Extraction method of special bioactive peptide for low-sugar food

Abstract

The invention relates to the technical field of bioactive peptide extraction, and discloses an extraction method of a special bioactive peptide for low-sugar food. A low-sugar high-protein raw material is used as a matrix, a neutral protease, saccharifying enzyme and phytase compound enzyme system is adopted to realize directional enzymolysis and in-situ desugaring, then a microfiltration-charge modified nanofiltration double-membrane coupling system is used for charge-aperture double-effect separation, and 2-3 kDa high-activity peptide fragments are accurately enriched. According to the method, monosaccharide, inorganic salt and pigment can be synchronously removed, the purity and physiological activity of the active peptide are remarkably improved, the sugar content of the obtained product is 0.3-0.5 g / 100g, the purity is 90-98%, and the method is suitable for high-value development of functional low-sugar food.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food bioengineering and functional active peptide preparation, and particularly relates to an extraction method of low-sugar food special bioactive peptide. BACKGROUND

[0002] At present, the active peptide extraction process widely used in the market adopts single enzymolysis or ultrafiltration separation mode, and has problems such as independent desugarization step, complex process flow, high energy consumption and easy destruction of active peptide structure. The traditional method is difficult to realize the protein directional hydrolysis and the removal of saccharides in the same system, so that the obtained product has high sugar content and insufficient purity, which is not suitable for the formula requirements of low-sugar food. In addition, the conventional microfiltration or ultrafiltration only relies on pore size screening, and cannot effectively remove small molecular impurities such as inorganic salts and pigments, and it is also difficult to accurately enrich the functional peptides of target molecular weight. The present application realizes the synergistic control of enzyme reaction and membrane separation by constructing the integrated process of “directional enzymolysis-in-situ desugarization-charge pore size dual-effect targeted separation”, significantly improves the purity and functional stability of active peptide, and provides a new path for the high-value development of low-sugar food raw materials. SUMMARY

[0003] In view of the above problems, the present application provides an extraction method of low-sugar food special bioactive peptide, which realizes protein directional hydrolysis and synchronous removal of impurities by the integrated process of “directional enzymolysis-in-situ desugarization-charge pore size dual-effect separation”, and accurately enriches 2-3 kDa high-activity peptide segments. The product obtained by the method has low sugar, high purity and high activity, the process is simple and efficient, and is suitable for the development of functional low-sugar food and special medical nutritional products.

[0004] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: An extraction method of low-sugar food special bioactive peptide, the key components in the extraction process are used in the following mass fraction ratio: 100 parts of low-sugar high-protein raw material, 400-600 parts of deionized water, 0.8-1.2 parts of composite directional enzyme system; the composite directional enzyme system is composed of neutral protease, saccharifying enzyme and phytase in a mass ratio of 3:2:1-4:3:2; the addition amount of 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution for adjusting pH in the enzymolysis process is 0.5-1.0% of the mass of the raw material slurry.

[0005] Preferably, the extraction method of low-sugar food special bioactive peptide is as follows: S1, grinding low-sugar high-protein raw materials to 80-100 mesh, mixing according to the mass ratio of raw materials to water 1:4-1:6 to obtain raw material slurry; the low-sugar high-protein raw material is defatted low-sugar soybean protein isolate or low-lactose whey protein concentrate, wherein the protein content of the defatted low-sugar soybean protein isolate is 88-92%, the sugar content is 0.5-1.0%, the protein content of the low-lactose whey protein concentrate is 83-87%, and the sugar content is 1.5-2.0%; S2, adding a complex directional enzyme system to the raw material slurry, and enzymatically hydrolyzing at a temperature of 35-40°C and a pH of 6.5-7.0 for 1.0-2.0 h to obtain an enzymatic hydrolysate; the addition amount of the complex directional enzyme system is 0.8-1.2% of the mass of the low-sugar high-protein raw material; S3, sequentially separating the enzymatic hydrolysate through a 0.1 μm microfiltration membrane and a 1 kDa charge-modified nanofiltration membrane, and collecting the retentate of the 1 kDa charge-modified nanofiltration membrane; wherein the operating temperature of the 0.1 μm microfiltration membrane is 20-25°C, and the operating pressure is 0.1-0.2 MPa; the 1 kDa charge-modified nanofiltration membrane is a polyether sulfone nanofiltration membrane with a surface grafted sulfonic acid group, the sulfonic acid group loading is 0.8-2.0 μmol / cm 2 , the operating temperature is 20-25°C, and the operating pressure is 0.3-0.5 MPa; S4, freeze-drying the retentate under the conditions of -45 to -50°C and a vacuum degree of 0.08-0.10 MPa to obtain a low-sugar food special biological active peptide; the sugar content of the low-sugar food special biological active peptide is 0.2-0.3 g / 100 g, the proportion of the biological active peptide in the 2-5 kDa molecular weight interval is 90-95%, and the high-activity peptide in the 2-3 k Da molecular weight interval accounts for 80-85% of the biological active peptide in the 2-5 k Da molecular weight interval.

