A preparation process for deodorized and decolorized pumpkin seed protein isolate

Abstract

This invention relates to the field of plant protein deep processing technology, specifically a preparation process for deodorized and decolorized pumpkin seed protein isolate. This process involves low-temperature cold pressing combined with supercritical CO2 for deep degreasing and deodorization; viscosity reduction extraction using a combination of cellulase and pectinase; solid-liquid separation using a horizontal screw centrifuge; decolorization of the supernatant through macroporous adsorption resin; physical concentration and purification of the protein using an ultrafiltration membrane module to replace the traditional acid precipitation process; and finally, deep deodorization through high-pressure homogenization and vacuum flash evaporation followed by spray drying. Through the synergistic effect of whole-membrane purification, enzymatic hydrolysis, and physical deodorization techniques, the resulting pumpkin seed protein isolate powder is milky white, with a protein content ≥90%, a nitrogen solubility index of over 90%, excellent water solubility and emulsifying properties, and no residual odor, making it suitable for the development of high-end plant-based foods and nutritional supplements.

Description

Technical Field

[0001] This invention relates to the field of plant protein deep processing technology, specifically a preparation process for deodorized and decolorized pumpkin seed protein isolate. Background Technology

[0002] Pumpkin seeds are rich in plant protein, with a balanced amino acid composition and free of common allergens, making them a high-quality plant protein source with significant development potential in the food industry. Modern food engineering often processes pumpkin seeds into pumpkin seed protein isolate to meet the market demand for high-end nutritional products and plant-based food ingredients. This protein product boasts excellent nutritional indicators and shows broad application potential in plant-based milk, protein bars, and special dietary foods.

[0003] Currently, the industrial production of pumpkin seed protein isolate generally follows the traditional alkaline extraction and acid precipitation process. This conventional process uses a strong alkaline solution to extract the protein, then adjusts the pH of the system to the isoelectric point to cause the protein to flocculate and precipitate, followed by decolorization with powdered activated carbon. To address the characteristic fishy odor of pumpkin seeds, existing technologies typically use high-concentration ethanol to repeatedly wash the protein gel, attempting to use organic solvents to remove fat-soluble pigments and volatile odor molecules.

[0004] The aforementioned existing technologies have revealed irreconcilable engineering flaws in actual large-scale production. Traditional strong acid and alkali environments combined with high-concentration ethanol washing cause irreversible and severe denaturation of the protein's spatial conformation, resulting in extremely poor water solubility in the final powder and loss of core functionalities such as emulsification and foaming. Large amounts of endogenous macromolecular polysaccharides and pectin from pumpkin seeds dissolve during the alkali extraction stage, causing an exponential increase in the viscosity of the liquid, severely hindering solid-liquid separation efficiency and easily clogging centrifuge equipment. Conventional powdered activated carbon decolorization methods are prone to particulate leakage, causing physical contamination of the product with black spots. The ethanol elution process generates massive amounts of difficult-to-treat, high-concentration biochemical wastewater, and if there are blind spots in the upstream degreasing step, the finished product is highly susceptible to lipid oxidation during its shelf life, leading to severe off-flavoring. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a preparation process for deodorized and decolorized pumpkin seed protein isolate. This process can effectively avoid protein denaturation, significantly reduce the viscosity of the extract, achieve thorough and purely physical deodorization and decolorization, and prepare high-quality protein powder with high water solubility and no odor residue.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a preparation process for deodorized and decolorized pumpkin seed protein isolate, comprising the following steps: S1: Pumpkin seed kernels are processed by low-temperature cold pressing to separate the basic oil. The resulting preliminary pulp is placed into a supercritical fluid extraction system to perform deep degreasing. By utilizing the dissolving properties of supercritical fluid, fat-soluble pigments and volatile fishy odor precursors are removed simultaneously, and defatted pumpkin seed cake with low residual oil is collected. S2: The defatted pumpkin seed cake produced in the previous process is micronized. The pulverized material is mixed with pure water according to the set material-liquid ratio, and an alkaline reagent is added to adjust the pH value of the system. A compound enzyme preparation composed of cellulase and pectinase is added to the mixture. The extraction reaction is carried out under constant temperature and stirring to promote the enzymatic degradation of macromolecular polysaccharides and colloidal networks. S3: A horizontal screw centrifuge is used to perform continuous solid-liquid separation of the extracted slurry, discharge the solid residue, and collect the light phase supernatant enriched with the target plant protein. S4: Drain the aforementioned supernatant through a chromatography purification column filled with macroporous adsorption resin. The resin matrix is ​​physically sieved and directionally adsorbed by van der Waals forces to remove coloring substances and polyphenolic oxides from the aqueous solution, resulting in a clear and transparent decolorized protein solution. S5: Pump the decolorized protein solution into the ultrafiltration membrane separation unit, set the transmembrane pressure difference to force water, inorganic salts and oligosaccharide molecules produced by enzymatic hydrolysis to pass through the membrane, and retain the high molecular weight natural pumpkin seed protein solution segment in the system to achieve protein concentration and purification that does not rely on acid precipitation regulation. S6: The high-pressure homogenizer applies high-frequency shearing to the concentrated protein solution to break up the aggregated state of large protein molecules. The solution is heated to a predetermined temperature threshold and then instantly injected into the flash tank chamber set to a high vacuum state. The violent boiling effect caused by the sudden drop in pressure is used to forcibly vaporize and strip away the residual low-boiling-point fishy-odor molecules in the liquid phase. S7: The fluid material that has undergone deep deodorization and decolorization treatment is transported into the spray drying tower, where the droplets are instantly dehydrated in a hot air convection environment, and the settled high-purity pumpkin seed protein isolate powder is collected.

