Extraction process of chenopodium quinoa polypeptide with low antinutritional factor content and high activity

Abstract

The invention discloses an extraction process of chenopodium quinoa polypeptide with low antinutritional factor content and high activity, and belongs to the technical field of plant extraction. The extraction process comprises the following steps: anti-nutritional factor removal pretreatment, directional protein extraction, enzymolysis, multi-stage membrane separation, activity enhancement and drying to obtain the quinoa polypeptide with low anti-nutritional factor content and high activity. According to the preparation method disclosed by the invention, anti-nutritional factors such as phytic acid in the chenopodium quinoa willd can be efficiently removed, and the dissolution rate of protein is increased to 95% or above; by adopting multi-enzyme synergistic hydrolysis, the generation of bitter peptides is reduced, and meanwhile, the proportion of active peptides with the molecular weight of 1000-2500Da is increased; a low-temperature process chain is optimized, the antioxidant and muscle repair activity of the quinoa polypeptide is reserved to the maximum extent, and the method is suitable for industrial continuous production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction, in particular to an extraction process of low-anti-nutritional factor and high-activity quinoa polypeptide. BACKGROUND

[0002] Quinoa is a genus of chenopodiaceae dicotyledon, the plant height varies from tens of centimeters to three meters due to different growth conditions, the plant is broom-shaped, the root system is shallow, the flower sequence is in order, self-pollination, the main and side shoots can all form seeds, the seeds are circular and flaky, the diameter is 1.5mm to 2mm. The appearance color is white, milk yellow, purple red and black. Among them, the black and purple red seeds are small, and the white and milk yellow seeds have better taste.

[0003] Although quinoa is a plant, it has complete protein that only animals have, which is very rare. The endosperm accounts for 68% of the planting, and has nutritional activity, the protein content is as high as 16%-22%, and the quality is equivalent to milk powder and meat; it is rich in various amino acids, among which there are all 9 kinds of essential amino acids necessary for human body, the proportion is appropriate and easy to absorb, especially rich in lysine, calcium, magnesium, phosphorus, potassium, iron, zinc, selenium, manganese, copper and other mineral nutrients, rich in unsaturated fatty acids, flavonoids, B vitamins and vitamin E, choline, betaine, folic acid, alpha-linolenic acid, beta-glucan and other beneficial compounds, the dietary fiber content is as high as 7.1%, the cholesterol is 0, it contains no gluten, is low in fat and low in calories (305kcal / 100g), and has low glycemic index (GI glycemic index value 35, low glycemic standard is 55). Quinoa is a whole grain and whole nutrient complete protein alkaline food, has high nutritional value, and is commonly known as "nutritional gold", "super grain" and "future food".

[0004] Bioactive peptides refer to peptide substances with special physiological functions, which are peptides with a length of about 2-20 amino acids and specific biological activities prepared by enzyme hydrolysis of proteins, and can be released from parent proteins to play specific physiological functions through in vivo or in vitro enzymolysis. Compared with chemical synthetic drugs, food-derived protein peptides have the advantages of safety, no toxic side effects, etc. Quinoa contains 16%-22% high-quality protein (containing all essential amino acids), and the polypeptide has the activities of antioxidant, immune enhancement and muscle repair, and has great market application potential.

[0005] However, the existing quinoa polypeptide extraction technology has the following key problems: 1. Anti-nutritional factor interference: Phytic acid (2.0-3.5 mg / g) in quinoa binds with protein to form a complex, resulting in low protein dissolution rate and low peptide yield by traditional enzymatic hydrolysis method; for example, Chinese patent application CN106819356A discloses a highland quinoa peptide and its preparation method and a medicine-food dual-purpose product, and specifically discloses that neutral protease is used for enzymatic hydrolysis treatment of quinoa powder, thereby extracting quinoa peptide from quinoa raw material, and the molecular weight of the quinoa peptide is small, which is beneficial to human body absorption. However, the preparation method has the following problems: the pulverized quinoa kernels are directly used as the substrate for protease hydrolysis, which affects the enzymatic efficiency and the yield of quinoa peptide, and the protein hydrolysis degree or peptide yield and other related indicators are not disclosed, and a large amount of impurities are contained, according to the experimental results, the extraction rate of quinoa protein by direct enzymatic hydrolysis is only 54% of the total quinoa protein, and it is difficult to remove or remove small molecular impurities at high cost.

