A method for simultaneous dual-enzyme extraction of quinoa oligopeptides

Abstract

This invention relates to the field of small molecule peptide technology, and more particularly to a method for simultaneous dual-enzyme extraction of quinoa oligopeptides. The method involves dividing the extract from defatted quinoa flour after alkali extraction into two parts, processing them separately in tanks, and introducing ultrasound assistance to enhance enzymatic hydrolysis efficiency. After hydrolysis, the hydrolysates are combined and the enzymes are inactivated. The small molecule peptides are then separated by ultrafiltration and fed into a reaction apparatus composed of an immobilized flavor protease and a regenerated cellulose membrane for cyclic debittering. Finally, the quinoa oligopeptide powder is obtained by freeze-drying. This invention utilizes separate tank operation to allow each protease to exert its full activity under optimal conditions, enriching the peptide spectrum diversity; it improves hydrolysis efficiency through ultrasonic cavitation effect; and it achieves continuous debittering and enzyme reuse using an immobilized flavor protease membrane, while avoiding the impact of free enzyme protein residue on product purity. The final product is a quinoa oligopeptide product with concentrated molecular weight, low bitterness, and high purity.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, and in particular to a method for simultaneous extraction of quinoa oligopeptides using two enzymes. Background Technology

[0002] Quinoa contains a large amount of high-quality protein, with a protein content of approximately 12% to 23%, and a balanced amino acid composition, rich in lysine, which is generally lacking in grains. Controlled enzymatic hydrolysis of quinoa protein into oligopeptides can transform it from a large storage protein into an active peptide product with functional properties such as antioxidant, blood pressure lowering, and easy digestibility and absorption, which has significant value in the fields of functional foods and special medical foods. Current technologies for preparing quinoa oligopeptides mainly use protease hydrolysis; a common method is to extract quinoa powder with alkali to obtain a protein solution, then add alkaline protease, hydrolyze under alkaline conditions for several hours, and after enzyme inactivation, separate to obtain peptides of different molecular weights.

[0003] In existing quinoa proteolytic peptide production technologies, a common method involves extracting quinoa flour into a protein solution using alkali, followed by hydrolysis with an alkaline protease. The cleavage sites of alkaline proteases are primarily concentrated at the carboxyl termini of hydrophobic amino acid residues. While this method offers high hydrolysis efficiency, the limited range of cleavage sites restricts the variety of peptides produced, which is dependent on the enzyme's substrate specificity. Quinoa protein contains a rich variety of amino acid sequence fragments, and a single protease cannot adequately cover and release the functional peptides corresponding to these sequences. This results in insufficient peptide diversity in the product, preventing the effective release of some potentially bioactive peptide sequences from the protein chain.

[0004] To improve peptide diversity, some processes employ stepwise hydrolysis using two proteases. This involves hydrolyzing with the first enzyme for a period, inactivating the enzyme, adjusting the pH and temperature, and then adding the second enzyme to continue hydrolysis. Theoretically, this stepwise approach increases the variety of cleavage sites, allowing enzymes with different specificities to act sequentially on different positions within the protein chain. However, in actual production, stepwise hydrolysis requires repeated adjustments of pH and temperature between the optimal conditions of the two enzymes, and adds an enzyme inactivation and temperature / cooling step. This process is lengthy, increases energy consumption, and the protein is prone to denaturation and aggregation during repeated temperature and pH adjustments, leading to loss and reduced final peptide yield.

[0005] Furthermore, during the enzymatic hydrolysis of quinoa protein, the breakage of peptide bonds exposes hydrophobic amino acid residues that were originally encapsulated within the protein molecule to the peptide terminus, resulting in low-molecular-weight hydrophobic peptides that exhibit a distinct bitter taste. Whether using a single protease or a stepwise two-enzyme hydrolysis, these hydrophobic bitter peptides generated in the initial stages of enzymatic hydrolysis negatively impact the flavor of the final product, leading to an unpleasant taste. Some existing processes employ activated carbon adsorption or the addition of free flavor proteases in the later stages of enzymatic hydrolysis for debittering. However, activated carbon has poor adsorption selectivity, resulting in the loss of some functional oligopeptides while adsorbing bitter peptides. The addition of free flavor proteases introduces additional enzymes, requiring subsequent enzyme inactivation and separation steps, increasing the purification burden. Moreover, free enzymes cannot be reused, leading to high costs. Therefore, a suitable extraction method for quinoa oligopeptides is needed. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for simultaneous dual-enzyme extraction of quinoa oligopeptides. This method involves dividing the extract from defatted quinoa flour after alkali extraction into two parts, processing them separately in tanks, and introducing ultrasound assistance to enhance enzymatic hydrolysis efficiency. After hydrolysis, the hydrolysates are combined and the enzymes are inactivated. The small molecule peptides are then separated by ultrafiltration and fed into a reaction apparatus composed of an immobilized flavor protease and a regenerated cellulose membrane for cyclic debittering. Finally, quinoa oligopeptide powder is obtained by freeze-drying. This invention utilizes separate tank operation to allow each protease to exert its full activity under optimal conditions, avoiding enzyme activity loss under conditions of simultaneous tank operation and enriching the peptide spectrum diversity. The ultrasonic cavitation effect improves protein extraction rate and hydrolysis efficiency. The immobilized flavor protease membrane enables continuous debittering and enzyme reuse, while avoiding the impact of free enzyme protein residues on product purity. Ultimately, a quinoa oligopeptide product with concentrated molecular weight, low bitterness, and high purity is obtained.

