A method for extracting protein from red beans and purifying kqs-1 peptide

Abstract

The present application relates to the field of bioactive peptide extraction and purification, and particularly relates to a method for extracting protein from red beans and separating and purifying KQS-1 peptide, which comprises the following steps: step one, raw material pretreatment, step two, red bean total protein extraction, step three, proteolysis, step four, KQS-1 peptide separation and purification, etc. The method for extracting protein from red beans and separating and purifying KQS-1 peptide of the present application realizes efficient and stable preparation of high-purity KQS-1 peptide from red bean raw materials through process optimization and combined purification strategy. The present application adopts a combined process route of ultrafiltration refining, ion exchange chromatography rough separation and reverse phase high performance liquid chromatography refining, which completely changes the situation of low purity and low yield caused by relying on single chromatography technology. According to different properties such as molecular size, charge and hydrophobicity, various impurities are removed step by step and systematically, so that directional and efficient capture and fine separation of KQS-1 peptide are realized, and the purity of the final product can be stably above 95%, and the yield is more than 6%, which is much better than the level of 60%-70% in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide extraction and purification, specifically relating to a method for extracting protein from red beans and separating and purifying KQS-1 peptide. Background Technology

[0002] Red beans, a precious traditional food and medicinal resource, are rich in protein, with a content as high as 20% to 25%, making them an important source of high-quality plant protein. In recent years, with the deepening research on bioactive peptides, researchers have gradually focused on the precise isolation of bioactive peptides with specific functions from plant proteins. Research in this field is not only of great significance in food science, but has also attracted widespread attention in the biomedical field.

[0003] KQS-1 peptide, a bioactive peptide with a specific amino acid sequence, exhibits great potential in food preservation, nutritional supplementation, and pharmaceutical applications due to its unique properties. In food preservation, KQS-1 peptide can effectively inhibit microbial growth and extend the shelf life of food. In nutritional supplementation, it helps promote the absorption of nutrients and enhances immunity. In pharmaceutical applications, research shows that KQS-1 peptide possesses various biological activities such as antioxidant and anti-inflammatory effects, which are of great significance for the prevention and treatment of certain diseases.

[0004] However, the technology for efficiently extracting protein from red beans and further purifying KQS-1 peptides is still immature. Traditional processes often use alkaline extraction and acid precipitation to extract red bean protein. While this method is simple to operate, it suffers from low protein extraction rates (usually below 75%), insufficient product purity, and poor process stability. Furthermore, in the peptide separation stage, existing processes often rely on single chromatographic purification methods (such as ion exchange or gel filtration), making it difficult to achieve efficient capture and fine separation of the target peptides. This results in a final product purity that is often only 60%-70%, with a low yield, failing to meet the quality requirements of high-purity peptide products in high-end foods, health supplements, and pharmaceutical preparations. Therefore, overcoming these technical problems and deficiencies is a key issue that needs to be addressed. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects described in the background art. By optimizing the extraction and purification process and the purification strategy of ultrafiltration + ion exchange + reversed-phase high-performance liquid chromatography, the targeted separation of KQS-1 peptide is achieved, thereby solving the problems of low extraction rate of red bean protein and insufficient purity and yield of KQS-1 peptide in the prior art.

[0006] To achieve the above-mentioned objectives, the technical solution of this invention is: a method for extracting protein from red beans and separating and purifying KQS-1 peptide, mainly comprising the following steps: Step 1, raw material pretreatment: The red bean raw material is subjected to impurity removal, low-temperature drying, crushing and grinding in sequence to obtain red bean slurry; Step 2, extraction of total protein from red beans: The red bean powder slurry obtained in Step 1 is extracted at a constant temperature under alkaline conditions, followed by solid-liquid separation, clarification, concentration and drying to obtain total protein powder from red beans; Step 3, enzymatic hydrolysis: Dissolve the total protein powder from red beans obtained in step 2, adjust to neutral conditions, add neutral protease for enzymatic hydrolysis, inactivate the enzyme after hydrolysis and separate solid and liquid to obtain crude peptide solution. Step 4, KQS-1 peptide separation and purification: The crude peptide solution obtained in Step 3 is subjected to ultrafiltration, ion exchange chromatography, reversed-phase high-performance liquid chromatography purification and freeze drying to obtain KQS-1 peptide dry powder.

[0007] Specifically, in step one, the protein content of the red bean raw material used is ≥22%, the temperature of the low-temperature drying is 50-80℃, and the material is dried until the moisture content is ≤13%; the particle size range of the crushed material is 80-100 mesh; the material-liquid ratio of the grinding process is red bean powder: deionized water = 1g: 10mL.

[0008] Specifically, in step two, the alkaline condition is to adjust the pH value of the slurry to 8.5-9.0; the constant temperature extraction temperature is 50℃ and the time is 2 hours.