[0006] 2. Insufficient enzymatic specificity: when a single protease (such as flavor protease) is used to hydrolyze quinoa protein, bitter peptides (such as hydrophobic amino acid fragments) are easily produced, the sensory qualified rate is low, and additional debittering treatment is required; for example, Chinese patent application CN111197066A discloses an enzymatic method for quinoa active peptide with bile salt adsorption, including the following steps: step 1, quinoa protein is extracted from quinoa by alkali dissolution and acid precipitation; step 2, quinoa protein is used as the object, and flavor protease is added for enzymatic hydrolysis; step 3, after the completion of the enzymatic reaction, the product is placed in a 95℃ water bath for 8-12 min for inactivation, then taken out and cooled, the pH value is adjusted to 7.0, the supernatant is collected by centrifugation, and freeze-drying is performed to obtain quinoa active peptide.

[0007] 3. Poor activity retention: high-temperature pretreatment causes the DPPH free radical scavenging rate of quinoa polypeptide to decrease, and amino acid oxidation is easily caused; for example, Chinese patent application CN110951809A provides a preparation process of red quinoa polypeptide powder, including the following steps: S1, high-temperature pretreatment of red quinoa substrate; S2, red quinoa protease enzymatic hydrolysis; S3, refined quinoa peptide solution; S4, concentration of quinoa peptide solution; S5, sterilization, drying and granulation to obtain red quinoa polypeptide powder.

[0008] Therefore, it is urgent to develop a green process for efficiently removing anti-nutritional factors and directionally preparing low-bitterness high-activity quinoa polypeptide to solve the efficiency and quality contradiction of the prior art. SUMMARY

[0009] The purpose of the present application is to provide a green process for efficiently removing anti-nutritional factors and directionally preparing low-bitterness high-activity quinoa polypeptide.

[0010] In a first aspect, the present application provides an extraction process of low anti-nutritional factor high-activity quinoa polypeptide, including the following steps: S1: Anti-nutritional factor removal pretreatment: quinoa seed is dried and crushed to obtain quinoa powder, which is mixed with water and phytase to remove phytic acid; water is added for soaking to obtain a pretreatment solution; S2: Directional protein extraction: extracted by alkali dissolution and acid precipitation to obtain quinoa protein; S3: Enzymatic hydrolysis: quinoa protein is dissolved in water and then subjected to enzymatic hydrolysis by adding a complex enzyme, and then inactivated to obtain a crude quinoa polypeptide extract; the complex enzyme is a mixture of alkaline protease, neutral protease and flavor protease with a mass ratio of (1-3):(2-4):(0.5-1.5); S4: Multistage membrane separation and activity enhancement: the crude quinoa polypeptide extract is subjected to membrane filtration, ultrafiltration with a 2500 Da membrane, nanofiltration concentration, and drying to obtain low anti-nutritional factor and high activity quinoa polypeptide.

[0011] Preferably, in step S1, the drying temperature is 40-45℃, and the drying is performed until the water content is 8%-10%.

[0012] Preferably, in step S1, the crushing size is 120-150 mesh.

[0013] Preferably, in step S1, the phytic acid removal includes the following steps: quinoa seeds are mixed with water at a solid-liquid ratio of 1g:12-15mL, phytase is added, and the mixture is incubated at a temperature of 50-55℃ and a pH of 5.0-5.5 for 1.5-2h to obtain a phytic acid-removed solution. Preferably, the phytase has an enzyme activity of 4500-5500U / g, and the addition amount of the phytase is 0.3%-0.5% of the mass of the quinoa powder.