[0007] To achieve the above objectives, the present invention employs the following technical solution: a method for simultaneous dual-enzyme extraction of quinoa oligopeptides, comprising the following steps: Step S1: Pretreatment: Wash quinoa with water and dry it at 45°C for 24 hours, then crush it and pass it through a 100-mesh sieve to obtain quinoa powder; mix the quinoa powder with n-hexane, stir at room temperature for 3 hours, and centrifuge at 3000 rpm for 5 minutes to obtain defatted quinoa powder. Step S2: Alkaline extraction treatment: Mix defatted quinoa powder and deionized water, adjust the pH to 10 with sodium hydroxide solution, stir at 40°C for 3 hours, adjust the pH to 8.5 with hydrochloric acid solution, centrifuge at 6000 rpm for 10 minutes, filter, collect the liquid and insoluble matter to obtain alkaline extract and alkaline insoluble substances. Step S3: Double enzyme treatment: The protein content of the alkaline extract was determined and controlled to be 5 wt% by concentration or addition of deionized water. The extract was then divided and added to tank A for alkaline protease treatment and tank B for neutral protease treatment, resulting in hydrolysate from tank A and hydrolysate from tank B. The hydrolysate from tank A and tank B were mixed and kept at 95°C for 10 min to obtain a mixed double enzyme hydrolysate, and the pH was adjusted to 7. Step S4: Ultrafiltration treatment: Centrifuge the double enzyme hydrolysate at 4000 rpm for 15 min, collect liquid 2 to obtain the double enzyme hydrolysate extract, send the double enzyme hydrolysate extract into the ultrafiltration circulation device for ultrafiltration, collect the permeate and adjust the protein content to 5 wt% by concentration or by adding deionized water to obtain the ultrafiltration permeate. Step S5: Flavor enzyme treatment: The ultrafiltration permeate is preheated and fed into a multi-layer stacked reaction device composed of enzyme-immobilized regenerated cellulose membranes. Liquid 3 is collected to obtain the reaction treatment solution. Step S6: Post-processing: The reaction solution was centrifuged at 6000 rpm for 10 min, pre-cooled, and then freeze-dried to obtain quinoa oligopeptide powder.

[0008] Furthermore, in step S1, the ratio of quinoa powder to n-hexane is 1:10.

[0009] In step S2: the ratio of defatted quinoa powder to deionized water is 1:10, the molar concentration of sodium hydroxide solution is 2 mol / L, and the molar concentration of hydrochloric acid solution is 1 mol / L.

[0010] In step S3: In tank A: the pH value is first adjusted to 10 using sodium hydroxide / hydrochloric acid solution and stirred at 50℃ for 60 min, then alkaline protease is added, and an ultrasonic auxiliary device is introduced simultaneously. The mixture is kept at this temperature for 8 h, and the pH is kept constant during the reaction. In tank B: the pH value is first adjusted to 7 using sodium hydroxide / hydrochloric acid solution and stirred at 45℃ for 60 min, then neutral protease is added, and an ultrasonic auxiliary device is introduced simultaneously. The mixture is kept at this temperature for 8 h, and the pH is kept constant during the reaction. The ultrasound-assisted device is a 20kHz probe-type ultrasound with a power density of 80W / L, a pulse mode of 3s / 5s, and a total effective ultrasound time of 15min; the amount of alkaline protease added is 5000 U / g protein, and the amount of neutral protease added is 4000 U / g.

[0011] In step S4: the ultrafiltration membrane used is a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 5000U; during the ultrafiltration process: the ultrafiltration pressure is 0.2MPa, the temperature is 30℃, and the flow rate is 5L / min.

[0012] In step S5: the preheating temperature is 50℃; the reaction device consisting of multiple stacked enzyme-immobilized regenerated cellulose films is obtained by stacking 4 enzyme-immobilized regenerated cellulose films; the reaction pH is 7, the reaction temperature is 52℃, and the reaction residence time is 2h.

[0013] In step S6: pre-cooling is carried out at -40℃ for 2 hours; freeze drying: cold trap temperature ≤ -50℃, vacuum degree < 20Pa, drying time 48 hours.

[0014] Furthermore, in step S5: the enzyme immobilization membrane used in this step covalently immobilizes the flavor protease on the regenerated cellulose porous membrane matrix; and since step S4 uses an ultrafiltration membrane with a rejection capacity of 5000U, the risk of large protein molecules clogging the enzyme membrane pores is avoided; when the ultrafiltration permeate flows on the membrane surface and in the membrane pores, it comes into full contact with the immobilized enzyme, and the aminopeptidase and carboxypeptidase in the flavor protease remove amino acid residues one by one from both ends of the peptide chain; since there is no free enzyme protein in the reaction solution, there is no need to set up a separate enzyme inactivation and removal step, thus eliminating the heat denaturation protein precipitation and filtration process caused by enzyme inactivation in traditional processes.

[0015] The enzyme-immobilized regenerated cellulose film was prepared through the following steps: Step A1: Mix 1-butyl-3-methylimidazolium chloride ion liquid, microcrystalline cellulose and polyethylene glycol. Stir for 3 hours under nitrogen protection, stirring rate of 300 rpm and temperature of 90℃. Vacuum degassing for 3 hours, pour onto a glass plate and coat with a doctor blade, controlling the gap to be 1.5±0.05 mm. Let stand at 15℃ for 1 hour, soak and wash with deionized water, peel off to obtain regenerated cellulose film. Furthermore, in step A1, the ratio of 1-butyl-3-methylimidazolium chloride ion liquid, microcrystalline cellulose, and polyethylene glycol is 100g:15g:1.5-2g; the polyethylene glycol is PEG-400.