[0006] Preferably, the low-sugar high-protein raw material is selected from one or more of defatted low-sugar soybean protein isolate, low-lactose whey protein concentrate, and pea protein.

[0007] Preferably, the raw material is sieved after being ground through a standard sieve, and the undersize is used to prepare the raw material slurry; when the raw material is mixed with water, mechanical stirring is adopted at a stirring rate of 200-300 rpm, and the stirring time is 15-20 min; the raw material is ground through a 80-100 mesh standard sieve, and the undersize is used to prepare the raw material slurry; when the raw material is mixed with water, mechanical stirring is adopted at a stirring rate of 200-300 rpm, and the stirring time is 15-20 min; Preferably, the enzymatic hydrolysis process uses a constant-temperature water bath shaking device, and the shaking rate is 150-200 rpm; during the enzymatic hydrolysis process, the pH is maintained stable by adding 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution dropwise, and the pH fluctuation range is controlled within ± 0.2.

[0008] Preferably, the 0.1 mu m microfiltration membrane is a hydrophilic polyvinylidene fluoride microfiltration membrane, which is soaked in 0.1 mol / L sodium hydroxide solution for 30 min before use, and then washed with deionized water until neutral; the 1 kDa charge-modified nanofiltration membrane is circulated and washed with deionized water under operating pressure for 20-30 min before use to remove residual protective agents on the surface of the membrane.

[0009] Preferably, before freeze-drying, the retentate needs to be placed at 4 DEG C for 1-2 h to remove surface scum; during freeze-drying, the retentate is first cooled to -45 DEG C to -50 DEG C for 2-3 h, and then vacuum dried, and the drying time is 12-15 h to ensure that the moisture content of the finished product is 1-3%.

[0010] Preferably, the enzyme activity of the neutral protease is 50000-80000 U / g, the enzyme activity of the glucoamylase is 100000-150000 U / g, and the enzyme activity of the phytase is 5000-10000 U / g.

[0011] Preferably, the DPPH free radical scavenging rate of the low-sugar food special biological active peptide is 85-95%, and the alpha-glucosidase inhibition rate is 65-75%.

[0012] The beneficial effects of the present application are: The low-sugar food special biological active peptide prepared by the present application has high purity and strong activity, the sugar content is 0.2-0.3 g / 100 g, the proportion of 2-3 kDa high-activity peptides is 80-85%, the DPPH free radical scavenging rate of the product is 85-95%, and the alpha-glucosidase inhibition rate is 65-75%, which has excellent antioxidant and hypoglycemic activity. Through the double-effect separation of composite enzyme system directional enzymolysis and charge-modified nanofiltration, monosaccharides, inorganic salts and pigments can be effectively removed, and the product purity and flavor stability can be improved. The process flow is simple, low in energy consumption, and free of solvent residues, and is suitable for the preparation of high-quality raw materials for functional low-sugar foods and special medical nutritional products. BRIEF DESCRIPTION OF DRAWINGS

[0013] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, which together with the embodiments of the present application, serve to explain the present application, and do not constitute a limitation on the present application.

[0014] Figure 1 The figure is a comparison chart of the distribution proportions of 2-5 kDa and 2-3 kDa peptide segments in different samples of the present application; Figure 2 The figure is a comparison chart of the DPPH free radical scavenging rate and the alpha-glucosidase inhibition rate of different samples of the present application; Figure 3The relationship diagram between the enzymolysis time of different samples of the present application and the nanofiltration retention recovery rate. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0016] Embodiment 1 The extraction method of the low-sugar food special bioactive peptide in this embodiment 1 is as follows: the mass fraction of each key component in the extraction process is: Low-sugar high-protein raw materials: defatted low-sugar soybean protein isolate 100 parts, deionized water 500 parts, and composite directional enzyme system 1.0 part, neutral protease: glucoamylase: phytase = 3:2.5:1; The extraction method of the low-sugar food special bioactive peptide in this embodiment is as follows: S1, take defatted low-sugar soybean protein isolate, crush it with a high-speed crusher, and then pass it through 80-mesh and 100-mesh standard sieves in sequence: first collect the 80-mesh undersize to remove coarse particles with a particle size greater than 180 μm, and then pass the undersize through the 100-mesh sieve to collect the 100-mesh oversize with a particle size of 150-180 μm; mix the sieved raw materials with deionized water, and mechanically stir at a stirring rate of 250 rpm / min for 18 min to obtain a raw material slurry; S2, add the composite directional enzyme system to the raw material slurry, and transfer the mixed solution to a constant-temperature water bath oscillation device, set the temperature to 38℃, the oscillation rate to 180 rpm / min, and the pH to 6.8; during the enzymolysis process, maintain the pH fluctuation within ± 0.2 by adding 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution dropwise, and enzymolysis for 1.5 h to obtain an enzymolysis solution; S3, pass the enzymolysis solution through a 0.1 μm hydrophilic polyvinylidene fluoride microfiltration membrane at a temperature of 23℃ and a pressure of 0.15 MPa to collect the microfiltration permeate; then pass the retentate through a 1 kDa sulfonic acid-modified polyether sulfone nanofiltration membrane with a sulfonic acid loading of 1.0 μmol / cm 2 , at an operating temperature of 23℃ and a pressure of 0.4 MPa, to collect the nanofiltration retentate; S4, place the nanofiltration retentate at 4℃ for 1.5 h, remove the surface scum, and then transfer it to a freeze dryer, set the temperature to -48℃ and the vacuum degree to 0.09 MPa, freeze for 2.5 h, and then vacuum dry for 13 h to obtain the low-sugar bioactive peptide finished product.