[0009] The solid-liquid separation is performed by plate and frame filtration, the clarification is performed by centrifugation, and the concentration is performed by vacuum concentration under the following conditions: vacuum degree ≤ -0.09MPa and temperature 60-70℃, until the volume is concentrated to 1 / 5-1 / 6 of the original volume.

[0010] The drying process is spray drying, with an inlet air temperature of 180-200℃ and an outlet air temperature of 80-90℃.

[0011] Specifically, in step three, the ratio of dissolved material to liquid is 1g of total red bean protein powder to 20mL of deionized water; the neutral condition is to adjust the pH of the solution to 7.0-7.5; the enzymatic hydrolysis temperature is 50℃; and the time is 4 hours.

[0012] The amount of neutral protease added is 2% of the total protein powder mass of red beans, and its enzyme activity is ≥50000U / g.

[0013] The enzyme inactivation conditions were maintained at 121°C for 20 minutes.

[0014] Specifically, in step four, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, operates at a pressure of 0.2-0.3 MPa, and collects the permeate.

[0015] Specifically, in step four, the ion exchange chromatography uses a DEAE-52 cellulose column; before loading the sample, the column is equilibrated with 0.05 mol / L, pH 7.0 phosphate buffer, and the loading flow rate is 5 mL / min.

[0016] First, impurities are eluted using the aforementioned equilibration buffer, and then gradient elution is performed using a phosphate buffer containing 0.1-0.5 mol / L NaCl. The target elution peak with UV absorption at a wavelength of 220 nm is collected.

[0017] Specifically, in step four, the reversed-phase high-performance liquid chromatography purification uses a C18 column; Before loading the sample, an acetonitrile-water solution with a volume ratio of 10:90 was used as the mobile phase to balance the chromatographic system, and the mobile phase flow rate was 10 mL / min. The target component collected by ion exchange chromatography was concentrated and then loaded onto the sample at a flow rate of 2 mL / min. Gradient elution was performed using acetonitrile-water solution, with the acetonitrile volume ratio increasing from 10% to 40% within 30 minutes; the elution peaks with retention times consistent with those of the KQS-1 peptide standard were collected and monitored at a wavelength of 220 nm.

[0018] Specifically, in step four, the freeze-drying conditions are as follows: first, pre-freeze at -40℃ for 4 hours, and then dry at -55℃ and vacuum degree ≤10Pa for 12 hours.

[0019] Preferably, in step one, 10 kg of red bean raw material is taken, and after removing impurities by vibrating screen, it is dried at a low temperature of 60℃ for 5 hours until the moisture content is 12.5%. It is then pulverized to 90 mesh using a pulverizer. 5 kg of red bean powder is added to 50 L of deionized water at a material-to-liquid ratio of 1:10, and then processed by a colloid mill at 3000 r / min for 8 minutes to obtain a uniform slurry.

[0020] In step two, the slurry obtained in step one is pumped into an extraction tank and stirred at a speed of 80 r / min. The pH is adjusted to 8.8 with 1 mol / L sodium hydroxide, and the extraction is carried out at a constant temperature of 50℃ for 2 hours. Sodium hydroxide is added every 20 minutes to maintain pH stability. The extract is filtered by plate and frame press to obtain filtrate. The soybean residue after plate and frame pressing is washed twice with 10L of deionized water, and the washing liquid is added to the filtrate. The combined filtrate is centrifuged at 4500 r / min for 15 minutes, and the clear filtrate is collected. The clear liquid is vacuum concentrated to 12L under vacuum conditions of -0.09 MPa and 65℃ to obtain a concentrated solution with a protein concentration of 22%. Then, it is spray dried with an inlet air temperature of 190℃ and an outlet air temperature of 85℃ to obtain 0.65 kg of red bean total protein powder.

[0021] In step three, 0.5 kg of the protein powder obtained in step two was dissolved in 10 L of deionized water. The pH was adjusted to 7.2 with 1 mol / L hydrochloric acid. 10 g of neutral protease was added at 50 °C and the mixture was kept at a constant temperature for 4 hours, with stirring for 5 minutes every 30 minutes. After the enzymatic hydrolysis was completed, the hydrolysate was pumped into a sterilization tank and inactivated at 121 °C for 20 minutes. After cooling to room temperature, it was filtered to obtain 9.8 L of crude peptide solution.

[0022] In step four, the crude peptide solution obtained in step three is ultrafiltered through a 10 kDa ultrafiltration membrane at a pressure of 0.25 MPa, and 8.5 L of permeate is collected. The permeate is loaded at a flow rate of 5 mL / min onto a DEAE-52 cellulose ion exchange column equilibrated with 0.05 mol / L, pH 7.0 phosphate buffer. The column is eluted first with equilibration buffer, and then with a gradient elution of phosphate buffer containing 0.1-0.5 mol / L NaCl. The target peak is collected at a wavelength of 220 nm to obtain 1.2 L of crude pure solution.