[0014] Preferably, in step S1, the water soaking is performed by adding water to the phytic acid-removed solution to a solid-liquid ratio of 1g:18-22mL and soaking at 30-35℃ for 3-4h.

[0015] Preferably, in step S2, the alkali dissolution and acid precipitation method includes the following steps: (1) Alkali dissolution: alkali is added to the pretreatment solution to adjust the pH to 9.0-9.5, and the mixture is stirred at 40-45℃ for 40-60min to fully dissolve the protein; (2) Acid precipitation purification: add acid to adjust the pH to 4.0-4.5, and after standing for 1-2h, centrifuge at a speed of 4500-5500r / min for 15-25min to collect the precipitate to obtain quinoa protein.

[0016] Further preferably, in step (1), the alkali is sodium hydroxide, and in step (2), the acid is hydrochloric acid.

[0017] Preferably, in step S3, the amount of the complex enzyme is 1.2%-1.8% of the mass of the protein.

[0018] Preferably, in step S3, the enzymatic hydrolysis is carried out in two stages: 0-1.5h, first stage, temperature 55-60℃, pH 8.0-8.5; 1.5-3h, second stage, under nitrogen atmosphere, temperature is lowered to 50-55℃, pH is adjusted to 6.5-7.0.

[0019] Preferably, in step S3, the inactivation is: temperature is raised to 80-85℃ for 10-20min, the enzymatic hydrolysis is terminated, and a crude quinoa polypeptide solution is obtained.

[0020] Preferably, in step S4, the membrane for membrane filtration is a 0.1μm inorganic ceramic membrane.

[0021] Preferably, in step S4, the nanofiltration concentration is: nanofiltration under pressure 2.0-2.5MPa and temperature 38-42℃, and the concentration is up to 30%-35% (v / v) of the polypeptide concentration.

[0022] Preferably, in step S4, the drying is spray drying or freeze drying. Further preferably, the parameters for spray drying are: inlet air temperature 180-200℃, outlet air temperature 80-90℃, and feed rate 20-25mL / min.

[0023] Further preferably, the parameters for freeze drying are: temperature -50℃~-60℃, and vacuum degree 5-10Pa.

[0024] The present application has at least the following beneficial effects: 1. The present application efficiently removes interfering protein dissolution factors such as phytic acid through specific anti-nutritional factor removal pretreatment, with a phytic acid removal rate ≥90%, a protein dissolution rate of 95%-97%, and an increase of 10%-12% compared to traditional processes; and further improves the yield and activity of quinoa polypeptides.

[0025] 2. The present application simultaneously improves the activity and sensory value of the prepared quinoa polypeptides through specific extraction processes, including anti-nutritional factor removal pretreatment, directional protein extraction, multi-enzyme synergistic hydrolysis and bitterness control, multi-stage membrane separation and activity enhancement, and low-temperature drying, with a proportion of active peptides (1000-2500Da) ≥92%, a DPPH free radical scavenging rate ≥90%, an ACE inhibition rate ≥86%, and a bitterness value (sensory score) ≤2 points (on a 10-point scale), without the need for additional debittering.

[0026] 3. The extraction process of the present application has strong industrialization adaptability: the whole process of pretreatment-enzymatic hydrolysis-drying is low-temperature controllable (maximum temperature ≤85℃), the spray drying capacity reaches 500kg / d, is suitable for large-scale continuous production, and the energy consumption is reduced by 40% compared to freeze drying. Detailed Implementation

[0027] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0028] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0029] Quinoa seeds: Zhangjiakou Youguxiang Food Co., Ltd.; Phytase: Novozymes (China) Biotechnology Co., Ltd.; Alkaline protease: Novozymes (China) Biotechnology Co., Ltd.; Neutral protease: Novozymes (China) Biotechnology Co., Ltd.; Flavor protease: Novozymes (China) Biotechnology Co., Ltd.

