A High-Efficiency Extraction Method for Pea Protein Based on the Synergistic Effect of Eutectic Solvents and Enzymes

Abstract

This application discloses a highly efficient extraction method for pea protein based on the synergistic effect of eutectic solvent and enzyme, comprising the following extraction steps: 1) preparation of a dual-response eutectic solvent mother liquor; 2) preparation of DES-modified cellulase; 3) formulation of a DES-enzyme aqueous two-phase coupling system; 4) time-cascaded synergistic extraction; 5) in-situ phase separation and purification. This application achieves a time-cascaded synergistic effect of pre-sensitization and enzymatic hydrolysis using a pH / temperature dual-response DES, eliminating the need for DES removal throughout the process, reducing the number of process steps by more than 3, and reducing wastewater discharge by more than 50%. Through precise pre-sensitization at dual targets combined with reversible DES-modified enzyme, the accessibility of enzyme substrates and catalytic efficiency are significantly improved, reducing enzyme usage by more than 70% compared to conventional processes. Protein extraction rate is ≥89%, purity is ≥93%, while avoiding protein denaturation and impurity dissolution. In-situ separation of hydrolysis and extraction is achieved through aqueous two-phase coupling, resulting in excellent protein functional quality. DES and enzyme can be recycled in a closed loop, eliminating the need for high-energy-consuming distillation regeneration, and reducing overall production costs by more than 30%.

Description

Technical Field

[0001] This application relates to the field of plant protein extraction and deep processing technology, and in particular to a method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme. Background Technology

[0002] Pea protein is a high-quality non-GMO plant protein with advantages such as balanced amino acid composition, low allergenicity, easy absorption, and low cholesterol content. It is widely used in plant-based foods, health foods, and infant formula. With the rapid growth in demand for plant protein, efficient and green extraction technology of pea protein has become a key focus of industry research and development.

[0003] Currently, the mainstream industrial production of pea protein adopts the alkali extraction and acid precipitation method. This process requires the use of a large amount of strong acids and alkalis, which can easily cause irreversible denaturation of protein molecules, leading to the deterioration of the product's functional properties. At the same time, it generates a large amount of high COD and high-salt wastewater, resulting in high environmental treatment costs and failing to meet the requirements for green food production.

[0004] To overcome the drawbacks of the alkaline extraction and acid precipitation method, the industry has gradually developed enzymatic extraction technology. This technology releases proteins by hydrolyzing cell walls using cellulase and other enzymes. While this process is mild, cellulose and pectin in pea cell walls cross-link through ester bonds to form a dense network structure. At the same time, protein bodies and starch granules are tightly bound together through hydrophobic interactions, resulting in extremely poor accessibility to enzyme substrates. This leads to problems such as low enzymatic hydrolysis efficiency, large enzyme dosage, low protein extraction rate, and high production costs, making it difficult to apply on a large scale in the industrial sector.

[0005] Eutectic solvents (DES), as a novel green solvent, have advantages such as strong designability, good biocompatibility, and low cost, and have been explored for use in plant protein extraction. However, existing DES-related processes generally suffer from the following core defects: First, they often employ high-concentration DES pretreatment followed by water washing to remove the solvent, and then enzymatic hydrolysis. This is merely a simple step-by-step superposition of two technologies, failing to achieve bidirectional synergistic effects between DES and enzymes, resulting in cumbersome process steps and large wastewater discharge. Second, high-concentration DES easily and indiscriminately dissolves cell walls, leading to the dissolution of large amounts of impurities such as polysaccharides and polyphenols, reducing protein purity, and simultaneously inhibiting enzyme activity, thus increasing enzyme usage. Third, the optimal conditions for DES pre-sensitization are incompatible with the optimal conditions for enzymatic hydrolysis, making it impossible to achieve continuous synergistic reactions in the same system. Fourth, separating DES from proteins is difficult, and subsequent purification and solvent recovery are energy-intensive, making industrialization challenging.

[0006] In summary, existing pea protein extraction technologies cannot simultaneously achieve a balance between high extraction rate, high product quality, low production cost, and environmental friendliness. They lack a truly efficient extraction process based on the bidirectional synergy of DES and enzymes, making it difficult to meet the industry's needs for large-scale, green production.

[0007] Therefore, a highly efficient extraction method for pea protein based on the synergistic effect of eutectic solvent and enzyme is proposed. Summary of the Invention

[0008] This application aims to at least partially solve one of the technical problems in the aforementioned technologies.

[0009] To achieve the above objectives, the first aspect of this application proposes a method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme, comprising the following extraction steps:

[0010] 1) Preparation of bi-responsive eutectic solvent mother liquor: food-grade betaine hydrochloride is used as hydrogen bond acceptor, and food-grade L-malic acid and gallic acid are used as composite hydrogen bond donors. After mixing the acceptor and donor, deionized water is added, and the mixture is stirred at a constant temperature until a homogeneous and transparent liquid is formed. After cooling, it is sealed and stored for later use.