[0023] After concentrating the crude pure solution to 0.2 L, it was loaded onto a C18 reversed-phase high-performance liquid chromatography column equilibrated with an acetonitrile-water solution of volume ratio 10:90 at a flow rate of 2 mL / min. The column was eluted with a 10%-40% acetonitrile-water gradient for 30 min, and the elution peak with the retention time of the KQS-1 peptide standard was collected at a wavelength of 220 nm to obtain 0.15 L of purified solution.

[0024] After the purified liquid was pre-frozen at -40℃ for 4 hours, it was freeze-dried at -55℃ and 10Pa for 12 hours to obtain 32g of KQS-1 peptide powder.

[0025] Preferably, in step one, 20 kg of red bean raw material is taken, and after removing impurities by vibrating screen, it is dried at a low temperature of 60°C for 4 hours until the moisture content is 12%. It is then pulverized to 80 mesh using a pulverizer. 10 kg of red bean powder is taken and added to 100 L of deionized water at a material-to-liquid ratio of 1:10. The mixture is then processed by a colloid mill at 3000 r / min for 10 minutes to obtain a uniform slurry.

[0026] In step two, the slurry obtained in step one is pumped into an extraction tank and stirred at a stirring speed of 80 r / min. The pH is adjusted to 9.0 with 1 mol / L sodium hydroxide and extracted at a constant temperature of 50℃ for 2 hours. Sodium hydroxide is added every 20 minutes to maintain pH stability. The extract is then filtered by plate and frame filter, centrifuged and clarified, and vacuum concentrated to 25 L. Finally, it is spray dried at an inlet air temperature of 200℃ and an outlet air temperature of 90℃ to obtain 1.32 kg of total red bean protein powder.

[0027] In step three, 1 kg of the protein powder obtained in step two was dissolved in 20 L of deionized water, the pH was adjusted to 7.5, 20 g of neutral protease was added and enzymatically hydrolyzed at 50 °C for 4 hours, and after inactivation and filtration, 19.5 L of crude peptide solution was obtained.

[0028] In step four, after ultrafiltration, ion exchange chromatography, and reversed-phase high-performance liquid chromatography, 65g of KQS-1 peptide powder was obtained by freeze drying.

[0029] The method for extracting protein from red beans and purifying KQS-1 peptide according to the present invention has the following beneficial effects: 1. The method for extracting protein from red beans and purifying KQS-1 peptide of the present invention, through process optimization and combined purification strategies, achieves efficient and stable preparation of high-purity KQS-1 peptide from red bean raw materials. Its technical indicators significantly surpass those of traditional methods and can meet the strict quality requirements of peptide products in high-end application fields.

[0030] 2. This invention significantly improves the extraction rate of red bean protein by optimizing the alkaline extraction process parameters and employing an enhanced separation method combining plate and frame filtration and centrifugal clarification, overcoming the bottleneck of traditional alkaline extraction and acid precipitation methods where the extraction rate is typically below 75%. Simultaneously, optimized spray drying parameters ensure the high purity and good functionality of the resulting protein powder, providing a high-quality and stable substrate for subsequent enzymatic hydrolysis, thus guaranteeing the quality and yield of the final product from the source.

[0031] 3. This invention employs a combined process route of ultrafiltration purification → ion exchange chromatography rough separation → reversed-phase high-performance liquid chromatography purification, completely changing the situation where relying on a single chromatographic technology leads to low purity and yield. Based on different properties such as molecular size, charge, and hydrophobicity, various impurities are removed step-by-step and systematically, thereby achieving targeted, efficient capture and fine separation of KQS-1 peptides. The final product purity can stably reach over 95%, and the yield exceeds 6%, far superior to the purity level of only 60%-70% in existing technologies. Detailed Implementation

[0032] The present invention will now be described in more detail through specific embodiments.

[0033] In the description of this invention, it should be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship shown, and are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention. Example

[0034] This embodiment discloses a method for extracting protein from red beans and separating and purifying KQS-1 peptide. By optimizing the extraction and purification process and using an ultrafiltration + ion exchange + reversed-phase high-performance liquid chromatography purification strategy, the method achieves the directional separation of KQS-1 peptide, thereby solving the problems of low extraction rate of red bean protein and insufficient purity and yield of KQS-1 peptide in the prior art.

[0035] The method for extracting protein from red beans and purifying KQS-1 peptide in this embodiment mainly includes the following steps: Step 1, raw material pretreatment: The red bean raw materials are subjected to impurity removal, low-temperature drying, crushing and grinding in sequence to obtain red bean slurry.

[0036] In this step, the protein content of the red bean raw material used is ≥22%, the low-temperature drying temperature is 50-80℃, and the material is dried until the moisture content is ≤13%; the particle size range of the crushed material is 80-100 mesh. The material-to-liquid ratio of the grinding process is red bean powder:deionized water = 1g:10mL.