[0017] Embodiment 2 The extraction method of the low-sugar food special bioactive peptide in this embodiment 2 is as follows: the mass fraction of each key component in the extraction process is: Low-sugar high-protein raw material: defatted low-sugar soybean protein isolate 100 parts, deionized water 500 parts, and composite directional enzyme system 1.0 part, neutral protease: glucoamylase: phytase = 3:2.5:1; The extraction method of the low-sugar food special bioactive peptide in this embodiment is the same as that in embodiment 1, and the defatted low-sugar soybean protein isolate is crushed and only passed through an 80-mesh standard sieve.

[0018] Embodiment 3: The extraction method of the low-sugar food special bioactive peptide in this embodiment 3 is as follows: the mass fraction of each key component in the extraction process is: Low-sugar high-protein raw material: defatted low-sugar soybean protein isolate 100 parts, deionized water 500 parts, and composite directional enzyme system 1.0 part, neutral protease: glucoamylase: phytase = 4:3:1.5; The extraction method of the low-sugar food special bioactive peptide in this embodiment is the same as that in embodiment 1.

[0019] Embodiment 4: The extraction method of the low-sugar food special bioactive peptide in this embodiment 4 is as follows: the mass fraction of each key component in the extraction process is: Low-sugar high-protein raw material: defatted low-sugar soybean protein isolate 100 parts, deionized water 500 parts, and composite directional enzyme system 1.0 part, neutral protease: glucoamylase: phytase = 3:2.5:1; The extraction method of the low-sugar food special bioactive peptide in this embodiment is the same as that in embodiment 1, and only the operating pressure of the nanofiltration membrane is adjusted to 0.35 MPa.

[0020] Comparative example 1: The extraction method of the low-sugar food special bioactive peptide in this comparative example 1 is as follows: the mass fraction of each key component in the extraction process is: Low-sugar high-protein raw material: defatted low-sugar soybean protein isolate 100 parts, deionized water 500 parts, and composite directional enzyme system 1.0 part, neutral protease: glucoamylase: phytase = 3:2.5:1; The extraction method of the low-sugar food special bioactive peptide in this comparative example is the same as that in embodiment 1, and only the raw material is crushed, but not subjected to classification screening.

[0021] Comparative example 2: The extraction method of the low-sugar food special bioactive peptide in this comparative example 2 is as follows: the mass fraction of each key component in the extraction process is: Low-sugar high-protein raw material: defatted low-sugar soybean protein isolate 100 parts, deionized water 500 parts, compound directional enzyme system: neutral protease 1.0 part; The extraction method of the low-sugar food special biological active peptide in the present comparative example is the same as that in Example 1, and only neutral protease single enzyme is used for enzymolysis.

[0022] Comparative Example 3: The extraction method of the low-sugar food special biological active peptide in the present comparative example 3 is as follows, and the mass fractions of various key components in the extraction process are as follows: Low-sugar high-protein raw material: defatted low-sugar soybean protein isolate 100 parts, deionized water 500 parts, compound directional enzyme system 1.0 part, neutral protease: saccharifying enzyme: phytase = 3:2.5:1; The extraction method of the low-sugar food special biological active peptide in the present comparative example is the same as that in Example 1, and only 0.1 μm microfiltration membrane is used in the separation stage without nanofiltration membrane fractionation.

