount of β-cyclodextrin added is 2%-4% of the dry weight of fresh sunflower heads.

3. The method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S1, the hydrophobic Schiff base ligand is N-(2-hydroxybenzylmethyl)dodecane-1-amine.

4. The method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S2, during phase change extraction and in-situ chelation, the temperature of the stirring reaction is 25℃-35℃, the stirring speed is 300rpm to 500rpm, and the pressure is maintained for 40min-60min.

5. The method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S3, the hydrofluoroether is methoxynonafluorobutane, and the injection volume is 10%-15% of the total liquid volume of the system. During the shearing, the shearing rate is 2000rpm-3000rpm, lasting for 10min-15min.

6. The method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S3, the periodic pressure pulsation is based on the pressure value set in step S2, and the pressure is controlled to fluctuate within the range of ±0.05MPa of the base pressure, with a fluctuation frequency of 0.05Hz-0.2Hz, and the pulsation lasts for 10-20 minutes.

7. The method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S3, after the pressure pulsation ends, the microemulsion fractionation also includes a pressurized centrifugation step, with a centrifugation speed of 3000rpm-5000rpm and a centrifugation time of 5min-10min.

8. The method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S4, the processing steps after separating and collecting the middle layer pectin-enriched phase include: adding a 75% ethanol solution with a volume of 2 to 3 times the volume of the middle layer pectin-enriched phase to break the emulsion, filtering and collecting the solid, and vacuum drying at 40℃-50℃.

9. A method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S4, the carbon dioxide removal process includes the following steps: Nitrogen gas is introduced into the lower protein-containing liquid phase to purge it, or the temperature is heated to 50°C to 60°C until the system becomes turbid and separates into an upper hydrophobic solvent phase and a lower aqueous precipitate layer.

10. A method for extracting pectin and protein from sunflower heads according to claim 1, characterized in that, In step S4, after the carbon dioxide removal treatment, a solid-liquid separation step is also included: the layered system is centrifuged to remove the upper hydrophobic solvent phase containing hydrophobic Schiff base ligands, and the lower aqueous precipitate layer containing protein precipitates is retained.

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