[0037] In this step, red bean raw materials with a protein content of ≥22% are used to provide the necessary material basis and quality assurance for subsequent efficient extraction. The low-temperature drying temperature is controlled within the range of 50-80℃ and dried to a moisture content of ≤13%. This condition can effectively remove moisture, facilitate long-term storage and pulverization, and minimize protein denaturation or material mold growth caused by excessive temperature or humidity. The particle size of the crushed material is 80-100 mesh. This particle size can provide sufficient specific surface area to promote mass transfer and dissolution in the subsequent alkali extraction process, and can effectively prevent problems such as excessive viscosity and poor flowability of the slurry caused by excessively fine particles.

[0038] The use of a fixed material-liquid ratio of 1g:10mL for grinding red bean powder and deionized water is to form a suspension system with uniform solid-liquid mixing, excellent stability and fluidity. This ratio ensures sufficient contact between the solvent and the material.

[0039] Step 2, extraction of total protein from red beans: The red bean powder slurry obtained in Step 1 is extracted at a constant temperature under alkaline conditions, followed by solid-liquid separation, clarification, concentration and drying to obtain total protein powder from red beans.

[0040] In this step, the alkaline condition is to adjust the pH value of the slurry to 8.5-9.0; the constant temperature extraction temperature is 50℃ and the time is 2 hours.

[0041] The solid-liquid separation is performed using plate and frame filtration, the clarification is performed using centrifugation, and the concentration is performed using vacuum concentration under the conditions of vacuum degree ≤ -0.09MPa and temperature 60-70℃, until the volume is concentrated to 1 / 5-1 / 6 of the original volume. The drying is performed using spray drying, with an inlet air temperature of 180-200℃ and an outlet air temperature of 80-90℃.

[0042] In this embodiment, based on the biochemical characteristic that red bean protein has significantly increased solubility in alkaline conditions, the pH of the slurry was adjusted to 8.5-9.0 to promote its transfer from the solid phase to the liquid phase. The extraction temperature and time were set to 50°C and 2 hours, respectively, to provide the necessary environment for the full dissolution of the protein through mild isothermal conditions, while minimizing protein denaturation and adverse side reactions caused by high heat.

[0043] A combination of plate and frame filtration and centrifugal clarification is used for separation, efficiently removing solid impurities of different particle sizes in a stepwise manner. This provides a highly clarified protein extract for subsequent processes, ensuring the continuity of the overall process. Vacuum concentration is performed under conditions of vacuum ≤ -0.09 MPa and temperature 60-70℃, concentrating the extract to 1 / 5-1 / 6 of its original volume. Utilizing negative pressure to lower the boiling point achieves low-temperature evaporation, effectively protecting the native conformation and activity of the protein and reducing the liquid volume to an ideal concentration range suitable for spray drying. Spray drying is then performed with an inlet air temperature of 180-200℃ and an outlet air temperature of 80-90℃, using a high-temperature airflow to instantly remove moisture and form powder.

[0044] The above key process parameters were verified through experiments, and the experimental verification data are shown in Table 1 below: Table 1 Step 3, enzymatic hydrolysis: Dissolve the total protein powder from red beans obtained in step 2, adjust to neutral conditions, add neutral protease for enzymatic hydrolysis, inactivate the enzyme after hydrolysis, and separate the solid and liquid to obtain crude peptide solution.

[0045] In this step, the ratio of dissolved material to liquid is 1g of total red bean protein powder to 20mL of deionized water; the neutral condition is to adjust the pH of the solution to 7.0-7.5; the enzymatic hydrolysis temperature is 50℃ and the time is 4 hours; the amount of neutral protease added is 2% of the mass of the total red bean protein powder, and its enzyme activity is ≥50000U / g; the enzyme inactivation condition is to maintain at 121℃ for 20 minutes.

[0046] In this embodiment, red bean total protein powder is dissolved in deionized water at a ratio of 1g:20mL to ensure complete dissolution and the formation of a stable solution system, providing a material basis for subsequent homogenization and enzymatic hydrolysis. The pH of the solution is precisely adjusted and maintained within a neutral range of 7.0-7.5, which is the optimal pH range for the selected neutral protease, effectively ensuring the enzyme's catalytic efficiency and stability, and driving the reaction to proceed efficiently. During the enzymatic hydrolysis process, a temperature of 50°C provides a suitable environment to maintain enzyme activity, and a 4-hour reaction cycle allows for thorough and complete enzymatic hydrolysis.

[0047] The amount of neutral protease added is 2% of the total protein powder mass of red beans, and its enzyme activity is ≥50000U / g. A fixed addition ratio ensures sufficient catalyst input. Enzyme inactivation is carried out at 121℃ for 20 minutes to completely deactivate the protease, thereby precisely terminating the reaction and preventing subsequent changes in the properties of the product.

[0048] Finally, the trace amounts of insoluble matter produced during the enzyme inactivation process were removed by solid-liquid separation to obtain a clear crude peptide solution.