[0007] Preferably, in step S1, the supercritical fluid extraction system uses supercritical carbon dioxide as the extraction medium; the extraction pressure is set to 20MPa~30MPa, the extraction temperature to 35℃~45℃, and the extraction time to 2~4 hours; the residual oil content of the obtained defatted pumpkin seed cake is less than 0.5% of the total mass of the material.

[0008] Preferably, in step S2, the material-to-water ratio is 1:8-15 by mass-to-volume; the alkaline reagent is an aqueous sodium hydroxide solution, adjusting the pH of the system to 7.5-8.5; the total mass of the compound enzyme preparation is 0.1%-0.3% of the dry basis mass of the material, and the ratio of cellulase to pectinase activity units in the compound enzyme preparation is 1-2:1; the reaction temperature of the constant temperature stirring is 40℃-50℃, and the extraction time is 1-2 hours.

[0009] Preferably, in step S3, the operating speed of the horizontal screw centrifuge is set to 3500-4500 r / min, and the solid-liquid separation factor is maintained between 2500g and 3500g to meet the continuous slag discharge requirements of the high-viscosity polysaccharide degradation liquid.

[0010] Preferably, in step S4, the macroporous adsorption resin is a weakly polar or non-polar food-grade macroporous resin; the volume hourly space velocity of the feed solution through the chromatography purification column is set to 1 BV / h to 3 BV / h, and the operating temperature is kept constant at 20℃ to 30℃.

[0011] Preferably, in step S5, the membrane material of the ultrafiltration membrane separation component has a molecular weight cutoff of 20kDa to 50kDa; the transmembrane operating pressure during system operation is set to 0.2MPa to 0.5MPa; the feed solution is circulated within the membrane system until its volume is concentrated to 1 / 5 to 1 / 3 of the initial feed volume.

[0012] Preferably, in step S5, the dialysis solution discharged through the ultrafiltration membrane is concentrated and guided into the reverse osmosis membrane device for interception and purification, and the produced pure water is returned to step S2 for water recycling.

[0013] Preferably, in step S6, the pressure of the high-pressure homogenization process is set to 30MPa to 50MPa; the heating temperature to the predetermined temperature is 60℃ to 75℃; and the absolute vacuum inside the vacuum flash tank is maintained at -0.08MPa to -0.095MPa.

[0014] Preferably, in step S7, the hot air inlet temperature of the spray drying tower is set to 170℃~190℃, and the exhaust gas outlet temperature is set to 80℃~90℃; the rotation speed of the atomizing disc is maintained at 15000~20000r / min, so as to ensure the integrity of the powder particles and the moisture content meets the standards.