[0030] Example 1 An extraction process for quinoa polypeptides with low anti-nutritional factors and high activity includes the following steps: S1: Antibiotic-free nutritional factor pretreatment: Take 200g of quinoa seeds (protein content 20.5%), dry at 42℃ to 9% moisture content, and grind to 140 mesh; add deionized water at a ratio of 1g:14mL, add 0.4% phytase (enzyme activity 5000U / g) of the quinoa seed weight, keep warm at 52℃ and pH 5.2 for 1.8h, the phytate removal rate is 91.3%; add water to make a solid-liquid ratio of 1g:20mL, soak at 32℃ for 3.5h to obtain the pretreated solution; S2: Targeted protein extraction: The pretreatment solution was adjusted to pH 9.2, stirred at 43℃ for 50 min, then adjusted to pH 4.2 and allowed to stand for 1.5 h. After centrifugation at 5000 r / min for 20 min, quinoa protein (purity 93.1%, phytic acid residue 0.08 mg / g) was obtained. S3: Enzymatic hydrolysis: Under a nitrogen atmosphere, dissolve quinoa protein in deionized water at a ratio of 1g:15mL, add 1.5% compound enzyme (alkaline protease, neutral protease and flavor protease in a mass ratio of 2:3:1), incubate at 58℃ and pH 8.3 for 1.5h in the first stage, and at 52℃ and pH 6.8 for 1.5h in the second stage. S4: multistage membrane separation and activity enhancement: the crude extract was filtered by 0.1 μm inorganic ceramic membrane to remove unhydrolyzed protein and enzyme residue; then the active peptide segment with a molecular weight of 1000-2500 Da (the peptide in this interval has the highest DPPH clearance rate) was intercepted by 2500 Da hollow fiber membrane ultrafiltration, and the permeate was reused; the polypeptide concentrate with a concentration of 32% (v / v) was obtained by nanofiltration under a pressure of 2.3 MPa and a temperature of 40℃; low-temperature spray drying (the inlet air temperature was 190℃, the outlet air temperature was 85℃, the feeding rate was 22 mL / min, and the water content was <3%) was performed to obtain 31.2 g of quinoa polypeptide product.

[0031] Example 2 An extraction process of low-antinutritional factor high-activity quinoa polypeptide includes the following steps: S1: antinutritional factor removal pretreatment: 200 g of quinoa seeds (protein content 20.5%) was dried at 40℃ to a water content of 10%, and then crushed to 120 mesh; 1 g of the quinoa seeds was added to 15 mL of deionized water, and 0.3% of phytase (enzyme activity 5500 U / g) was added based on the mass of the quinoa seeds; the mixture was incubated at 50℃ and pH 5.0 for 2 h, and the phytate removal rate was 90.5%; water was added to a solid-liquid ratio of 1 g:18 mL, and the mixture was soaked at 35℃ for 3 h to obtain a pretreatment liquid; S2: directional protein extraction: the pH of the pretreatment liquid was adjusted to 9.0, and the mixture was stirred at 45℃ for 40 min; then the pH was adjusted to 4.5, and the mixture was allowed to stand for 2.0 h; the mixture was centrifuged at 4500 r / min for 25 min to obtain quinoa protein (purity 92.5%, phytate residue 1%); S3: enzymolysis: under a nitrogen atmosphere, 1 g of quinoa protein was dissolved in 15 mL of deionized water, and 1.2% of a composite enzyme (mass ratio of alkaline protease, neutral protease, and flavor protease was 3:2:1.5) was added; the first stage was incubated at 58℃ and pH 8.3 for 1.5 h, and the second stage was incubated at 52℃ and pH 6.8 for 1.5 h; S4: multistage membrane separation and activity enhancement: the crude extract was filtered by 0.1 μm inorganic ceramic membrane to remove unhydrolyzed protein and enzyme residue; then the active peptide segment with a molecular weight of 1000-2500 Da (the peptide in this interval has the highest DPPH clearance rate) was intercepted by 2500 Da hollow fiber membrane ultrafiltration, and the permeate was reused; the polypeptide concentrate with a concentration of 32% (v / v) was obtained by nanofiltration under a pressure of 2.3 MPa and a temperature of 40℃; low-temperature spray drying (the inlet air temperature was 190℃, the outlet air temperature was 85℃, the feeding rate was 22 mL / min, and the water content was <3%) was performed to obtain 31.2 g of quinoa polypeptide product.