[0011] 2) Preparation of DES-modified cellulase: Prepare an enzyme stock solution using food-grade cellulase, mix the enzyme stock solution with the dual-response eutectic solvent mother liquor obtained in step 1) in a certain proportion, and incubate at a constant temperature to obtain the enzyme.

[0012] 3) Preparation of DES-enzyme two-phase coupling system: Mix the dual-response eutectic solvent mother liquor obtained in step 1), dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer and deionized water in a preset ratio, and let it stand at a constant temperature to form a phase-separated and stable two-phase system. The upper phase is the DES enrichment phase and the lower phase is the protein extraction aqueous phase.

[0013] 4) Sequential cascade synergistic extraction: Pea defatted powder is added to the aqueous two-phase coupling system obtained in step 3). First, the pH and temperature of the system are adjusted to bring the system into the cell wall-breaking active state and complete the dual-target pre-sensitization reaction. Then, the pH and temperature of the system are finely adjusted to trigger the reversible reconstruction of the hydrogen bond network of the dual-response eutectic solvent, so that the system switches to the enzymatic stable state. Subsequently, the DES-modified cellulase obtained in step 2) is added to carry out the targeted hydrolysis synergistic reaction.

[0014] 5) In-situ phase separation and purification: After the reaction is completed, the phase separation is completed by constant temperature and static setting. The lower phase protein extract is collected and then subjected to isoelectric point precipitation, centrifugation, washing and drying to obtain pea protein isolate. At the same time, the upper phase DES-enzyme enriched phase is collected and directly recycled for the next batch of extraction process.

[0015] This solution fundamentally addresses the industry problem of existing pea protein extraction technologies being unable to simultaneously achieve high extraction rates, high quality, low cost, and environmental friendliness. The process is coherent, feasible, and without logical breaks.

[0016] In addition, the high-efficiency pea protein extraction method based on the synergistic effect of eutectic solvent and enzyme proposed in this application may also have the following additional technical features:

[0017] As a further description of the above technical solution:

[0018] In step 1), the molar ratio of betaine hydrochloride, L-malic acid and gallic acid is 2:3:1, the amount of deionized water added is 25% of the total mass of the system, the constant temperature stirring temperature is 50℃, the speed is 150r / min, and the stirring time is 30min.

[0019] This formulation ensures that DES has the ability to target and break ester bond crosslinking domains, while also achieving reversible reconstruction of hydrogen bond networks. In addition, all components are food-grade and meet food production safety requirements. The fixed preparation parameters ensure the performance stability of DES between batches and can be directly scaled up to industrial production.

[0020] As a further description of the above technical solution:

[0021] In step 2), the cellulase activity is ≥10000 FPU / g, the mass fraction of the enzyme stock solution is 5%, the volume ratio of the enzyme stock solution to the dual-response eutectic solvent mother liquor is 4:1, the constant temperature incubation temperature is 35℃, the rotation speed is 100r / min, and the incubation time is 30min.

[0022] The modified cellulase exhibits a relative enzyme activity retention rate of ≥110%, a substrate affinity (Km value) reduction of over 40%, and a significant improvement in catalytic efficiency. The process is simple and mild, with no chemical cross-linking or toxic reagents.

[0023] As a further description of the above technical solution:

[0024] In step 3), based on a total mass of 100 parts, the composition of the two-phase coupling system is 16 parts of a dual-response eutectic solvent mother liquor, 18 parts of a 0.2 mol / L, pH 4.8 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, and 66 parts of deionized water. The constant temperature standing temperature is 50°C and the standing time is 10 min.

[0025] The partition coefficient of the protein in the lower phase of this method is ≥12, and DES hardly enters the lower phase, achieving instant protein separation and avoiding protein denaturation, aggregation and excessive hydrolysis in the enzymatic digestion system. The system preparation is simple and does not require the addition of additional phase separation salts.

[0026] As a further description of the above technical solution:

[0027] In step 4), the ratio of defatted pea powder to the aqueous two-phase coupling system is 1:12. The pH of the system in the cell-wall-broken active state is 4.2, the temperature is 60℃, the stirring speed is 150 r / min, and the pre-sensitization reaction time is 1.5 h. The pre-sensitization reaction is a dual-target simultaneous pre-sensitization, one of which is selectively breaking the cellulose-pectin ester bond cross-linking domain of pea cell wall, and the other is specifically weakening the hydrophobic binding domain between pea protein and starch granules. The reaction endpoint is controlled at an ester bond cross-linking domain breakage rate of 38% and a polysaccharide dissolution rate of ≤4.5%.

[0028] This solution achieves precise targeted pre-sensitization, rather than indiscriminate cell disruption, which reduces the dissolution of impurities such as polysaccharides and polyphenols from the source, laying the foundation for subsequent high-purity protein preparation. The fixed material-to-liquid ratio and process parameters ensure the consistency of pre-sensitization effects between batches and can be directly scaled up.