[0049] The experimental verification data for the above protease hydrolysis process parameters are shown in Table 2 below: Table 2 Step 4, KQS-1 peptide separation and purification: The crude peptide solution obtained in Step 3 is subjected to ultrafiltration, ion exchange chromatography, reversed-phase high-performance liquid chromatography purification and freeze drying to obtain KQS-1 peptide dry powder.

[0050] In this step, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, operates at a pressure of 0.2-0.3 MPa, and collects the permeate. The ion exchange chromatography uses a DEAE-52 cellulose column; before loading, the column is equilibrated with 0.05 mol / L, pH 7.0 phosphate buffer, and the loading flow rate is 5 mL / min.

[0051] First, impurities are eluted using the aforementioned equilibration buffer, and then gradient elution is performed using a phosphate buffer containing 0.1-0.5 mol / L NaCl. The target elution peak with UV absorption at a wavelength of 220 nm is collected.

[0052] Meanwhile, the reversed-phase high-performance liquid chromatography purification uses a C18 column.

[0053] Before sample loading, an acetonitrile-water solution (10:90 v / v) was used as the mobile phase to balance the chromatographic system at a flow rate of 10 mL / min. The target fraction collected by ion exchange chromatography was concentrated and loaded at a flow rate of 2 mL / min. Gradient elution was performed using acetonitrile-water solution, with the acetonitrile volume percentage increasing from 10% to 40% within 30 minutes. Elution peaks with retention times consistent with the KQS-1 peptide standard were collected and monitored at 220 nm.

[0054] The freeze-drying conditions are as follows: first, pre-freeze at -40℃ for 4 hours, and then dry at -55℃ and vacuum degree ≤10Pa for 12 hours.

[0055] In this embodiment, purification is achieved using multi-stage, high-resolution separation techniques based on the differences in physicochemical properties between the target peptide and impurities. An ultrafiltration membrane with a molecular weight cutoff of 10 kDa is used at 0.2-0.3 MPa to efficiently remove residual large-molecule proteins and polymers from the enzymatic hydrolysate. This pressure range balances membrane flux and operational stability, achieving initial enrichment. Ion exchange chromatography is performed using a DEAE-52 cellulose column at pH 7.0, based on the difference in surface charge between the target peptide and impurities. The sample is equilibrated and loaded with 0.05 mol / L phosphate buffer (flow rate 5 mL / min), followed by gradient elution with 0.1-0.5 mol / L NaCl to selectively elute the target component. The characteristic absorption of peptide bonds is monitored at 220 nm to collect the target peak, effectively removing a large amount of charged impurities and significantly improving product purity.

[0056] Based on the subtle differences in hydrophobicity between the target peptide and impurities, reversed-phase high-performance liquid chromatography (RP-HPLC) purification was performed using a C18 column. A 10:90 acetonitrile-water equilibrium system (flow rate 10 mL / min) was used to concentrate the sample, loading it at a rate of 2 mL / min. Elution was then performed by gradually increasing the acetonitrile ratio from 10% to 40% over 30 minutes, maximizing the separation of key impurities and significantly enhancing the separation effect. The elution process was monitored at a wavelength of 220 nm, and the retention time was compared with that of the KQS-1 peptide standard to collect the target component.

[0057] Finally, water is crystallized by low-temperature pre-freezing, and then the ice crystals are sublimated under high vacuum and low temperature, thereby completely removing water without destroying the structure of heat-sensitive peptides and obtaining a loose, easily soluble, and stable dry powder.

[0058] Using the above methods, a purification strategy was constructed, which involves ultrafiltration to remove macromolecules → ion exchange to separate charge → reversed-phase chromatography to separate hydrophobicity → freeze drying to obtain the final product. This strategy can systematically remove various impurities and ensure the high purity and high yield of the final product.

[0059] It should be noted that the experimental data disclosed in this embodiment are experimental data of parameter values ​​near the preferred parameters. Experimental data of parameter values ​​that are too much or too little beyond the preferred parameters have relatively low reference value and will not be elaborated on here. Example

[0060] The similarities with the above embodiments will not be repeated, the differences are as follows: This embodiment discloses a method for extracting protein from red beans and separating and purifying KQS-1 peptide. The specific process steps are as follows: Take 10 kg of red bean raw material, remove impurities by vibrating screen, dry at 60℃ for 5 hours until the moisture content is 12.5%, pulverize to 90 mesh using a pulverizer, take 5 kg of red bean powder and add 50 L of deionized water at a material-to-liquid ratio of 1:10, and process it by colloid mill at 3000 r / min for 8 minutes to obtain a uniform slurry.