[0032] Example 3 An extraction process of low-antinutritional factor high-activity quinoa polypeptide includes the following steps: S1: Anti-nutritional factor removal pretreatment: 200 g of quinoa seeds (protein content 20.5%) was dried at 45°C to a moisture content of 8%, and then ground to 150 mesh; 1 g of the quinoa seeds was added to 12 mL of deionized water, and 0.5% of phytase (enzyme activity 4500 U / g) based on the mass of the quinoa seeds was added, and then incubated at 525°C and pH 5.5 for 1.5 h, and the phytate removal rate was 90.7%; the water was added to a solid-liquid ratio of 1 g:22 mL, and then soaked at 30°C for 4 h to obtain a pretreatment solution; S2: Directional protein extraction: the pH of the pretreatment solution was adjusted to 9.5, and then stirred at 40°C for 60 min, and then the pH was adjusted to 4.0 and left to stand for 1.0 h, and then centrifuged at 5500 r / min for 15 min to obtain quinoa protein (purity 91.8%, phytate residue 1.2%); S3: Enzymatic hydrolysis: under a nitrogen atmosphere, 1 g of the quinoa protein was dissolved in 15 mL of deionized water, and 1.8% of a complex enzyme (mass ratio of alkaline protease, neutral protease and flavor protease 1:4:0.5) was added, and then incubated at 58°C and pH 8.3 for 1.5 h in the first stage, and then incubated at 52°C and pH 6.8 for 1.5 h in the second stage; S4: Multistage membrane separation and activity enhancement: the crude extract solution was filtered through a 0.1 μm inorganic ceramic membrane to remove unhydrolyzed protein and enzyme residues; then the solution was ultrafiltered through a 2500 Da hollow fiber membrane to retain active peptide segments with a molecular weight of 1000-2500 Da (the highest DPPH clearance rate in this interval), and the permeate was reused; nanofiltration was performed at a pressure of 2.5 MPa and a temperature of 38°C to obtain a 30% (v / v) concentration of polypeptide concentrate; low-temperature spray drying (inlet air temperature 190°C, outlet air temperature 85°C, feed rate 22 mL / min, water content <3%) to obtain 30.2 g of quinoa polypeptide product.

[0033] Comparative Example 1 An extraction process for a low anti-nutritional factor and high activity quinoa polypeptide, which differs from Example 1 in that the parameters of the extraction process are changed, and the specific steps include the following: S1: Anti-nutritional factor removal pretreatment: 200 g of quinoa seeds (protein content 20.5%) was dried at 45°C to a moisture content of 8%, and then ground to 150 mesh; 1 g of the quinoa seeds was added to 12 mL of deionized water, and 0.5% of phytase (enzyme activity 4500 U / g) based on the mass of the quinoa seeds was added, and then incubated at 525°C and pH 5.5 for 1.5 h, and the phytate removal rate was 90.7%; the water was added to a solid-liquid ratio of 1 g:22 mL, and then soaked at 30°C for 4 h to obtain a pretreatment solution; S2: Directional protein extraction: the pH of the pretreatment solution was adjusted to 9.5, and then stirred at 40°C for 60 min, and then the pH was adjusted to 4.0 and left to stand for 1.0 h, and then centrifuged at 5500 r / min for 15 min to obtain quinoa protein (purity 91.8%, phytate residue 1.2%); S3: Enzymatic hydrolysis: under nitrogen atmosphere, dissolve the buckwheat protein in deionized water at 1 g:20 mL, add 2% complex enzyme (mass ratio of 1:1:1 of alkaline protease, neutral protease and flavor protease), first stage 50°C, pH 7.0, incubate for 1.5 h, second stage 60°C, pH 8.5, incubate for 1.5 h; S4: Multi-stage membrane separation and activity enhancement: filter the crude extract liquid with 0.1 μm inorganic ceramic membrane to remove unhydrolyzed protein and enzyme residue; then ultrafiltrate through 2500 Da hollow fiber membrane to retain active peptide segments with a molecular weight of 1000-2500 Da (the highest DPPH clearance rate in this interval), and reuse the permeate; under a pressure of 3.0 MPa and a temperature of 35°C, nanofiltration is performed to concentrate the polypeptide to a concentration of 40% (v / v); low-temperature spray drying (inlet air temperature 220°C, outlet air temperature 95°C, feed rate 22 mL / min, water content <3%) to obtain 25.8 g of buckwheat polypeptide product.