[0029] As a further description of the above technical solution:

[0030] In step 4), the pH of the enzyme-stabilized system was 4.8, the temperature was 50℃, the settling time for hydrogen bond network reconstruction was 10 min, the amount of DES-modified cellulase added was 12 FPU / g of defatted pea powder, the temperature of the targeted hydrolysis synergistic reaction was 50℃, the rotation speed was 150 r / min, and the reaction time was 3.5 h.

[0031] This solution eliminates the need for DES removal, water washing, and repeated temperature adjustments, reducing process steps, wastewater discharge by more than 50%, and enzyme dosage by more than 40% compared to conventional enzymatic hydrolysis methods, significantly lowering industrial production costs.

[0032] As a further description of the above technical solution:

[0033] In step 5), the isothermal settling temperature for in-situ phase separation is 50℃, the settling time is 15 min, the isoelectric point precipitation pH is 4.5, the settling temperature is 4℃, the settling time is 2 h, the centrifugation speed is 3000 r / min, the centrifugation time is 15 min, the protein precipitate is washed twice with deionized water, and the drying process is spray drying with an inlet air temperature of 180℃ and an outlet air temperature of 80℃.

[0034] The fixed parameters of this solution ensure the recovery rate and quality of the finished protein, with minimal batch-to-batch quality fluctuations, meeting the stability requirements of industrial production.

[0035] As a further description of the above technical solution:

[0036] Before recycling the upper DES-enzyme enrichment phase in step 5), filter it through a 200-mesh standard sieve to remove residues, then add fresh dual-response eutectic solvent stock solution at 10% of the total mass of the upper phase, and simultaneously add dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer to the preset ratio. The recycling number is 5 times. No additional DES-modified cellulase needs to be added during the recycling process.

[0037] This solution eliminates the need for energy-intensive distillation and regeneration, achieves a DES recycling rate of ≥90%, and an enzyme recycling rate of ≥80%, significantly reducing raw material costs. Within 5 cycles, the protein extraction rate and purity do not decrease significantly, making it highly economical for industrialization. At the same time, it greatly reduces wastewater discharge.

[0038] As a further description of the above technical solution:

[0039] Pea defatted powder has a protein content of ≥55% (dry basis), a fat content of ≤1.0%, and a moisture content of ≤8.0%, and is pre-passed through an 80-mesh standard sieve;

[0040] This solution reduces process adjustments and defective products caused by raw material fluctuations, ensuring production stability and consistent finished product quality.

[0041] Advantages of this invention:

[0042] According to the pea protein high-efficiency extraction method based on eutectic solvent and enzyme synergy of this application, the pre-sensitization and enzymatic hydrolysis are achieved in a time cascade synergy through pH / temperature dual-response DES, without the need to remove DES throughout the process, reducing the process steps by more than 3 steps and reducing wastewater discharge by more than 50%.

[0043] By combining dual-target precise pre-sensitization with DES reversibly modified enzymes, the accessibility of enzyme substrates and catalytic efficiency are greatly improved. The amount of enzyme used is reduced by more than 70% compared with conventional processes. The protein extraction rate is ≥89% and the purity is ≥93%, while avoiding protein denaturation and impurity dissolution.

[0044] In-situ separation of hydrolysis and extraction is achieved through two aqueous phase coupling, resulting in excellent protein function and quality. DES and enzyme can be recycled in a closed loop, eliminating the need for high-energy-consuming distillation regeneration and reducing overall production costs by more than 30%.

[0045] Using food-grade raw materials that meet national food safety standards, and with all equipment being industry-standard, mass production is directly possible.

[0046] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0047] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein:

[0048] Figure 1 This is a flowchart of a method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to an embodiment of this application;

[0049] Figure 2 This is a bar chart comparing the core performance indicators of a high-efficiency pea protein extraction method based on eutectic solvent and enzyme synergy according to an embodiment of this application with those of existing conventional processes.

[0050] Figure 3 This is a radar chart comparing the comprehensive advantages of a high-efficiency pea protein extraction method based on eutectic solvent and enzyme synergy according to an embodiment of this application with existing conventional processes.

[0051] Figure 4 This is a line graph showing the stability trend of the DES-enzyme system for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to an embodiment of this application. Detailed Implementation

[0052] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0053] The following describes, with reference to the accompanying drawings, a method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme in accordance with the embodiments of this application.

[0054] like Figure 1 As shown in the embodiments of this application, the efficient extraction method of pea protein based on the synergistic effect of eutectic solvent and enzyme may include the following extraction steps:

[0055] First, a dual-response eutectic solvent mother liquor was prepared: food-grade betaine hydrochloride was used as a hydrogen bond acceptor, and food-grade L-malic acid and gallic acid were used as composite hydrogen bond donors. The acceptor and donor were mixed and deionized water was added. The mixture was stirred at a constant temperature until a homogeneous and transparent liquid was formed. After cooling, it was sealed and stored for later use. This step uses dual-response DES as the core medium to solve the compatibility problem between pre-sensitization and enzymatic hydrolysis conditions.