[0016] Step A2: The regenerated cellulose film is dried and immersed in a silane coupling agent solution. It is immersed for 15 minutes at room temperature, then heated to 60°C and reacted for 1 hour. It is washed with ethanol and dried at 40°C to obtain the modified regenerated cellulose film. Furthermore, in step A2: the silane coupling agent solution is a mixture of γ-aminopropyltriethoxysilane, ethanol and deionized water, wherein the volume fraction of γ-aminopropyltriethoxysilane is 10% and the volume fraction of deionized water is 2%; the ratio of regenerated cellulose film to silane coupling agent solution is 2g:30-40mL.

[0017] Step A3: Prepare a flavor protease solution using phosphate buffer and a glutaraldehyde solution using phosphate buffer. Immerse the modified regenerated cellulose film in the glutaraldehyde solution at room temperature for 1 hour, then remove it and wash it with phosphate buffer. Transfer it to the flavor protease solution and shake it at 15°C for 12 hours. Finally, transfer it to the glycine solution and soak it at room temperature for 1 hour. Wash it with deionized water to obtain the enzyme-immobilized regenerated cellulose film. Further, in step A3: the phosphate buffer has a pH of 7 and a molar concentration of 0.1 mol / L, the flavor protease solution has a concentration of 3-5 mg / mL, the glutaraldehyde solution has a volume fraction of 2.5%, and the glycine solution has a pH of 7.5 and a molar concentration of 0.1 mol / L; the ratio of the modified regenerated cellulose film, glutaraldehyde solution, flavor protease solution, and glycine solution is 2 g: 30-35 mL: 35 mL: 20 mL.

[0018] Compared with existing technologies, the beneficial effects of this invention are: a method for simultaneous extraction of quinoa oligopeptides using two enzymes. This method divides the extract from defatted quinoa flour after alkali extraction into two parts, processes them separately in tanks, and introduces ultrasound assistance to enhance enzymatic hydrolysis efficiency. After hydrolysis, the hydrolysates are combined and the enzymes are inactivated. Small molecule peptides are then separated by ultrafiltration and fed into a reaction device composed of an immobilized flavor protease and regenerated cellulose membrane for cyclic debittering. Finally, quinoa oligopeptide powder is obtained by freeze-drying. This invention, through separate tank operation, allows each of the two proteases to exert its full activity under optimal conditions, avoiding enzyme activity loss under conditions of same tank conditions and enriching peptide diversity. The ultrasonic cavitation effect improves protein extraction rate and hydrolysis efficiency. The use of an immobilized flavor protease membrane enables continuous debittering and enzyme reuse, while avoiding the impact of free enzyme protein residues on product purity, ultimately obtaining a quinoa oligopeptide product with concentrated molecular weight, low bitterness, and high purity.

[0019] This invention utilizes separate enzymatic hydrolysis in two tanks to maximize the activity of both enzymes: Traditional single-tank dual-enzyme hydrolysis requires selecting a compromise between the optimal pH values ​​of the two enzymes, resulting in the inhibition of the activity of both enzymes. This invention divides the alkaline extract into two tanks: Tank A uses an alkaline environment of pH 10 specifically suited for alkaline protease, and Tank B uses a neutral environment of pH 7 specifically suited for neutral protease. Under their respective optimal pH conditions, the peptide bond cleavage rate of alkaline protease reaches its maximum activity, while neutral protease is also in the pH range where its native conformation is most stable. The respective cleavage site preferences of the two enzymes are fully revealed: alkaline protease mainly attacks the carboxyl-terminal peptide bonds of hydrophobic amino acids, producing a large number of small to medium molecular weight peptides ending in hydrophobic amino acids; neutral protease tends to cleave peptide bonds in hydrophilic regions, producing peptides with a milder bitterness. After combining the two hydrolysates, the resulting peptide profile is significantly broader in terms of molecular weight distribution and amino acid sequence variety than that of single protease or dual-enzyme co-hydrolysis under compromise pH conditions. The functional activities of different peptides can be preserved and complemented in the same product.

[0020] Ultrasonic-assisted hydrolysis improves enzymatic hydrolysis efficiency and peptide yield: This invention introduces a probe-type ultrasonic auxiliary device simultaneously during the dual-enzyme hydrolysis process. The cavitation effect generated by ultrasound in the feed solution is the core mechanism for efficiency improvement. From generation and growth to instantaneous collapse, cavitation bubbles release strong shock waves and microjets in microscopic local areas. The microjets impact the surface of substrate protein particles at speeds up to hundreds of meters per second, effectively scouring the barrier layer composed of polysaccharides and insoluble fibers on the outside of the protein particles, accelerating the mass transfer rate of enzyme molecules from the liquid phase to the solid-liquid interface. At the same time, the shear force generated by cavitation causes partial delamination of the compact structure of quinoa protein, further exposing the hydrophobic core region and enzyme cleavage sites that were still partially buried inside the protein aggregates after alkali extraction. The degree of protein unfolding is further increased, allowing alkaline proteases and neutral proteases to obtain more accessible cleavage sites.

[0021] A targeted substrate pool is constructed by ultrafiltration membrane fractionation: The peptide molecular weight distribution range is relatively wide in the mixture after dual-enzyme hydrolysis and enzyme inactivation. In this invention, an ultrafiltration membrane with a cutoff of 5000U is set up for fractionation. Incompletely hydrolyzed protein fragments and peptides larger than 5000U are retained in the retentate and can be recovered for secondary enzymatic hydrolysis or used for other products. The ultrafiltration permeate collected on the permeate side of this ultrafiltration membrane is equivalent to a targeted small molecule substrate pool. All peptides in the pool already have the basic molecular weight characteristics of the target product. During subsequent flavor protease treatment, there are no useless large molecule substrates competing with and consuming the enzyme's active site.