[0034] Comparative Example 2 A low-antinutritional factor high-activity buckwheat polypeptide extraction process, comprising the following steps: S1: Take 200 g of buckwheat seeds (protein content 20.5%), crush to 140 mesh, add deionized water at 1 g:14 mL to obtain a pretreatment liquid; S2: Directional protein extraction: adjust the pH of the pretreatment liquid to 9.2, stir at 43°C for 50 min, then adjust the pH to 4.2 and stand for 1.5 h, centrifuge at 5000 r / min for 20 min to obtain buckwheat protein (purity 72%, phytic acid residue 8.33%); S3: Enzymatic hydrolysis: dissolve the buckwheat protein in 10 times the weight of deionized water, adjust the pH to 9.3 with 0.1 mol / L HCL and 0.1 mol / L NaOH, add 3000 U of alkaline protease per gram of initial buckwheat protein, and water bath at 46°C for 2.5 h. Then adjust the pH to 6.7 with 0.1 mol / L HCL and 0.1 mol / L NaOH, add 2500 U of papain per gram of initial buckwheat protein, and water bath at 58°C for 1.5 h. Cool to 45°C, adjust the pH to 3.1 with 0.1 mol / L HCL and 0.1 mol / L NaOH, add 4000 U of acid protease per gram of initial buckwheat protein, and water bath at 45°C for 1.5 h to obtain an enzymatic hydrolysate; S4: Multistage membrane separation and activity enhancement: The crude extract was filtered by 0.1 μm inorganic ceramic membrane to remove unhydrolyzed proteins and enzyme residues; then, the active peptide segments with a molecular weight of 1000-2500 Da (the highest DPPH clearance rate) were retained by 2500 Da hollow fiber membrane ultrafiltration, and the permeate was reused; the polypeptide concentrate with a concentration of 32% (v / v) was obtained by nanofiltration at a pressure of 2.3 MPa and a temperature of 40℃; and quinoa polypeptide product 22.8 g was obtained by low-temperature spray drying (inlet air temperature 190℃, outlet air temperature 85℃, feed rate 22 mL / min, water content <3%).