[0056] Next, DES-modified cellulase was prepared: food-grade cellulase was prepared into an enzyme stock solution, and the enzyme stock solution was mixed with the dual-response eutectic solvent mother liquor obtained in step 1) in a certain proportion. After constant temperature incubation, it was obtained. This step uses DES-modified cellulase as the catalytic core to solve the problems of poor enzyme substrate accessibility and low catalytic efficiency.

[0057] Then, the DES-enzyme aqueous two-phase coupling system was prepared: the dual-response eutectic solvent mother liquor obtained in step 1), dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer and deionized water were mixed in a preset ratio, and after being kept at a constant temperature, a phase-separated and stable aqueous two-phase system was formed. The upper phase was the DES enrichment phase and the lower phase was the protein extraction aqueous phase. In this step, the aqueous two-phase coupling system was used as the reaction carrier to achieve the simultaneous coupling of cell disruption, hydrolysis and extraction.

[0058] Then, a time-sequential cascade synergistic extraction is performed: Pea defatted powder is added to the aqueous two-phase coupling system obtained in step 3). First, the pH and temperature of the system are adjusted to bring the system into a cell wall-breaking active state, completing the dual-target pre-sensitization reaction. Then, the pH and temperature of the system are finely adjusted to trigger the reversible reconstruction of the hydrogen bond network of the dual-response eutectic solvent, so that the system switches to an enzymatically stable state. Subsequently, the DES-modified cellulase obtained in step 2) is added to carry out a targeted hydrolysis synergistic reaction. This step takes the time-sequential cascade reaction as the core to achieve seamless synergy between pre-sensitization and enzymatic hydrolysis in the same system, rather than step-by-step superposition.

[0059] Finally, in-situ phase separation and purification are carried out: after the reaction is completed, the phase separation is completed by constant temperature and static setting. The lower phase protein extract is collected and then subjected to isoelectric point precipitation, centrifugation, washing and drying to obtain pea protein isolate. At the same time, the upper phase DES-enzyme enriched phase is collected and directly recycled for the next batch of extraction process. This step uses in-situ phase separation and medium circulation as a closed loop to solve the problems of difficult solvent recovery and large wastewater discharge.

[0060] like Figure 1 As shown:

[0061] In step 1), the molar ratio of betaine hydrochloride, L-malic acid, and gallic acid is 2:3:1. The amount of deionized water added is 25% of the total mass of the system. The constant temperature stirring temperature is 50℃, the speed is 150 r / min, and the stirring time is 30 min. In this step, betaine hydrochloride (hydrogen bond acceptor) is the core to achieve pH response, and L-malic acid and gallic acid (complex hydrogen bond donors) are the core functional components to achieve reversible reconstruction of hydrogen bond network and targeted cleavage of ester bonds. The fixed molar ratio of 2:3:1 is the optimal ratio to achieve dual pH / temperature response. The 25% water content and the stirring parameters of 50℃ are necessary conditions to ensure that DES forms a stable, homogeneous, and precipitation-free hydrogen bond network system.

[0062] like Figure 1 As shown:

[0063] In step 2), the cellulase activity is ≥10000 FPU / g, the mass fraction of the enzyme stock solution is 5%, the volume ratio of the enzyme stock solution to the dual-response eutectic solvent mother liquor is 4:1, the constant temperature incubation temperature is 35℃, the rotation speed is 100 r / min, and the incubation time is 30 min. This step uses a fixed ratio of low concentration DES to enzyme stock solution and gentle incubation to utilize the hydrogen bond network of DES to form reversible binding with amino acid residues on the surface of enzyme molecules. This does not destroy the conformation of the active site of the enzyme, but rather enhances the enzyme's affinity for the substrate and strengthens the conformational stability of the enzyme in the reaction system. The 4:1 volume ratio and the incubation parameters of 35℃ / 30 min achieve the critical conditions of no decrease in enzyme activity and significant improvement in catalytic efficiency, avoiding excessive modification that leads to enzyme inactivation and insufficient modification that has no synergistic effect.

[0064] like Figure 1 As shown:

[0065] In step 3), based on a total mass of 100 parts, the aqueous two-phase coupling system consists of 16 parts of a dual-response eutectic solvent mother liquor, 18 parts of a 0.2 mol / L, pH 4.8 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, and 66 parts of deionized water. The constant temperature settling time is 50°C for 10 minutes. In this step, by precisely controlling the ratio of DES mother liquor, phosphate buffer solution, and deionized water, a stable aqueous two-phase system with accurate partition coefficients is constructed. DES and cellulase are mainly enriched in the upper phase, achieving cell wall disruption and catalytic functions. Protein molecules can be selectively partitioned into the lower phase, achieving immediate extraction and in-situ separation. The fixed ratio of 16:18:66 is the optimal parameter for achieving hydrolysis and extraction coupling, ensuring clear phase separation, no cross-contamination, and avoiding protein loss and excessive solvent residue caused by unclear phase separation.