[0015] This invention provides a process for preparing deodorized and decolorized pumpkin seed protein isolate. It has the following beneficial effects: 1. This invention replaces the traditional isoelectric point acid precipitation process with a physical concentration method that uses an ultrafiltration membrane separation component to retain large protein molecules. This avoids the destruction of the spatial conformation of protein molecules by drastic acid-base changes. In addition, the high-frequency shear force applied by high-pressure homogenization technology breaks down protein aggregates, maintains the high physiological activity of natural proteins and achieves excellent water solubility, with the nitrogen solubility index (NSI) of the finished powder jumping to over 90%.

[0016] 2. This invention uses supercritical carbon dioxide extraction at the front end to remove easily oxidizable precursors such as linoleic acid and fat-soluble pigments, and macroporous adsorption resin at the back end to selectively intercept polyphenol oxides and combine vacuum flash evaporation to instantly vaporize low-boiling-point odor molecules. This completely eliminates the need for powdered activated carbon and organic solvent elution processes, achieving a clean label standard with no black spot contamination and no chemical residues, and effectively preventing lipid oxidation and fishy reversion during shelf life.

[0017] 3. This invention targets and adds a compound enzyme preparation composed of cellulase and pectinase to the alkaline extract solution. During the extraction stage, the endogenous gum network and macromolecular polysaccharides in pumpkin seeds are simultaneously degraded by enzyme-catalyzed bond breaking. This releases the polysaccharide polymers from the severe binding of water, thereby significantly reducing the dynamic viscosity of the extract solution, increasing the dissolution rate of plant protein, and ensuring that the horizontal screw centrifuge can continuously discharge slag at high load without clogging.

[0018] 4. This invention collects the oligosaccharide-rich dialysis solution from the ultrafiltration membrane system and guides it to the reverse osmosis membrane equipment for deep physical sieving. The reverse osmosis removes impurities and produces pure water that is directly reused in the water recycling network of the extraction process. This completely cuts off the source of discharge of high-salt, high-chemical oxygen demand wastewater generated by the traditional acid precipitation method, thereby significantly reducing the cost of biochemical wastewater treatment for enterprises and achieving green and low-carbon production. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the preparation process of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1: like Figure 1 As shown in the figure, this embodiment of the invention provides a preparation process for deodorized and decolorized pumpkin seed protein isolate, including the following steps: S1. Take 100 kg of pumpkin seed kernels, and after removing some of the basic oil through conventional low-temperature cold pressing, the resulting cake is transported to a supercritical fluid extraction system. Supercritical carbon dioxide is used as the extraction medium, and the extraction working pressure is set to 25 MPa, the extraction temperature to 40℃, and the extraction is carried out continuously for 3 hours. After the operation is completed, the material is discharged, and the residual oil content of the defatted pumpkin seed cake is found to be 0.3%.

[0022] S2. The low-residual-oil pumpkin seed cake was micro-pulverized using a universal grinder and passed through an 80-mesh sieve. The pulverized material was weighed and added to pure water at a mass-to-volume ratio of 1:10. The pH of the system was adjusted to 8.0 using a 0.1 mol / L sodium hydroxide aqueous solution. A compound enzyme preparation, composed of a 1:1 ratio of cellulase and pectinase active units, was weighed and added to the tank at 0.2% of the dry weight of the material. The stirring device was turned on, and the temperature was kept constant at 45℃ for 1.5 hours for continuous extraction.

[0023] S3. The feed solution is pumped from the extraction tank into a horizontal screw centrifuge, with the speed set at 4000 r / min and the internal solid-liquid separation factor maintained at 3000 g. The semi-dry solid residue is discharged, and the light phase supernatant containing protein is collected. The feed solution flows smoothly throughout the process, and no blockage is observed.

[0024] S4. The collected supernatant is directed to a macroporous adsorption resin chromatography column (the packing material is AB-8 food-grade macroporous resin). The breakthrough volume hourly space velocity is controlled at 2 BV / h, and the operating temperature is kept constant at 25℃. The originally dark green turbid liquid transforms into a clear and transparent light yellow decolorized protein solution after flowing through the resin bed.