[0022] Continuous debittering and enzyme reuse are achieved through immobilized regenerated cellulose membranes: Flavor proteases are covalently immobilized on a regenerated cellulose membrane matrix via glutaraldehyde crosslinking, and the membranes are stacked to form a reaction device. Small molecule substrates in the ultrafiltration permeate can diffuse rapidly from the membrane surface into the microenvironment of the immobilized enzyme within the membrane pores. After contacting the enzyme's active site, the terminal hydrophobic amino acids are cleaved off. The cleaved hydrophobic amino acids and modified small molecule peptides are carried away with the feed solution, and the immediate removal of the products reduces the feedback inhibition of the enzyme by the products. Traditional free flavor enzyme processes require the addition of enzymes for each batch and heat inactivation after treatment. The enzyme protein is denatured and discarded after only one use. The immobilized flavor proteases are retained on the membrane matrix and can be reused continuously for multiple batches. The amount of substrate treated per unit of enzyme protein is several times that of free enzymes, and the cost of enzyme preparations is significantly reduced. Regarding product purity, the immobilized enzyme does not dissolve in the liquid phase, and the reaction solution does not contain free enzyme protein or enzyme protein fragments that have been denatured after enzyme inactivation; high-purity products can be obtained by nanofiltration purification and freeze drying, eliminating the precipitation, centrifugation or chromatographic separation steps required to remove enzyme protein. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the process proposed in this invention. Detailed Implementation

[0024] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0025] In the preparation examples, embodiments, and comparative examples of this invention: the alkaline protease used was from Amjad Microbiota, with an enzyme activity of 2.4 AU / g; the neutral protease was from Amjad Microbiota, with an enzyme activity of 1.5 AU / g; the flavor protease was from Nanjing Pangbo Biotechnology Co., Ltd., with an enzyme activity of 500 LAPU / g; and the microcrystalline cellulose was from Merck, model Avicel PH 101.

[0026] Preparation Example 1: The enzyme-immobilized regenerated cellulose film was prepared by the following steps: Step A1: Mix 1-butyl-3-methylimidazolium chloride ion liquid, microcrystalline cellulose and polyethylene glycol. Stir for 3 hours under nitrogen protection, stirring rate of 300 rpm and temperature of 90℃. Vacuum degassing for 3 hours, pour onto a glass plate and coat with a doctor blade, controlling the gap to be 1.5±0.05 mm. Let stand at 15℃ for 1 hour, soak and wash with deionized water, peel off to obtain regenerated cellulose film. Furthermore, in step A1, the ratio of 1-butyl-3-methylimidazolium chloride ion liquid, microcrystalline cellulose, and polyethylene glycol is 100g:15g:2g; the polyethylene glycol is PEG-400.

[0027] Step A2: The regenerated cellulose film is dried and immersed in a silane coupling agent solution. It is immersed for 15 minutes at room temperature, then heated to 60°C and reacted for 1 hour. It is washed with ethanol and dried at 40°C to obtain the modified regenerated cellulose film. Furthermore, in step A2: the silane coupling agent solution is a mixture of γ-aminopropyltriethoxysilane, ethanol and deionized water, wherein the volume fraction of γ-aminopropyltriethoxysilane is 10% and the volume fraction of deionized water is 2%; the ratio of regenerated cellulose film to silane coupling agent solution is 2g:40mL.

[0028] Step A3: Prepare a flavor protease solution using phosphate buffer and a glutaraldehyde solution using phosphate buffer. Immerse the modified regenerated cellulose film in the glutaraldehyde solution at room temperature for 1 hour, then remove it and wash it with phosphate buffer. Transfer it to the flavor protease solution and shake it at 15°C for 12 hours. Finally, transfer it to the glycine solution and soak it at room temperature for 1 hour. Wash it with deionized water to obtain the enzyme-immobilized regenerated cellulose film. Further, in step A3: the phosphate buffer has a pH of 7 and a molar concentration of 0.1 mol / L, the flavor protease solution has a concentration of 5 mg / mL, the glutaraldehyde solution has a volume fraction of 2.5%, and the glycine solution has a pH of 7.5 and a molar concentration of 0.1 mol / L; the ratio of the modified regenerated cellulose film, glutaraldehyde solution, flavor protease solution, and glycine solution is 2 g: 35 mL: 35 mL: 20 mL.

[0029] Preparation Example 2: Compared with Preparation Example 1, the amount of polyethylene glycol used in step A1 was adjusted to 1.5g, while other steps remained unchanged.

[0030] Preparation Example 3: Compared with Preparation Example 1, the amount of silane coupling agent solution used in step A2 was adjusted to 30 mL, while the other steps remained unchanged.

[0031] Preparation Example 4: Compared with Preparation Example 1, the concentration of the flavor protease solution in step A3 was adjusted to 3 mg / mL, while the other steps remained unchanged.

[0032] Preparation Example 5: Compared with Preparation Example 1, the amount of glutaraldehyde solution used in step A3 in Preparation Example 5 was adjusted to 30 mL, while the other steps remained unchanged.

[0033] Comparative Preparation Example 1: Compared with Preparation Example 1, the silane modification step in step A2 is removed in Comparative Preparation Example 1, while the other steps remain unchanged.

[0034] Comparative Preparation Example 2: Compared with Comparative Preparation Example 1, the amount of polyethylene glycol used in step A1 was adjusted to 0.2g, while other steps remained unchanged.