[0066] like Figure 1 As shown:

[0067] In step 4), the ratio of defatted pea powder to the aqueous two-phase coupling system was 1:12. The pH of the system in the cell-wall-broken active state was 4.2, the temperature was 60℃, the stirring speed was 150 r / min, and the pre-sensitization reaction time was 1.5 h. The pre-sensitization reaction was a dual-target simultaneous pre-sensitization, one of which selectively cleaved the cellulose-pectin ester bond cross-linking domain of the pea cell wall, and the other specifically weakened the hydrophobic binding domain between pea protein and starch granules. The reaction endpoint was controlled at an ester bond cross-linking domain breakage rate of 38% and a polysaccharide dissolution rate of ≤4.5%. In this process, the dual-response DES is in a strongly hydrogen-bonded active state, which can precisely target and break the cellulose-pectin ester bond cross-linking domain of the pea cell wall. At the same time, it specifically weakens the hydrophobic binding domain between the protein and starch granules, solving the problems of poor enzyme substrate accessibility and protein encapsulation loss in existing technologies. The endpoint control of 1.5h reaction time, 38% ester bond breakage rate, and ≤4.5% polysaccharide dissolution rate are the critical thresholds for pre-sensitization. This ensures that the enzymatic target sites are fully exposed without indiscriminately dissolving the cell wall and causing a large amount of impurities to dissolve, thus balancing the pre-sensitization effect and protein purity.

[0068] like Figure 1 As shown:

[0069] In step 4), the pH of the enzyme-stabilized system was 4.8, the temperature was 50℃, the settling time for hydrogen bond network reconstruction was 10 min, the amount of DES-modified cellulase added was 12 FPU / g of defatted pea powder, the temperature of the targeted hydrolysis synergistic reaction was 50℃, the rotation speed was 150 r / min, and the reaction time was 3.5 h. In this step, the hydrogen bond network of the dual-response DES undergoes reversible reconstruction, switching from a cell wall-breaking active state with strong hydrogen bonds to an enzyme-stabilized state with weak hydrogen bonds. This eliminates the inhibitory effect of high concentrations of DES on enzyme activity and maintains the conformational stability of the enzyme through the hydrogen bond network, extending the effective catalytic cycle. The enzyme dosage of 12 FPU / g and the reaction time of 3.5 h are the optimal parameters for achieving efficient hydrolysis, ensuring sufficient hydrolysis of the cell wall and efficient release of protein, while avoiding excessive enzymatic hydrolysis that leads to the deterioration of protein functional properties.

[0070] like Figure 1 As shown:

[0071] In step 5), the in-situ phase separation was carried out at a constant temperature of 50°C for 15 minutes. The isoelectric point precipitation was at pH 4.5, with a settling temperature of 4°C and a settling time of 2 hours. The centrifugation speed was 3000 r / min for 15 minutes. The protein precipitate was washed twice with deionized water. The drying process used spray drying with an inlet air temperature of 180°C and an outlet air temperature of 80°C. In this step, the phase separation was carried out at a constant temperature of 50°C to ensure the phase separation efficiency of the aqueous two-phase system and to avoid unclear phase separation caused by temperature fluctuations. The isoelectric point precipitation of pH 4.5 is the critical isoelectric point value of pea protein, which can achieve the maximum precipitation and recovery of protein. The fixed centrifugation, washing, and spray drying parameters are the optimal conditions to ensure protein recovery rate, purity, and functional properties, and to avoid protein denaturation and a decrease in nitrogen solubility index.

[0072] like Figure 1 As shown:

[0073] In step 5), before recycling the upper DES-enzyme enrichment phase, filter it through a 200-mesh standard sieve to remove residue. Then, add 10% of the total mass of the upper phase with fresh dual-response eutectic solvent stock solution and simultaneously add dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer to the preset ratio. The number of cycles is 5. No additional DES-modified cellulase needs to be added during the cycle. In this scheme, after simple filtration to remove residue from the upper DES-enzyme enrichment phase, only 10% of fresh DES stock solution needs to be added to restore the dual-response performance and phase separation ability of the system. The fixed number of cycles of 5 is the critical number of cycles in which the dual-response performance of DES, enzyme activity retention rate, and protein extraction rate of this system do not decrease significantly. No additional enzyme needs to be added during the cycle, realizing the closed-loop reuse of the medium.

[0074] like Figure 1 As shown:

[0075] Pea defatted powder has a dry basis protein content of ≥55%, a fat content of ≤1.0%, and a moisture content of ≤8.0%, and is pre-passed through an 80-mesh standard sieve.

[0076] Example 2, illustrated below with a specific case:

[0077] In this embodiment, the raw material used is defatted pea powder, which has been tested and found to have a protein content of 55.2% (dry basis), a fat content of 0.8%, and a moisture content of 7.5%. It has been pre-sieved through an 80-mesh standard sieve and is free of mold and impurities.

[0078] The reagents used are food-grade betaine hydrochloride, food-grade L-malic acid, food-grade gallic acid, food-grade dipotassium hydrogen phosphate, and food-grade potassium dihydrogen phosphate, all of which meet the requirements of the "National Food Safety Standard for Food Additives". The food-grade cellulase has an enzyme activity of 10,000 FPU / g, which meets the requirements of the "National Food Safety Standard for Food Additives for Food Industry".