[0025] S4. Pump the above-mentioned decolorized protein solution into an ultrafiltration membrane separation unit with a molecular weight cutoff of 30 kDa. Adjust the transmembrane operating pressure to 0.3 MPa. Water and small molecule impurities permeate through the membrane and are discharged. The feed solution is circulated and concentrated in the pipeline until the total volume of the retentate is reduced to one-quarter of the initial feed volume. The permeate waste liquid is sent to a reverse osmosis membrane device, and the produced purified water is introduced into a storage tank for use in the next batch of production.

[0026] S5. The ultrafiltration-concentrated protein solution is fed into a high-pressure homogenizer and subjected to a working pressure of 40 MPa for crushing. The material then flows through a tubular heat exchanger and is heated to 68°C. It is then instantaneously injected through a special nozzle into a vacuum flash tank where the absolute vacuum is maintained at -0.09 MPa. Volatile odor components are removed by the vacuum pump along with the rapid boiling of moisture within the material.

[0027] S6. The homogenized liquid material treated by flash evaporation is continuously pumped into the spray drying tower. The hot air inlet temperature is set to 180℃, the exhaust gas outlet temperature is set to 85℃, and the atomizing disc speed is maintained at 18000 r / min. The bottom cyclone separator collects the clean-colored pumpkin seed protein powder.

[0028] Example 2: This invention provides a process for preparing deodorized and decolorized pumpkin seed protein isolate. The overall steps are the same as in Example 1, with some operational parameters adjusted as follows: In the supercritical carbon dioxide extraction process, the working pressure was set at 20 MPa, the temperature at 35°C, and the extraction time was 2 hours, with a measured residual oil content of 0.48% in the cake. In the extraction reaction section, the material-to-liquid ratio was set at 1:8, the system pH was calibrated to 7.5, and 0.1% of a compound enzyme preparation (cellulase and pectinase activity ratio 2:1) was added. The extraction temperature was 40°C, and the reaction time was 1 hour. The volume hourly space velocity (VHSV) of the macroporous resin column was set to 3 BV / h. The ultrafiltration membrane was selected with a molecular weight cutoff of 50 kDa, and the transmembrane pressure was set at 0.2 MPa, concentrating to one-third of the initial volume. The high-pressure homogenization pressure was set at 30 MPa, the preheating temperature before flash evaporation was set at 60°C, and the absolute vacuum of the vacuum flash evaporator was maintained at -0.08 MPa. The spray drying parameters were adjusted to an inlet air temperature of 170°C and an outlet air temperature of 80°C. The remaining steps and unspecified parameters were consistent with those in Example 1.

[0029] Example 3: This invention provides a process for preparing deodorized and decolorized pumpkin seed protein isolate. The overall steps are the same as in Example 1, with some operational parameters adjusted as follows: The supercritical carbon dioxide extraction working pressure is increased to 30 MPa, the temperature is 45°C, and the extraction time is 4 hours, resulting in an extremely low residual oil content of 0.12% in the cake. In the extraction reaction section, the material-to-liquid ratio is increased to 1:15, the system pH is calibrated to 8.5, 0.3% of a compound enzyme preparation (cellulase and pectinase activity ratio 1:1) is added, the extraction temperature is 50°C, and the reaction time is extended to 2 hours. The volume hourly space velocity (VHSV) of the macroporous resin column is slowed to 1 BV / h. The molecular weight cutoff of the ultrafiltration membrane is tightened to 20 kDa, the transmembrane pressure is set to 0.5 MPa, and the concentration is reduced to one-fifth of the initial volume. The high-pressure homogenization pressure is increased to 50 MPa, the preheating temperature before flash evaporation is increased to 75°C, and the absolute vacuum of the vacuum flash evaporator is reduced to -0.095 MPa. The spray drying parameters are adjusted to an inlet air temperature of 190°C and an outlet air temperature of 90°C. The remaining process flow is the same as in Example 1.