[0035] Example 1: A method for simultaneous dual-enzyme extraction of quinoa oligopeptides, comprising the following steps: Step S1: Pretreatment: Wash quinoa with water and dry it at 45°C for 24 hours, then crush it and pass it through a 100-mesh sieve to obtain quinoa powder; mix the quinoa powder with n-hexane, stir at room temperature for 3 hours, and centrifuge at 3000 rpm for 5 minutes to obtain defatted quinoa powder. Step S2: Alkaline extraction treatment: Mix defatted quinoa powder and deionized water, adjust the pH to 10 with sodium hydroxide solution, stir at 40°C for 3 hours, adjust the pH to 8.5 with hydrochloric acid solution, centrifuge at 6000 rpm for 10 minutes, filter, collect the liquid and insoluble matter to obtain alkaline extract and alkaline insoluble substances. Step S3: Double enzyme treatment: The protein content of the alkaline extract was determined and controlled to be 5 wt% by concentration or addition of deionized water. The extract was then divided and added to tank A for alkaline protease treatment and tank B for neutral protease treatment, resulting in hydrolysate from tank A and hydrolysate from tank B. The hydrolysate from tank A and tank B were mixed and kept at 95°C for 10 min to obtain a mixed double enzyme hydrolysate, and the pH was adjusted to 7. Step S4: Ultrafiltration treatment: Centrifuge the double enzyme hydrolysate at 4000 rpm for 15 min, collect liquid 2 to obtain the double enzyme hydrolysate extract, send the double enzyme hydrolysate extract into the ultrafiltration circulation device for ultrafiltration, collect the permeate and adjust the protein content to 5 wt% by concentration or by adding deionized water to obtain the ultrafiltration permeate. Step S5: Flavor enzyme treatment: The ultrafiltration permeate is preheated and fed into a multi-layer stacked reaction device composed of enzyme-immobilized regenerated cellulose membranes. Liquid 3 is collected to obtain the reaction treatment solution. Step S6: Post-processing: The reaction solution was centrifuged at 6000 rpm for 10 min, pre-cooled, and then freeze-dried to obtain quinoa oligopeptide powder.

[0036] Furthermore, in step S1, the ratio of quinoa powder to n-hexane is 1g:10mL.

[0037] In step S2: the ratio of defatted quinoa powder to deionized water is 1g:10mL, the molar concentration of sodium hydroxide solution is 2mol / L, and the molar concentration of hydrochloric acid solution is 1mol / L.

[0038] In step S3: In tank A: the pH value is first adjusted to 10 using sodium hydroxide / hydrochloric acid solution and stirred at 50℃ for 60 min, then alkaline protease is added, and an ultrasonic auxiliary device is introduced simultaneously. The mixture is kept at this temperature for 8 h, and the pH is kept constant during the reaction. In tank B: the pH value is first adjusted to 7 using sodium hydroxide / hydrochloric acid solution and stirred at 45℃ for 60 min, then neutral protease is added, and an ultrasonic auxiliary device is introduced simultaneously. The mixture is kept at this temperature for 8 h, and the pH is kept constant during the reaction. The ultrasound-assisted device is a 20kHz probe-type ultrasound with a power density of 80W / L, a pulse mode of 3s / 5s, and a total effective ultrasound time of 15min; the amount of alkaline protease added is 5000 U / g protein, and the amount of neutral protease added is 4000 U / g.

[0039] In step S4: the ultrafiltration membrane used is a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 5000U; during the ultrafiltration process: the ultrafiltration pressure is 0.2MPa, the temperature is 30℃, and the flow rate is 5L / min.

[0040] In step S5: the preheating temperature is 50℃; the reaction device consisting of four stacked enzyme-immobilized regenerated cellulose films prepared in Example 1 is obtained by stacking four enzyme-immobilized regenerated cellulose films; the reaction pH is 7, the reaction temperature is 52℃, and the reaction residence time is 2h.

[0041] In step S6: pre-cooling is carried out at -40℃ for 2 hours; freeze drying: cold trap temperature ≤ -50℃, vacuum degree < 20Pa, drying time 48 hours.

[0042] Example 2: Compared with Example 1, the enzyme-immobilized regenerated cellulose film in Example 2 was modified to be the same as that obtained in Example 2, while the other steps remained unchanged.

[0043] Example 3: Compared with Example 1, the enzyme-immobilized regenerated cellulose film in Example 3 was modified to be the same as that obtained in Example 3, while the other steps remained unchanged.

[0044] Example 4: Compared with Example 1, the enzyme-immobilized regenerated cellulose film in Example 4 was modified to be the same as that obtained in Example 4, while the other steps remained unchanged.

[0045] Example 5: Compared with Example 1, the enzyme-immobilized regenerated cellulose film in Example 5 was modified to be the same as that obtained in Example 5, while the other steps remained unchanged.

[0046] Example 6: Compared with Example 1, the enzyme-immobilized regenerated cellulose film in Example 6 was modified to be the one prepared in Comparative Preparation Example 1, while other steps remained unchanged.

[0047] Example 7: Compared with Example 1, the enzyme-immobilized regenerated cellulose film in Example 7 was modified to be the one prepared in Comparative Preparation Example 2, while other steps remained unchanged.

[0048] Comparative Example 1: Compared with Example 1, in step S3, the alkaline extract was placed in the same tank, the pH was adjusted to 8.5, the temperature was 50°C, and 5000 U / g of alkaline protease and 4000 U / g of neutral protease were added. The mixture was stirred and hydrolyzed for 8 hours. Other steps remained unchanged.

[0049] Comparative Example 2: Compared with Example 1, Comparative Example 2 does not introduce an ultrasound-assisted device in step S3, while other steps remain unchanged.

[0050] Comparative Example 3: Compared with Example 1, Comparative Example 3 does not use an immobilized enzyme membrane reactor in step S5. Instead, 2000 U / g of free flavor protease is added to the ultrafiltration permeate, stirred at 50°C for 2 hours, and then inactivated at 95°C for 15 minutes. The remaining steps are the same as in Example 1.