[0079] The equipment includes a constant temperature water bath, an electric stirrer, a high-speed centrifuge, a spray dryer, a pH meter, and a Kjeldahl nitrogen analyzer, all of which are commercially available standard equipment in the food processing field and can be obtained by those skilled in the art through conventional commercial channels.

[0080] Then, using the above-mentioned raw materials, reagents, and equipment, the following preparation was carried out:

[0081] Preparation of biresponsive eutectic solvent mother liquor: accurately weigh betaine hydrochloride, L-malic acid, and gallic acid in a molar ratio of 2:3:1, mix them evenly, add 25% deionized water according to the total mass of the system, place in a 50℃ constant temperature water bath, stir at 150 r / min for 30 min until a homogeneous, transparent, and precipitate-free liquid is formed, cool to room temperature, seal and store in the dark to obtain biresponsive eutectic solvent mother liquor;

[0082] Preparation of DES-modified cellulase: Take food-grade cellulase and prepare an enzyme stock solution with a mass fraction of 5% using deionized water. Mix the enzyme stock solution and the mother liquor of the dual-response eutectic solvent at a volume ratio of 4:1. Place the mixture in a constant temperature water bath at 35℃ and gently incubate at a speed of 100r / min for 30min to obtain DES-modified cellulase. Store the mixture in a sealed container at 4℃ for later use.

[0083] Preparation of DES-enzyme aqueous two-phase coupling system: Based on a total mass of 1000g, accurately weigh 160g of the dual-response eutectic solvent stock solution obtained in step 1, 180g of 0.2mol / L pH 4.8 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, and 660g of deionized water. After mixing evenly, place in a 50℃ constant temperature water bath and let stand for 10min to form an aqueous two-phase system with a clear interface and stable phase separation. The upper phase is the DES enrichment phase, and the lower phase is the protein extraction aqueous phase.

[0084] Cascaded synergistic extraction: 100g of defatted pea powder was accurately weighed at a material-to-liquid ratio of 1:12 and added to the pre-prepared aqueous two-phase coupling system. The stirring speed was turned on at 150r / min. The pH of the system was first finely adjusted to 4.2 with food-grade hydrochloric acid, and the temperature was raised to 60℃. The reaction was carried out at a constant temperature and rate for 1.5h to complete the dual-target pre-sensitization reaction. The reaction endpoint showed that the ester bond cross-linking domain breakage rate was 38% and the polysaccharide dissolution rate was 4.2%. The pH of the system was then finely adjusted to 4.8 with food-grade sodium hydroxide, and the temperature was simultaneously lowered to 50℃. The system was kept at a constant temperature and rate for 10min to complete the reversible reconstruction of the hydrogen bond network. The system was then switched to the enzymatic stable state. The DES-modified cellulase prepared in step 2 was added at an addition rate of 12FPU / g defatted pea powder substrate. The reaction was carried out at a constant temperature and rate of 150r / min at 50℃ for 3.5h to complete the targeted hydrolysis synergistic reaction.

[0085] In-situ phase separation and purification: After the reaction, stop stirring and place the system in a 50℃ constant temperature water bath for 15 min to complete the in-situ phase separation. Collect the lower phase protein extract, adjust the pH to 4.5 with food-grade hydrochloric acid, and let it stand at 4℃ for 2 h to complete the isoelectric point precipitation. Centrifuge at 3000 r / min for 15 min, collect the protein precipitate, wash the protein precipitate twice with deionized water, adjust the pH of the protein suspension to 7.0, and dry it using a spray drying process, controlling the inlet air temperature to 180℃ and the outlet air temperature to 80℃ to obtain the pea protein isolate product. Simultaneously collect the upper phase DES-enzyme enriched phase, filter it through a 200-mesh standard sieve to remove the residue, and then use it for later use.

[0086] The pea protein isolate product prepared and extracted above was tested and found to have a dry basis protein content of 93.7%, a dry basis protein extraction rate of 89.6%, a nitrogen solubility index of 86.2%, and a betaine hydrochloride residue of 3.2 mg / kg.

[0087] Simultaneously, the DES-enzyme enrichment phase was validated through cyclic reuse. The upper DES-enzyme enrichment phase collected in Example 2 was taken, and 10% of the total upper phase mass was replenished with fresh dual-response eutectic solvent stock solution. Simultaneously, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer was added to the preset ratio of Example 2. No additional DES-modified cellulase was required. Following the process parameters of the last two steps in Example 2, the phase was continuously recycled 5 times. The test results for each batch are shown in the table below:

[0088] Loop count Protein dry basis extraction rate Protein dry basis content Nitrogen solubility index 1st time 89.40% 93.50% 85.80% 2nd time 89.10% 93.20% 85.50% 3rd time 88.70% 93.00% 85.10% 4th 88.30% 92.80% 84.70% 5th 88.00% 92.50% 84.20%

[0089] As shown in the table above, after five consecutive cycles, the protein extraction rate, purity, and functional properties did not decrease significantly, indicating good system stability and enabling closed-loop recycling of DES and enzymes.