[0030] Comparative example: Pumpkin seed protein isolate was prepared using the industry's traditional alkaline extraction and acid precipitation method. The specific steps are as follows: Pumpkin seed kernels from the same batch were cold-pressed and defatted. The pressed pulp was pulverized and then extracted with n-hexane for a second defatting. The resulting powder was mixed with water at a ratio of 1:10, and the pH was adjusted to 8.0 with alkali. The mixture was stirred at 45°C for 1.5 hours (without any enzyme preparation). Due to its extremely high viscosity, a disc centrifuge was used, which caused severe clogging. After cleaning, a turbid supernatant was obtained by forced separation. Powdered activated carbon (2%) was added for decolorization for 1 hour, and then the mixture was filtered. Hydrochloric acid was added dropwise to the filtrate to adjust the pH to the isoelectric point of 4.5, causing protein gel to precipitate. The precipitate was collected by centrifugation. The precipitate was washed twice with 75% ethanol solution to remove the odor, centrifuged to reconstitute, and then the pH was adjusted back to 7.0 with alkali. The precipitate was then dried in a spray drying tower under the same conditions to obtain comparative protein powder.

[0031] Performance testing and analysis The physicochemical and functional properties of the pumpkin seed protein isolates produced in Examples 1-3 and the comparative examples were tested.

[0032] Appearance and color: The products in Examples 1, 2 and 3 all exhibit a uniform milky white to very light yellow color, with no impurities visible to the naked eye; the comparative product is grayish-green and black activated carbon fine particles are visible under a microscope.

[0033] Odor evaluation: Examples 1, 2, and 3, after brewing, exhibited a light and elegant nutty aroma, without any grassy or bean-like smell; the comparative examples had a noticeable solvent residue odor and a residual astringent and fishy smell.

[0034] Nitrogen solubility index (NSI): This is a measure of water solubility. Example 1 showed an NSI of 93.4%, Example 2 showed 91.2%, and Example 3 showed 94.1%. The comparative example, due to the dual denaturation of acid, alkali, and ethanol, had an NSI of only 28.5%, and after reconstitution, it showed obvious sand-like precipitate.

[0035] Protein content (dry basis): 91.5% in Example 1, 90.2% in Example 2, and 92.8% in Example 3; the comparative example had only 83.6% protein content due to polysaccharide complexation inhibition.

[0036] Accelerated shelf-life testing: The products were incubated at 40°C and 75% humidity for 6 months. The example product group showed no odor variation; the comparative product developed a significant fishy odor in the 4th month, indicating that lipid oxidation produced volatiles.

[0037] The test results objectively confirm that the process system disclosed in this invention completely reverses the situation where existing technologies cause protein function loss, achieving the technical expectation of high purity, high water solubility, and long-lasting odorlessness.

[0038] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A preparation process of deodorized and decolored pumpkin seed protein isolate, characterized in that, Includes the following steps: S1: Pumpkin seed kernels are processed by low-temperature cold pressing to separate the basic oil. The resulting preliminary pulp is placed into a supercritical fluid extraction system to perform deep degreasing. By utilizing the dissolving properties of supercritical fluid, fat-soluble pigments and volatile fishy odor precursors are removed simultaneously, and defatted pumpkin seed cake with low residual oil is collected. S2: The defatted pumpkin seed cake produced in the previous process is micronized. The pulverized material is mixed with pure water according to the set material-liquid ratio, and an alkaline reagent is added to adjust the pH value of the system. A compound enzyme preparation composed of cellulase and pectinase is added to the mixture. The extraction reaction is carried out under constant temperature and stirring to promote the enzymatic degradation of macromolecular polysaccharides and colloidal networks. S3: A horizontal screw centrifuge is used to perform continuous solid-liquid separation of the extracted slurry, discharge the solid residue, and collect the light phase supernatant enriched with the target plant protein. S4: Drain the aforementioned supernatant through a chromatography purification column filled with macroporous adsorption resin. The resin matrix is ​​physically sieved and directionally adsorbed by van der Waals forces to remove coloring substances and polyphenolic oxides from the aqueous solution, resulting in a clear and transparent decolorized protein solution. S5: Pump the decolorized protein solution into the ultrafiltration membrane separation unit, set the transmembrane pressure difference to force water, inorganic salts and oligosaccharide molecules produced by enzymatic hydrolysis to pass through the membrane, and retain the high molecular weight natural pumpkin seed protein solution segment in the system to achieve protein concentration and purification that does not rely on acid precipitation regulation. S6: The high-pressure homogenizer applies high-frequency shearing to the concentrated protein solution to break up the aggregated state of large protein molecules. The solution is heated to a predetermined temperature threshold and then instantly injected into the flash tank chamber set to a high vacuum state. The violent boiling effect caused by the sudden drop in pressure is used to forcibly vaporize and strip away the residual low-boiling-point fishy-odor molecules in the liquid phase. S7: The fluid material that has undergone deep deodorization and decolorization treatment is transported into the spray drying tower, where the droplets are instantly dehydrated in a hot air convection environment, and the settled high-purity pumpkin seed protein isolate powder is collected.