[0051] The performance was tested according to the steps of the embodiments and comparative examples: 1) The degree of hydrolysis in step S3 and step S5 of the alkaline hydrolysis processes in the examples and comparative examples was determined using the pH-stat method: The calculation formula is as follows: Where: B is the volume of alkali solution consumed in the reaction (mL); Nb is the molar concentration of alkali solution (NaOH) (mol / L); Mp is the mass of protein in the substrate (g); htot is the total number of peptide bonds in the protein (mmol / g); α is the degree of dissociation of α-NH2, which is a function of reaction temperature.

[0052] 2) The peptide molecular weight distribution of the samples was measured using a Waters 1525 high-performance liquid chromatograph with a TSK gel2000 SW column. XL 300 mm × 7.8 nm; Weigh 100 mg of the sample to be tested into a 10 mL volumetric flask, dilute to volume with acetonitrile / water / trifluoroacetic acid (10 / 90 / 0.1, v / v) as the mobile phase, filter through a microporous membrane, and inject the sample. The column temperature is 30 ℃, the flow rate is 0.5 mL / min, and the detection is performed at 220 nm. The standards used for the molecular weight calibration curve are: cytochrome C, bacitracin, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen-acetaminophen. Calculate the percentage of peak area occupied by peptides with a molecular weight less than 2000 Da.

[0053] 3) Prepare solutions of quinine at different concentrations and use these as a standard for scoring; the concentrations of the standard solutions are 0 and 8 × 10⁻⁶. -6 1.6×10 -5 2.4×10 -5 3.2×10 -5 and 4×10 -5The g / mL concentration corresponds to scores of 0, 1, 2, 3, 4, and 5, with higher scores indicating stronger bitterness. The sample was prepared as a 1% (w / v) solution with pH=6.5 and compared with the standard solution at room temperature. The samples were scored according to the standard scores, and a total of 10 sensory evaluators participated in the evaluation. 4) The TS-5000Z intelligent taste system is used to evaluate the astringency, bitterness, and other taste indicators of samples in an objective and digital form. It can also analyze the astringent and bitter aftertastes of the flavor compounds. The specific process is as follows: Weigh 2g of sample and dissolve it in 100mL of water. Pour 35mL of the test solution into a sample cup and let it stand for 5 minutes. Immerse the two taste sensors in the reference solution and the sample solution successively to obtain the potential Vr of the reference solution and the potential Vs of the sample solution. Then, immerse the taste sensor in the reference solution for 3s to clean it, and then immerse the sensor in the sample solution to detect the potential Vr'. After the measurement, immerse the two taste sensors in ethanol solution for 330s to clean them and remove the adsorbed substances on the sensors before testing the next sample. Use the potential difference between Vs and Vr to evaluate astringency and bitterness. Use the potential difference between Vr' and Vr to evaluate the aftertaste of astringency and bitterness. Take the measurement data of the product in Example 1 as the zero point, and compare the potential difference of other experimental samples with the results of the product in Example 1.

[0054] The test results are shown in the table below: Table 1. Results of Hydrolysis Degree, Peak Area Percentage, and Sensory Evaluation Table 2. Results of tests for astringency, bitterness, astringent aftertaste, and bitter aftertaste. The test results shown in the table indicate that the examples and comparative examples are compared as follows: Compared to Example 1, Comparative Example 1, which involved separate enzymatic hydrolysis in different tanks, placed all alkaline extracts in the same tank and underwent simultaneous hydrolysis with two enzymes at a compromise pH of 8.5. This lacked the optimal cleavage of the carboxyl-terminal peptide bonds of hydrophobic amino acids by alkaline protease at pH 10 and the specific hydrolysis of hydrophilic peptide bonds by neutral protease at pH 7. The activity of alkaline protease at pH 8.5 was only about 80% to 85% of its optimal activity at pH 10. The neutral protease deviates from its optimal pH of 7 at pH 8.5, resulting in an activity loss of approximately 25% to 30%. Neither enzyme can cleave the substrate at its maximum rate, leading to a significant number of unbroken peptide bonds within the protein molecule and a low degree of hydrolysis at the hydrolysis endpoint. Incomplete hydrolysis results in a large number of medium-sized peptide segments remaining in the molecule. These peptide segments retain many hydrophobic amino acid residue sequences, not only producing a strong bitter taste but also exhibiting non-specific binding of their terminal hydrophobic groups to astringent receptors on the tongue, leading to a simultaneous increase in astringency and aftertaste. This further demonstrates the comprehensive superiority of the independent optimal pH enzymatic hydrolysis process used in Example 1 in improving the depth of hydrolysis, increasing the proportion of small molecule peptides, and simultaneously reducing the bitterness and astringency of the product.

[0055] Compared to Example 1, Comparative Example 2, while maintaining independent optimal pH hydrolysis in separate tanks, relied solely on mechanical stirring, lacking the crucial role of ultrasonic cavitation. First, the high-speed microjet generated by cavitation bubble collapse disrupted the mass transfer barrier layer around quinoa protein particles, accelerating enzyme diffusion to the protein surface. Second, cavitation shear force partially disintegrated the compact structure of quinoa protein, exposing the internal hydrophobic core region and peptide bonds, increasing the number of cleavage sites accessible to both proteases. Third, the transient local turbulence generated by cavitation increased the frequency of enzyme-substrate complex formation. The absence of these effects resulted in more peptide bonds in the substrate protein not being fully contacted and cleaved during hydrolysis, leading to a generally larger peptide molecular weight. The tandem hydrophobic amino acid residues on the longer peptide chains continuously released bitter stimuli in the mouth, resulting in a significantly higher bitter aftertaste. Simultaneously, the exposure of some phenolic hydroxyl-containing amino acid side chains also contributed to an increase in astringency and its aftertaste. This further illustrates the irreplaceable role of the ultrasonic-assisted method used in Example 1 in enhancing the efficiency of dual-enzyme hydrolysis in separate tanks, reducing the bitterness and astringency of the product, and its aftertaste.