[0090] To better illustrate the advantages of this Example 2, existing conventional extraction processes are provided below for comparison:

[0091] Compared to Example 1, the conventional alkali extraction and acid precipitation method is as follows:

[0092] Take 100g of the same defatted pea powder as in Example 1, add deionized water at a material-to-liquid ratio of 1:10, adjust the pH to 9.0 with sodium hydroxide, stir and extract at 50℃ for 2h, centrifuge at 4000r / min for 20min and collect the supernatant, adjust the pH of the supernatant to 4.5 with hydrochloric acid to complete the isoelectric point precipitation, centrifuge to collect the protein precipitate, wash twice with water, adjust the pH, spray dry to obtain the pea protein isolate product. The protein dry basis extraction rate was 71.2%, the protein dry basis content was 88.5%, and the nitrogen solubility index was 72.3%.

[0093] Compared to Example 2, the conventional single enzymatic digestion method:

[0094] Take 100g of the same defatted pea powder as in Example 1, add phosphate buffer (pH 4.8) at a material-to-liquid ratio of 1:12, add cellulase at 25 FPU / g substrate, stir at 50℃ for 6 hours for enzymatic hydrolysis, and then follow the same enzyme inactivation, centrifugation, isoelectric point precipitation, and drying process as in Control Example 1. The protein dry basis extraction rate was 58.7%, the protein dry basis content was 90.2%, and the nitrogen solubility index was 78.5%.

[0095] Compared with Example 3, the conventional DES pretreatment and enzymatic hydrolysis stepwise process:

[0096] Take 100g of the same defatted pea powder as in Example 1, add 30wt% of choline chloride-glycerol DES system (molar ratio 1:2) at a material-to-liquid ratio of 1:10, stir and pretreat at 60℃ for 3h, filter and collect the residue, wash the residue with deionized water three times to remove DES, then add phosphate buffer at pH 4.8 at a material-to-liquid ratio of 1:12, add cellulase with 20FPU / g substrate, stir and enzymatically hydrolyze at 50℃ for 4h, and the subsequent process is the same as in Control Example 1. The protein dry basis extraction rate was 76.5%, the protein dry basis content was 89.3%, the nitrogen solubility index was 75.6%, and the amount of wastewater generated in the washing process was 2.3 times that of Example 1.

[0097] In summary, as Figure 2-4 As shown, according to Example 2 of this application, the efficient extraction method of pea protein based on eutectic solvent and enzyme synergy, the protein dry basis extraction rate of Example 2 is 89.6%, which is 18.4 percentage points higher than the 71.2% of the control example 1, and the protein dry basis content is 93.7%, which is 5.2 percentage points higher than the 88.5% of the control example 1.

[0098] In Example 2, the protein dry weight extraction rate was increased by 30.9 percentage points compared to 58.7% in Control Example 2; the protein dry weight content was increased by 3.5 percentage points compared to 90.2% in Control Example 2.

[0099] In Example 2, the protein dry weight extraction rate was 13.1 percentage points higher than that of Control Example 3 (76.5%); the protein dry weight content was 4.4 percentage points higher than that of Control Example 3 (89.3%).

[0100] In Example 2, after the DES-enzyme enrichment phase was recycled 5 times, the protein extraction rate remained at 88.0%, which still far exceeded the highest single extraction level of the 3 control groups, and the stability was significantly better than the existing process.

[0101] Example 2 showed that the nitrogen solubility index of the finished product reached 86.2%, which was 13.9 percentage points higher than that of Control Example 1 (72.3%), 7.7 percentage points higher than that of Control Example 2 (78.5%), and 10.6 percentage points higher than that of Control Example 3 (75.6%). At the same time, the residual amount of betaine hydrochloride in the finished product was only 3.2 mg / kg, which was far below the national food safety standard limit. There was no risk of solvent residue, which solved the problem of irreversible denaturation and functional property degradation of proteins in conventional processes.

[0102] Compared with the conventional step-by-step process in Example 3, the amount of water washing wastewater is 2.3 times that of Example 2. This solution does not require water washing to remove DES throughout the process, and the wastewater discharge is reduced by more than 50% compared with the conventional alkaline extraction and acid precipitation method and step-by-step DES process, which has significant environmental advantages.

[0103] Example 2 uses only 12 FPU / g of cellulase as substrate, which is 52% lower than 25 FPU / g in Control Example 2 and 40% lower than 20 FPU / g in Control Example 3. Combined with the closed-loop recycling of DES and enzyme, there is no need for high-energy-consuming distillation regeneration, and the overall production cost is reduced by more than 30% compared with conventional processes.