2. The preparation process of the deodorized and decolored pumpkin seed protein isolate according to claim 1, characterized in that, In step S1, the supercritical fluid extraction system uses supercritical carbon dioxide as the extraction medium; the extraction pressure is set to 20MPa~30MPa, the extraction temperature to 35℃~45℃, and the extraction time to 2~4 hours; the residual oil content of the obtained defatted pumpkin seed cake is less than 0.5% of the total mass of the material.

3. The preparation process of the deodorized and decolored pumpkin seed protein isolate according to claim 1, characterized in that, In step S2, the material-to-water ratio of the pulverized material is 1:8-15 by mass-to-volume; the alkaline reagent is an aqueous sodium hydroxide solution, and the pH of the system is adjusted to 7.5-8.5; the total mass of the compound enzyme preparation added accounts for 0.1%-0.3% of the dry basis mass of the material, and the ratio of cellulase to pectinase activity units in the compound enzyme preparation is 1-2:1; the reaction temperature of the constant temperature stirring is 40℃-50℃, and the extraction time is 1-2 hours.

4. The preparation process of deodorized and decolorized pumpkin seed protein isolate according to claim 1, characterized in that, In step S3, the operating speed of the horizontal screw centrifuge is set to 3500-4500 r / min, and the solid-liquid separation factor is maintained between 2500g and 3500g.

5. The preparation process of deodorized and decolorized pumpkin seed protein isolate according to claim 1, characterized in that, In step S4, the macroporous adsorption resin is a weakly polar or non-polar food-grade macroporous resin; the volume hourly space velocity of the feed solution through the chromatography purification column is set to 1 BV / h to 3 BV / h, and the operating temperature is kept constant at 20℃ to 30℃.

6. The preparation process of deodorized and decolorized pumpkin seed protein isolate according to claim 1, characterized in that, In step S5, the membrane material of the ultrafiltration membrane separation component has a molecular weight cutoff of 20kDa to 50kDa; the transmembrane operating pressure during system operation is set to 0.2MPa to 0.5MPa; the feed solution is circulated within the membrane system until its volume is concentrated to 1 / 5 to 1 / 3 of the initial feed volume.

7. The preparation process of deodorized and decolorized pumpkin seed protein isolate according to claim 1, characterized in that, In step S5, the dialysis solution discharged through the ultrafiltration membrane is concentrated and guided into the reverse osmosis membrane equipment for interception and purification, and the produced pure water is returned to step S2 for water recycling.

8. The preparation process of deodorized and decolorized pumpkin seed protein isolate according to claim 1, characterized in that, In step S6, the pressure of the high-pressure homogenization process is set to 30MPa to 50MPa; the heating temperature to the predetermined temperature is 60℃ to 75℃; and the absolute vacuum inside the vacuum flash tank is maintained at -0.08MPa to -0.095MPa.

9. The preparation process of deodorized and decolorized pumpkin seed protein isolate according to claim 1, characterized in that, In step S7, the hot air inlet temperature of the spray drying tower is set to 170℃~190℃, and the exhaust gas outlet temperature is set to 80℃~90℃; the rotation speed of the atomizing disc is maintained at 15000~20000r / min.

Images