[0056] Compared with Example 1, Comparative Example 3, unlike Example 1 which uses a multi-layer stacked reaction device composed of enzyme-immobilized regenerated cellulose membranes for cyclic debittering, directly adds the flavor protease in free form to the ultrafiltration permeate for a one-time reaction. This lacks the solid-liquid interface enrichment effect and immediate product separation provided by the immobilized enzyme membrane. In the immobilized enzyme membrane reactor of Example 1, the feed solution flows through the enzyme membrane surface and internal pores multiple times in a circulating manner, providing multiple opportunities for contact with the active center of the immobilized flavor protease. The debittered product with its excised ends leaves the enzyme active area immediately with the mobile phase, reducing the product inhibition effect. In contrast, in Comparative Example 3, the contact between the enzyme and the substrate depends on the probability of random collisions generated by stirring. Some peptide molecules with hydrophobic ends do not fully contact the enzyme during the entire reaction process, failing to complete the excision of the hydrophobic amino acids at the ends. These residual hydrophobic peptides cause persistent bitterness and a slight astringency in the mouth, resulting in a relatively larger increase in the bitter aftertaste. Although the S5 degree of hydrolysis value of Comparative Example 3 is slightly higher than that of Example 1, this is because the free enzyme initially does not have the steric hindrance caused by immobilization and can freely contact the substrate. This further illustrates the superiority of the immobilized enzyme membrane circulating reactor used in Example 1 in terms of debittering uniformity and reduction of bitter aftertaste, as well as the cost advantage of the reusable immobilized enzyme compared to the single consumption of free enzyme.

[0057] Compared to Example 1, Example 2 used the enzyme immobilization membrane prepared in Example 2. Compared to Example 1, the amount of PEG-400 in step A1 was reduced from 2g to 1.5g, resulting in a decrease in pore-forming agent. This increased the permeation resistance of the feed solution within the membrane pores, reduced the probability of contact between some peptides and the immobilized enzyme inside the membrane, and slightly lowered the overall cleavage efficiency of the flavor protease on the hydrophobic peptide ends compared to Example 1. Compared to Example 1, Example 3 reduced the amount of silane coupling agent solution from 40 mL to 30 mL, resulting in a lower amino grafting density on the membrane surface, a smaller number of glutaraldehyde crosslinking arms, and a decrease in the amount of covalently immobilized flavor protease. The enzyme activity per unit area of ​​the membrane reactor was insufficient, and the bitterness and astringency indices were further increased compared to the actual example.

[0058] Compared with Example 1, Example 4 reduced the concentration of the flavor protease solution from 5 mg / mL to 3 mg / mL, weakening the driving force for enzyme molecule diffusion to the aldehyde groups on the membrane surface during coupling, thus reducing the amount of enzyme loaded on the membrane. The bitter aftertaste was relatively high in all examples, indicating that when the amount of immobilized enzyme is insufficient, the removal of some hydrophobic peptide ends is incomplete, leaving residues in the mouth and producing a persistent bitter aftertaste.

[0059] Compared with Example 1, Example 6 shows that because Comparative Preparation Example 1 completely eliminated the silanization modification in step A2, the membrane surface lacked a stable cross-linking bridging layer formed by primary amino groups and glutaraldehyde. Flavor proteases mainly relied on physical adsorption and hydrogen bonding. The shear force generated by the circulating flow caused the adsorbed enzyme protein to gradually detach, resulting in a continuous decrease in effective enzyme activity and a significant reduction in debittering effect. This indicates that the debittering ability of the enzyme membrane is greatly weakened without covalent immobilization. Compared with Example 1, Example 7 shows that because Comparative Preparation Example 2 drastically reduced the amount of PEG-400 from 2g to 0.2g, the membrane was nearly dense, and the feed solution could hardly penetrate into the membrane's internal pores. The enzymatic reaction only occurred on the outer surface of the membrane, resulting in a sharp reduction in the effective reaction area.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for simultaneous dual-enzyme extraction of quinoa oligopeptides, characterized in that: Includes the following steps: Step S1: Pretreatment: Quinoa was washed with water and dried at 45°C for 24 hours, then pulverized and passed through a 100-mesh sieve to obtain quinoa flour; the quinoa flour was mixed with n-hexane, stirred at room temperature for 3 hours, and centrifuged at 3000 rpm for 5 minutes to obtain defatted quinoa flour. Step S2: Alkaline extraction treatment: Mix defatted quinoa powder and deionized water, adjust the pH to 10 with sodium hydroxide solution, stir at 40°C for 3 hours, adjust the pH to 8.5 with hydrochloric acid solution, centrifuge at 6000 rpm for 10 minutes, filter, and collect liquid 1 and insoluble matter 1 to obtain alkaline extract and alkaline insoluble substance. Step S3: Double enzyme treatment: The protein content of the alkaline extract was determined and controlled to be 5 wt% by concentration or addition of deionized water. The extract was then divided and added to tank A for alkaline protease treatment and tank B for neutral protease treatment, resulting in hydrolysate from tank A and hydrolysate from tank B. The hydrolysate from tank A and tank B were mixed and kept at 95°C for 10 min to obtain a mixed double enzyme hydrolysate, and the pH was adjusted to 7. Step S4: Ultrafiltration treatment: Centrifuge the double enzyme hydrolysate at 4000 rpm for 15 min, collect liquid 2 to obtain the double enzyme hydrolysate extract, send the double enzyme hydrolysate extract into the ultrafiltration circulation device for ultrafiltration, collect the permeate and adjust the protein content to 5 wt% by concentration or by adding deionized water to obtain the ultrafiltration permeate. Step S5: Flavor enzyme treatment: The ultrafiltration permeate is preheated and fed into a multi-layer stacked reaction device composed of enzyme-immobilized regenerated cellulose membranes. Liquid 3 is collected to obtain the reaction treatment solution. Step S6: Post-processing: The reaction solution was centrifuged at 6000 rpm for 10 min, pre-cooled, and then freeze-dried to obtain quinoa oligopeptide powder.