[0035] Comparative Example 3 An extraction process of low-antinutritional factor and high-activity quinoa polypeptide, which is different from Example 1 in that S4 is different, specifically comprising the following steps: S1: Antinutritional factor removal pretreatment: Take 200 g of quinoa seeds (protein content 20.5%), dry at 42℃ to a moisture content of 9%, and crush to 140 mesh; add deionized water at a ratio of 1 g:14 mL, and add phytase at 0.4% of the mass of the quinoa seeds (enzyme activity 5000 U / g), and incubate at 52℃ and pH 5.2 for 1.8 h, with a phytate removal rate of 90.8%; add water to a solid-liquid ratio of 1 g:20 mL, and soak at 32℃ for 3.5 h to obtain a pretreatment solution; S2: Directional protein extraction: adjust the pH of the pretreatment solution to 9.2, stir at 43℃ for 50 min, then adjust the pH to 4.2 and stand for 1.5 h, centrifuge at 5000 r / min for 20 min to obtain quinoa protein (purity 93.1%, phytate residue 0.08 mg / g); S3: Enzymatic hydrolysis: dissolve the quinoa protein in deionized water at a ratio of 1 g:15 mL, add 1.5% of a composite enzyme (mass ratio of alkaline protease, neutral protease, and flavor protease is 2:3:1), incubate at 58℃ and pH 8.3 for 1.5 h in the first stage, and incubate at 52℃ and pH 6.8 for 1.5 h in the second stage; S4: Multistage membrane separation and activity enhancement: filter the crude extract by 0.1 μm inorganic ceramic membrane; obtain a polypeptide concentrate with a concentration of 32% by nanofiltration at a pressure of 2.3 MPa and a temperature of 40℃; and obtain quinoa polypeptide product 35.8 g by low-temperature spray drying (inlet air temperature 190℃, outlet air temperature 85℃, feed rate 22 mL / min, water content <3%).

[0036] Test Example 1 (1) Product yield: yield%=mass of quinoa polypeptide product / mass of quinoa seeds×100%.

[0037] (2) Peptide yield: The content of quinoa peptide in the quinoa polypeptide product was measured according to the Kjeldahl method in GB 5009.5-2025 "National Food Safety Standard-Determination of Protein in Foods": The TCA precipitation method was used to determine the content of quinoa peptide. 1.000 g of the quinoa peptide product prepared in Example 1-Example 3 or Comparative Example 1-Comparative Example 3 was accurately weighed, added with deionized water to make up to 10 mL, accurately pipetted 1 mL, and then mixed uniformly after adding a certain volume of ethanol and TCA precipitant. The mixture was centrifuged at 3000 r / min for 10 min.

[0038] The supernatant was determined according to the standard curve (equation Y = 0.01293X + 0.00306) and the od value of the sample was determined according to the following formula, and the content of quinoa peptide was calculated according to the following formula.

[0039] The peptide content of the quinoa peptide product = (A-0.00306) n x V x 1000 / 0.01293 x m; Wherein: A is the absorbance of the sample; n is the dilution multiple of the sample; V is the sample volume / m L; m is the sample mass / g.

[0040] Then the yield of quinoa peptide was calculated according to the peptide content: The yield of quinoa peptide = the peptide content of the quinoa peptide product / the protein content of the quinoa seed x 100%.

[0041] (3) Determination of the proportion of active peptides with a molecular weight of 1000-2500 Da in the quinoa peptide in the quinoa peptide product.

[0042] (4) DPPH free radical scavenging rate: determined according to T / SHRH 006-2018 Cosmetics- Free radical (DPPH) scavenging experiment method.

[0043] Table 1

[0044] Test Example 2 Bitterness score: The quinoa peptide product prepared by the application was subjected to sensory evaluation, and 30 trained food professionals (half male and half female) were selected to form a sensory evaluation team. The evaluation index was represented by taste, bitterness and odor. Different indexes were scored respectively. Taste 10 points was the best, taste 0 points was the worst, bitterness 0 points was the lowest bitterness, 10 points was the heaviest bitterness, odor 0 points was the lowest odor, 10 points was the heaviest odor. The specific scoring standards are shown in Table 2, the scoring results are shown in Table 3, and the average value of the scores is taken. The room temperature was controlled at 25℃±2℃, and the samples were rinsed with water to restore the original sensory ability.

[0045] Table 2

[0046] Table 3

[0047] Test Example 3 Health indicators: according to GB31611-2023 plant protein peptide national standard for detection.