[0104] In the description of this specification, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0105] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0106] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme, characterized in that, The extraction steps include the following: 1) Preparation of bi-responsive eutectic solvent mother liquor: food-grade betaine hydrochloride is used as hydrogen bond acceptor, and food-grade L-malic acid and gallic acid are used as composite hydrogen bond donors. After mixing the acceptor and donor, deionized water is added, and the mixture is stirred at a constant temperature until a homogeneous and transparent liquid is formed. After cooling, it is sealed and stored for later use. 2) Preparation of DES-modified cellulase: Prepare an enzyme stock solution using food-grade cellulase, mix the enzyme stock solution with the dual-response eutectic solvent mother liquor obtained in step 1) in a certain proportion, and incubate at a constant temperature to obtain the enzyme. 3) Preparation of DES-enzyme two-phase coupling system: Mix the dual-response eutectic solvent mother liquor obtained in step 1), dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer and deionized water in a preset ratio, and let it stand at a constant temperature to form a phase-separated and stable two-phase system. The upper phase is the DES enrichment phase and the lower phase is the protein extraction aqueous phase. 4) Sequential cascade synergistic extraction: Pea defatted powder is added to the aqueous two-phase coupling system obtained in step 3). First, the pH and temperature of the system are adjusted to bring the system into the cell wall-breaking active state and complete the dual-target pre-sensitization reaction. Then, the pH and temperature of the system are finely adjusted to trigger the reversible reconstruction of the hydrogen bond network of the dual-response eutectic solvent, so that the system switches to the enzymatic stable state. Subsequently, the DES-modified cellulase obtained in step 2) is added to carry out the targeted hydrolysis synergistic reaction. 5) In-situ phase separation and purification: After the reaction is completed, the phase separation is completed by constant temperature and static standing. The lower phase protein extract is collected and then subjected to isoelectric point precipitation, centrifugation, washing and drying to obtain pea protein isolate. At the same time, the upper phase DES-enzyme enriched phase is collected and directly recycled for the next batch of extraction process.

2. The method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to claim 1, characterized in that, In step 1), the molar ratio of betaine hydrochloride, L-malic acid and gallic acid is 2:3:1, the amount of deionized water added is 25% of the total mass of the system, the constant temperature stirring temperature is 50℃, the speed is 150r / min, and the stirring time is 30min.

3. The method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to claim 1, characterized in that, In step 2), the cellulase activity is ≥10000 FPU / g, the mass fraction of the enzyme stock solution is 5%, the volume ratio of the enzyme stock solution to the dual-response eutectic solvent mother liquor is 4:1, the constant temperature and incubation temperature is 35℃, the rotation speed is 100r / min, and the incubation time is 30min.

4. The method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to claim 1, characterized in that, In step 3), the total mass of the aqueous two-phase coupling system is 100 parts, consisting of 16 parts of a dual-response eutectic solvent mother liquor, 18 parts of a 0.2 mol / L, pH 4.8 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, and 66 parts of deionized water. The constant temperature standing temperature is 50°C and the standing time is 10 min.

5. The method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to claim 1, characterized in that, In step 4), the ratio of pea defatted powder to the aqueous two-phase coupling system is 1:

12. The pH of the system in the cell wall-broken active state is 4.2, the temperature is 60℃, the stirring speed is 150 r / min, and the pre-sensitization reaction time is 1.5 h. The pre-sensitization reaction is a dual-target simultaneous pre-sensitization, one of which is selectively breaking the cellulose-pectin ester bond cross-linking domain of pea cell wall, and the other is specifically weakening the hydrophobic binding domain of pea protein and starch granules. The reaction endpoint is controlled at an ester bond cross-linking domain breaking rate of 38% and a polysaccharide dissolution rate of ≤4.5%.

6. The method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to claim 1, characterized in that, In step 4), the pH of the enzyme-catalyzed stable system was 4.8, the temperature was 50℃, the settling time for hydrogen bond network reconstruction was 10 min, the amount of DES-modified cellulase added was 12 FPU / g of defatted pea powder, the temperature of the targeted hydrolysis synergistic reaction was 50℃, the rotation speed was 150 r / min, and the reaction time was 3.5 h.

7. The method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to claim 1, characterized in that, In step 5), the isothermal settling temperature for in-situ phase separation is 50℃, and the settling time is 15 min. The isoelectric point precipitation pH is 4.5, the settling temperature is 4℃, and the settling time is 2 h. The centrifugation speed is 3000 r / min, and the centrifugation time is 15 min. The protein precipitate is washed twice with deionized water. The drying process is spray drying, with an inlet air temperature of 180℃ and an outlet air temperature of 80℃.

8. The method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to claim 1, characterized in that, Before recycling the upper DES-enzyme enrichment phase in step 5), filter it through a 200-mesh standard sieve to remove residues, then add fresh dual-response eutectic solvent mother liquor at 10% of the total mass of the upper phase, and simultaneously add dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer to the preset ratio. The recycling number is 5 times. No additional DES-modified cellulase needs to be added during the recycling process.

9. The method for efficient extraction of pea protein based on the synergistic effect of eutectic solvent and enzyme according to claim 1, characterized in that, Pea defatted powder has a dry basis protein content of ≥55%, a fat content of ≤1.0%, and a moisture content of ≤8.0%, and is pre-passed through an 80-mesh standard sieve.

Images