2. The method for simultaneous dual-enzyme extraction of quinoa oligopeptides according to claim 1, characterized in that: In step S1: the ratio of quinoa powder to n-hexane is 1:10; In step S2: the ratio of defatted quinoa powder to deionized water is 1:10, the molar concentration of sodium hydroxide solution is 2 mol / L, and the molar concentration of hydrochloric acid solution is 1 mol / L.

3. The method for simultaneous dual-enzyme extraction of quinoa oligopeptides according to claim 1, characterized in that: In step S3: In tank A: the pH value is first adjusted to 10 using sodium hydroxide / hydrochloric acid solution and stirred at 50℃ for 60 min, then alkaline protease is added, and an ultrasonic auxiliary device is introduced simultaneously. The mixture is kept at this temperature for 8 h, and the pH is kept constant during the reaction. In tank B: the pH value is first adjusted to 7 using sodium hydroxide / hydrochloric acid solution and stirred at 45℃ for 60 min, then neutral protease is added, and an ultrasonic auxiliary device is introduced simultaneously. The mixture is kept at this temperature for 8 h, and the pH is kept constant during the reaction. The ultrasound-assisted device is a 20kHz probe-type ultrasound with a power density of 80W / L, a pulse mode of 3s / 5s, and a total effective ultrasound time of 15min; the amount of alkaline protease added is 5000 U / g protein, and the amount of neutral protease added is 4000 U / g.

4. The method for simultaneous dual-enzyme extraction of quinoa oligopeptides according to claim 1, characterized in that: In step S4: the ultrafiltration membrane used is a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 5000U; during the ultrafiltration process: the ultrafiltration pressure is 0.2MPa, the temperature is 30℃, and the flow rate is 5L / min; In step S5: the preheating temperature is 50℃; the reaction device consisting of multiple stacked enzyme-immobilized regenerated cellulose films is obtained by stacking 4 enzyme-immobilized regenerated cellulose films; the reaction pH is 7, the reaction temperature is 52℃, and the reaction residence time is 2h. In step S6: pre-cooling is carried out at -40℃ for 2 hours; freeze drying: cold trap temperature ≤ -50℃, vacuum degree < 20Pa, drying time 48 hours.

5. The method for simultaneous dual-enzyme extraction of quinoa oligopeptides according to claim 4, characterized in that: The enzyme-immobilized regenerated cellulose film was prepared through the following steps: Step A1: Mix 1-butyl-3-methylimidazolium chloride ion liquid, microcrystalline cellulose and polyethylene glycol. Stir for 3 hours under nitrogen protection, stirring rate of 300 rpm and temperature of 90℃. Vacuum degassing for 3 hours, pour onto a glass plate and coat with a doctor blade, controlling the gap to be 1.5±0.05 mm. Let stand at 15℃ for 1 hour, soak and wash with deionized water, peel off to obtain regenerated cellulose film. Step A2: The regenerated cellulose film is dried and immersed in a silane coupling agent solution. It is immersed for 15 minutes at room temperature, then heated to 60°C and reacted for 1 hour. It is washed with ethanol and dried at 40°C to obtain the modified regenerated cellulose film. Step A3: Prepare a flavor protease solution using phosphate buffer and a glutaraldehyde solution using phosphate buffer. Immerse the modified regenerated cellulose film in the glutaraldehyde solution at room temperature for 1 hour, then remove it and wash it with phosphate buffer. Transfer it to the flavor protease solution and shake it at 15°C for 12 hours. Finally, transfer it to the glycine solution and soak it at room temperature for 1 hour. Wash it with deionized water to obtain the enzyme-immobilized regenerated cellulose film.

6. The method for simultaneous dual-enzyme extraction of quinoa oligopeptides according to claim 5, characterized in that: In step A1: the ratio of 1-butyl-3-methylimidazolium chloride ion liquid, microcrystalline cellulose and polyethylene glycol is 100g:15g:1.5-2g; the polyethylene glycol is PEG-400.

7. The method for simultaneous dual-enzyme extraction of quinoa oligopeptides according to claim 5, characterized in that: In step A2: the silane coupling agent solution is a mixture of γ-aminopropyltriethoxysilane, ethanol and deionized water, wherein the volume fraction of γ-aminopropyltriethoxysilane is 10% and the volume fraction of deionized water is 2%; the ratio of regenerated cellulose film to silane coupling agent solution is 2g:30-40mL.

8. The method for simultaneous dual-enzyme extraction of quinoa oligopeptides according to claim 5, characterized in that: In step A3: phosphate buffer pH=7, molar concentration 0.1mol / L, flavor protease solution concentration 3-5mg / mL, glutaraldehyde solution volume fraction 2.5%, glycine solution pH=7.5, molar concentration 0.1mol / L; the ratio of modified regenerated cellulose film, glutaraldehyde solution, flavor protease solution and glycine solution is 2g:30-35mL:35mL:20mL.

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