[0061] The prepared slurry was pumped into an extraction tank and stirred at 80 rpm. The pH was adjusted to 8.8 with 1 mol / L sodium hydroxide, and the mixture was extracted at a constant temperature of 50°C for 2 hours. Sodium hydroxide was added every 20 minutes to maintain pH stability. The extract was filtered through a plate and frame filter press to obtain the filtrate. The soybean residue after plate and frame filtration was washed twice with 10 L of deionized water, and the washings were added to the filtrate. The combined filtrate was centrifuged at 4500 rpm for 15 minutes, and the clear filtrate was collected. The clear filtrate was vacuum concentrated to 12 L under a vacuum of -0.09 MPa and 65°C to obtain a concentrated solution with a protein concentration of 22%. The solution was then spray-dried at an inlet air temperature of 190°C and an outlet air temperature of 85°C to obtain 0.65 kg of total red bean protein powder.

[0062] Dissolve 0.5 kg of the protein powder prepared above in 10 L of deionized water. Adjust the pH to 7.2 with 1 mol / L hydrochloric acid. Add 10 g of neutral protease at 50 °C and hydrolyze for 4 hours at a constant temperature, stirring for 5 minutes every 30 minutes. After hydrolysis, pump the hydrolysate into a sterilization tank and inactivate it at 121 °C for 20 minutes. After cooling to room temperature, filter to obtain 9.8 L of crude peptide solution.

[0063] The crude peptide solution obtained above was ultrafiltered through a 10 kDa ultrafiltration membrane at a pressure of 0.25 MPa, and 8.5 L of permeate was collected. The permeate was loaded at a flow rate of 5 mL / min onto a DEAE-52 cellulose ion exchange column equilibrated with 0.05 mol / L, pH 7.0 phosphate buffer. The column was eluted first with equilibration buffer, and then with a gradient elution of phosphate buffer containing 0.1-0.5 mol / L NaCl. The target peak was collected at a wavelength of 220 nm to obtain 1.2 L of crude pure solution.

[0064] After concentrating the crude pure solution to 0.2 L, it was loaded onto a C18 reversed-phase high-performance liquid chromatography column equilibrated with an acetonitrile-water solution of volume ratio 10:90 at a flow rate of 2 mL / min. The column was eluted with a 10%-40% acetonitrile-water gradient for 30 min, and the elution peak with the retention time of the KQS-1 peptide standard was collected at a wavelength of 220 nm to obtain 0.15 L of purified solution.

[0065] After the purified liquid was pre-frozen at -40℃ for 4 hours, it was freeze-dried at -55℃ and 10Pa for 12 hours to obtain 32g of KQS-1 peptide powder.

[0066] By conducting pilot production using the process steps described in this embodiment and by precisely controlling the conditions and related parameters of each step, the purity of the final KQS-1 peptide obtained is as high as 98% or more. Example

[0067] The similarities with the above embodiments and their combinations will not be repeated, the differences being: This embodiment discloses a method for extracting protein from red beans and separating and purifying KQS-1 peptide. The specific process steps are as follows: Take 20kg of red bean raw material, remove impurities by vibrating screen, dry at 60℃ for 4 hours until the moisture content is 12%, and grind it to 80 mesh using a pulverizer. Take 10kg of red bean powder and add 100L of deionized water at a material-to-liquid ratio of 1:10. Process it with a colloid mill at 3000r / min for 10 minutes to obtain a uniform slurry.

[0068] The prepared slurry was pumped into an extraction tank and stirred at a speed of 80 r / min. The pH was adjusted to 9.0 with 1 mol / L sodium hydroxide and extracted at a constant temperature of 50℃ for 2 hours. Sodium hydroxide was added every 20 minutes to maintain pH stability. The extract was filtered by plate and frame filter, centrifuged and clarified, and vacuum concentrated to 25 L. Then it was spray dried at an inlet air temperature of 200℃ and an outlet air temperature of 90℃ to obtain 1.32 kg of red bean total protein powder.

[0069] Take 1 kg of the protein powder prepared above and dissolve it in 20 L of deionized water. Adjust the pH to 7.5, add 20 g of neutral protease and hydrolyze at 50 °C for 4 hours. After filtration, 19.5 L of crude peptide solution is obtained.

[0070] After processing by ultrafiltration, ion exchange chromatography, and reversed-phase high-performance liquid chromatography, 65g of KQS-1 peptide powder was obtained by freeze drying.

[0071] The scheme was implemented on a double-scale basis, demonstrating the stability and scalability of the process. Even after initial large-scale production, it remained stable and directly produced a high-purity final product, with the final KQS-1 peptide achieving a purity of 95.8%.

[0072] The process described in Example 3 allows for large-scale production of KQS-1 peptides while ensuring product purity, thus meeting market and production demands.

[0073] Furthermore, it should be noted that in specific implementations, the structures or processes described in this specification are not fixed or unchanging embodiments. The components of the embodiments of the present invention described and shown herein can be arranged and designed in various different configurations. These are all preferred embodiments of this application and are not intended to limit the scope of protection of this application. Additionally, this specification is for illustrative purposes only and does not represent the specific structure or actual quantity in a concrete implementation.