[0048] Table 4

[0049] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the scope of protection of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A low anti-nutritional factor high activity quinoa polypeptide extraction process, characterized in that, The method comprises the following steps: S1: Anti-nutritional factor pretreatment: after the quinoa seed is dried and crushed, quinoa powder is obtained, and phytic acid is removed by mixing the quinoa powder with water and phytase; water is added for soaking to obtain a pretreated solution; S2: Directional protein extraction: the protein is extracted by an alkali dissolution and acid precipitation method to obtain quinoa protein; S3: Enzymatic hydrolysis: after the quinoa protein is dissolved in water, a composite enzyme is added for enzymatic hydrolysis, and then inactivation is performed to obtain a quinoa polypeptide crude extract; the composite enzyme is a mixture of alkaline protease, neutral protease and flavor protease with a mass ratio of (1-3):(2-4):(0.5-1.5); S4: Multistage membrane separation and activity enhancement: the quinoa polypeptide crude extract is subjected to membrane filtration, ultrafiltration with a membrane with a cut-off of 2500 Da, nanofiltration concentration, and drying to obtain low-anti-nutritional factor and high-activity quinoa polypeptide.

2. The extraction process of claim 1, wherein, In step S1, the drying temperature is 40-45 DEG C, and the drying is performed until the water content is 8%-10%; the crushing size is 120-150 mesh.

3. The extraction process of claim 1, wherein, In step S1, the phytic acid removal comprises the following steps: the quinoa seed is mixed with water at a solid-liquid ratio of 1g:12-15 mL, and phytase is added, and the mixture is incubated at a temperature of 50-55 DEG C and a pH of 5.0-5.5 for 1.5-2 h to obtain a phytic acid-removed solution.

4. The extraction process according to claim 3, characterized in that, The phytase has an enzyme activity of 4500-5500 U / g, and the addition amount of the phytase is 0.3%-0.5% of the mass of the quinoa powder.

5. The extraction process of claim 1, wherein, In step S1, the water soaking is performed by adding water to the phytic acid-removed solution to a solid-liquid ratio of 1g:18-22 mL, and then the mixture is soaked at 30-35 DEG C for 3-4 h.

6. The extraction process of claim 1, wherein, In step S2, the alkali dissolution and acid precipitation method comprises the following steps: (1) alkali dissolution: alkali is added to the pretreated solution, the pH is adjusted to 9.0-9.5, and the mixture is stirred at 40-45 DEG C for 40-60 min to fully dissolve the protein; (2) acid precipitation purification: the pH is adjusted to 4.0-4.5 by adding acid, and the mixture is centrifuged at a speed of 4500-5500 r / min for 15-25 min after standing for 1-2 h to collect the precipitate to obtain quinoa protein.

7. The extraction process of claim 1, wherein, In step S3, the enzymatic hydrolysis is performed in two stages: 0-1.5 h, first stage, temperature 55-60 DEG C, pH 8.0-8.5; 1.5-3 h, second stage, under a nitrogen atmosphere, the temperature is reduced to 50-55 DEG C, the pH is adjusted to 6.5-7.0, and tea polyphenol is added in an amount of 0.04%-0.06% of the mass of the quinoa protein.

8. The extraction process of claim 1, wherein, In step S3, the inactivation is performed by increasing the temperature to 80-85 DEG C for 10-20 min to terminate the enzymatic hydrolysis, and a quinoa polypeptide crude extract is obtained.

9. The extraction process of claim 1, wherein, In step S4, the membrane for the membrane filtration is a 0.1 mu m inorganic ceramic membrane; the nanofiltration concentration is performed at a pressure of 2.0-2.5 MPa and a temperature of 38-42 DEG C, and the concentration is performed to a polypeptide concentration of 30%-35% by volume percentage.

10. The extraction process of claim 1, wherein, In step S4, the drying is performed by spray drying or freeze drying; the parameters for the spray drying are: an inlet air temperature of 180-200 DEG C, an outlet air temperature of 80-90 DEG C, and a feeding rate of 20-25 mL / min; the parameters for the freeze drying are: a temperature of-50 DEG C to-60 DEG C, and a vacuum degree of 5-10 Pa.

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