[0074] Unless otherwise defined, the technical or scientific terms used herein should be understood in their ordinary sense as would be understood by one of ordinary skill in the art to which this invention pertains. The use of terms such as "a" or "an" in this specification and claims does not necessarily indicate a limitation of quantity. Terms such as "comprising" or "including" mean that the element or component preceding the word encompasses the element or component listed following the word and its equivalents, without excluding other elements or components. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect.

[0075] The exemplary embodiments of the present invention have been described in detail above with reference to preferred embodiments. However, those skilled in the art will understand that various modifications and alterations can be made to the above specific embodiments without departing from the concept of the present invention, and various combinations can be made to the various technical features and structures proposed in the present invention without exceeding the protection scope of the present invention.

Claims

1. A method for extracting protein from red beans and isolating and purifying KQS-1 peptide, characterized in that, The steps are performed in the following order: Step 1, raw material pretreatment: The red bean raw material is subjected to impurity removal, low-temperature drying, crushing and grinding in sequence to obtain red bean slurry; Step 2, extraction of total protein from red beans: The red bean powder slurry obtained in Step 1 is extracted at a constant temperature under alkaline conditions, followed by solid-liquid separation, clarification, concentration and drying to obtain total protein powder from red beans; Step 3, enzymatic hydrolysis: After dissolving the total protein powder from red beans obtained in step 2, adjust to neutral conditions and add neutral protease for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment, and then separate solid and liquid to remove insoluble matter, and obtain a clear crude peptide solution. Step 4, KQS-1 peptide separation and purification: The crude peptide solution obtained in Step 3 is subjected to ultrafiltration, ion exchange chromatography, preparative reversed-phase high-performance liquid chromatography purification and freeze drying to obtain KQS-1 peptide dry powder.

2. The method for extracting protein from red beans and isolating and purifying KQS-1 peptide according to claim 1, characterized in that: In step one, the protein content of the red bean raw material used is ≥22%, the low-temperature drying temperature is 50-80℃, and the material is dried until the moisture content is ≤13%; the particle size range of the crushed material is 80-100 mesh. The ratio of material to liquid in the grinding process is red bean powder: deionized water = 1g: 10mL.

3. The method for extracting protein from red beans and isolating and purifying KQS-1 peptide according to claim 1, characterized in that: In step two, the alkaline condition is to adjust the pH of the slurry to 8.5-9.0; the constant temperature extraction temperature is 50℃ and the time is 2 hours. The solid-liquid separation is performed by plate and frame filtration, the clarification is performed by centrifugation, and the concentration is performed by vacuum concentration. The vacuum concentration conditions are vacuum degree ≤ -0.09MPa and temperature 60-70℃, until the volume is concentrated to 1 / 5-1 / 6 of the original volume. The drying process is spray drying, with an inlet air temperature of 180-200℃ and an outlet air temperature of 80-90℃.

4. The method for extracting protein from red beans and isolating and purifying KQS-1 peptide according to claim 1, characterized in that: In step three, the ratio of dissolved material to liquid is 1g of total red bean protein powder to 20mL of deionized water; the neutral condition is to adjust the pH of the solution to 7.0-7.5; the enzymatic hydrolysis temperature is 50℃; and the time is 4 hours. The amount of neutral protease added is 2% of the total protein powder mass of red beans, and its enzyme activity is ≥50000U / g; The enzyme inactivation conditions were maintained at 121°C for 20 minutes.

5. The method for extracting protein from red beans and isolating and purifying KQS-1 peptide according to claim 1, characterized in that: In step four, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, operates at a pressure of 0.2-0.3 MPa, and collects the permeate.

6. The method for extracting protein from red beans and separating and purifying KQS-1 peptide according to claim 1, characterized in that: In step four, the ion exchange chromatography uses a DEAE-52 cellulose column; before loading the sample, the column is equilibrated with 0.05 mol / L, pH 7.0 phosphate buffer, and the loading flow rate is 5 mL / min. First, impurities are eluted using the aforementioned equilibration buffer, and then gradient elution is performed using a phosphate buffer containing 0.1-0.5 mol / L NaCl. The target elution peak with UV absorption at a wavelength of 220 nm is collected.

7. The method for extracting protein from red beans and isolating and purifying KQS-1 peptide according to claim 1, characterized in that: In step four, the reversed-phase high-performance liquid chromatography purification uses a C18 column; Before loading the sample, an acetonitrile-water solution with a volume ratio of 10:90 was used as the mobile phase to balance the chromatographic system, and the mobile phase flow rate was 10 mL / min. The target component collected by ion exchange chromatography was concentrated and then loaded onto the sample at a flow rate of 2 mL / min. Gradient elution was performed using an acetonitrile-water solution, with the acetonitrile volume percentage increasing from 10% to 40% within 30 minutes. Elution peaks consistent with the retention time of KQS-1 peptide standards were collected by monitoring at a wavelength of 220 nm.

8. The method for extracting protein from red beans and isolating and purifying KQS-1 peptide according to claim 1, characterized in that, In step four, the freeze-drying conditions are as follows: first, pre-freeze at -40℃ for 4 hours, and then dry at -55℃ and vacuum degree ≤10Pa for 12 hours.

9. The method for extracting protein from red beans and isolating and purifying KQS-1 peptide according to any one of claims 1-8, characterized in that, In step one, take 10 kg of red bean raw material, remove impurities by vibrating screen, dry at 60℃ for 5 hours until the moisture content is 12.5%, grind to 90 mesh using a pulverizer, take 5 kg of red bean powder and add 50 L of deionized water at a material-to-liquid ratio of 1:10, and process it with a colloid mill at 3000 r / min for 8 minutes to obtain a uniform slurry. In step two, the slurry obtained in step one is pumped into an extraction tank and stirred at a speed of 80 r / min. The pH is adjusted to 8.8 with 1 mol / L sodium hydroxide, and the extraction is carried out at a constant temperature of 50℃ for 2 hours. Sodium hydroxide is added every 20 minutes to maintain pH stability. The extract is filtered by plate and frame press to obtain filtrate. The soybean residue after plate and frame pressing is washed twice with 10L of deionized water, and the washing liquid is added to the filtrate. The combined filtrate is centrifuged at 4500 r / min for 15 minutes, and the clear filtrate is collected. The clear liquid is vacuum concentrated to 12L under vacuum conditions of -0.09MPa and 65℃ to obtain a concentrated solution with a protein concentration of 22%. Then, it is spray dried with an inlet air temperature of 190℃ and an outlet air temperature of 85℃ to obtain 0.65 kg of red bean total protein powder. In step three, 0.5 kg of the protein powder obtained in step two was dissolved in 10 L of deionized water, and the pH was adjusted to 7.2 with 1 mol / L hydrochloric acid. 10 g of neutral protease was added at 50 °C and the mixture was kept at a constant temperature for 4 hours, with stirring for 5 minutes every 30 minutes. After the enzymatic hydrolysis was completed, the hydrolysate was pumped into a sterilization tank and inactivated at 121 °C for 20 minutes. After cooling to room temperature, it was filtered to obtain 9.8 L of crude peptide solution. In step four, the crude peptide solution obtained in step three is ultrafiltered through a 10 kDa ultrafiltration membrane at a pressure of 0.25 MPa, and 8.5 L of permeate is collected. The permeate is loaded at a flow rate of 5 mL / min onto a DEAE-52 cellulose ion exchange column equilibrated with 0.05 mol / L, pH 7.0 phosphate buffer. The column is eluted first with equilibration buffer, and then with a gradient elution of phosphate buffer containing 0.1-0.5 mol / L NaCl. The target peak is collected at a wavelength of 220 nm to obtain 1.2 L of crude pure solution. After concentrating the crude pure solution to 0.2 L, it was loaded onto a C18 reversed-phase high-performance liquid chromatography column equilibrated with acetonitrile-water solution at a flow rate of 2 mL / min. The column was eluted with a gradient of 10%-40% acetonitrile-water for 30 min, and the elution peak with the retention time of KQS-1 peptide standard was collected at a wavelength of 220 nm to obtain 0.15 L of purified solution. After the purified liquid was pre-frozen at -40℃ for 4 hours, it was freeze-dried at -55℃ and 10Pa for 12 hours to obtain 32g of KQS-1 peptide powder.

10. The method for extracting protein from red beans and isolating and purifying KQS-1 peptide according to any one of claims 1-8, characterized in that, In step one, take 20 kg of red bean raw material, remove impurities by vibrating screen, dry at 60℃ for 4 hours until the moisture content is 12%, and grind it to 80 mesh using a pulverizer. Take 10 kg of red bean powder and add 100 L of deionized water at a material-to-liquid ratio of 1:

10. Process it with a colloid mill at 3000 r / min for 10 minutes to obtain a uniform slurry. In step two, the slurry obtained in step one is pumped into an extraction tank and stirred at a stirring speed of 80 r / min. The pH is adjusted to 9.0 with 1 mol / L sodium hydroxide and extracted at a constant temperature of 50℃ for 2 hours. Sodium hydroxide is added every 20 minutes during the extraction to maintain pH stability. The extract was filtered by plate and frame filter, centrifuged and clarified, and vacuum concentrated to 25L; then spray dried at an inlet air temperature of 200℃ and an outlet air temperature of 90℃ to obtain 1.32kg of total red bean protein powder. In step three, 1 kg of the protein powder obtained in step two was dissolved in 20 L of deionized water, the pH was adjusted to 7.5, 20 g of neutral protease was added and enzymatically hydrolyzed at 50 °C for 4 hours, and after inactivation and filtration, 19.5 L of crude peptide solution was obtained. In step four, after ultrafiltration, ion exchange chromatography, and reversed-phase high-performance liquid chromatography, KQS-1 peptide powder was freeze-dried to obtain 65g of KQS-1 peptide powder with a purity of 95.8% and a yield of 6.5%.