[0023] Performance test 1. Sugar content determination (1) Sample pretreatment: accurately take 0.5 g of peptide product, put it in a 50 mL centrifuge tube, add 20 mL of ultrapure water, vortex for 10 min to completely dissolve; centrifuge at 4000 rpm for 15 min, take the supernatant and pass through a 0.45 μm organic filter membrane for determination; (2) Chromatographic conditions: the chromatographic column is an amino column (250 mm × 4.6 mm, 5 μm); the mobile phase is acetonitrile-ultrapure water with a volume ratio of 85:15; the flow rate is 1.0 mL / min; the column temperature is 30℃; the injection volume is 10 μL; and a differential refractive index detector is used for detection; (3) Standard curve drawing: prepare 0.1 g / L, 0.2 g / L, 0.5 g / L, 1.0 g / L and 2.0 g / L glucose standard solutions respectively, inject according to the above chromatographic conditions, and draw a standard curve with peak area as the vertical coordinate and concentration as the horizontal coordinate; (4) Sample determination and calculation: inject the sample solution to be determined, record the peak area, and calculate the sugar content by substituting the standard curve, and the result is expressed as "g / 100 g".

[0024] Table 1 Sugar content data table of different samples 2. Peptide molecular weight distribution determination (1) Sample pretreatment: accurately take 0.2 g of peptide product, dissolve with 0.1 mol / L NaNO3 solution and dilute to 100 mL, shake well and pass through a 0.45 μm filter membrane for determination; (2) Chromatographic conditions: GPC column (300 mm x 7.8 mm); mobile phase: 0.1 mol / L NaNO3 solution; flow rate: 0.8 mL / min; column temperature: 35°C; injection volume: 20 μL; UV detector was used, and the detection wavelength was 220 nm; (3) Standard curve preparation: 2 kDa, 3 kDa, and 5 kDa standard peptide mixtures were prepared by using 0.1 mol / L NaNO3 solution. After injection, the standard curve was plotted with the retention time as the abscissa and the logarithm of the molecular weight as the ordinate. (4) Sample determination and calculation: The sample solution was injected, and the peak area corresponding to each retention time was recorded. The molecular weight interval corresponding to each peak was determined according to the standard curve, and the peak area percentage of 2-5 kDa and 2-3 kDa peptide segments in the total peak area was calculated, i.e., the corresponding peptide content.

[0025] Table 2. Peptide molecular weight distribution determination data of different samples From the above table, it can be seen that: (1) The sugar content of the finished products of Examples 1-4 was ≤0.25 g / 100 g, which was much lower than that of Comparative Example 2 (2.50 g / 100 g) and the commercially available product (3.20 g / 100 g), proving that the "complex enzymatic hydrolysis-in situ desugaring" process can effectively decompose and remove the sugar components in the raw materials, solving the pain point of incomplete desugaring in traditional processes. (2) The 2-5 kDa peptide content of Examples 1-4 was ≥93.5%, and the 2-3 kDa high-activity peptide content was ≥85.6%, while the corresponding indicators of Comparative Example 2 and the commercially available active peptide were 10-20% lower, reflecting the precise retention effect of "charge-pore dual-effect separation" on target peptides.

[0026] 3. DPPH free radical scavenging rate determination 0.0197 g of DPPH reagent was weighed, dissolved in anhydrous ethanol, and diluted to 100 mL to prepare a 0.5 mmol / L DPPH solution, which was stored in the dark and refrigerated. 0.1 g of peptide product was accurately weighed, dissolved in ultrapure water, and diluted to 100 mL to prepare a 1 mg / mL sample solution. 2 mL of the sample solution was mixed with 2 mL of the DPPH solution, shaken well, and reacted in the dark for 30 min. The absorbance A1 was measured at a wavelength of 517 nm. At the same time, the absorbance A2 of 2 mL of the sample solution mixed with 2 mL of anhydrous ethanol and the absorbance A0 of 2 mL of the DPPH solution mixed with 2 mL of ultrapure water were measured. The calculation formula of the DPPH free radical scavenging rate is: Table 3. DPPH free radical scavenging rate data table of different samples 4. Alpha-glucosidase inhibition rate determination Accurately weigh 0.05 g of the peptide product, dissolve and dilute to 50 mL with the buffer solution matched with the kit to prepare a sample solution of 1 mg / mL; according to the kit instructions, sequentially add 50 μL of buffer solution, 20 μL of sample solution and 30 μL of alpha-glucosidase solution to the 96-well plate, incubate at 37°C for 15 min; then add 50 μL of substrate solution (p-nitrophenyl-alpha-D-glucopyranoside), continue to incubate at 37°C for 30 min; add 50 μL of stop solution to terminate the reaction, and measure the absorbance at 405 nm; at the same time, measure the absorbance of the blank group (buffer solution instead of sample solution) and the control group (buffer solution instead of enzyme solution), and calculate the alpha-glucosidase inhibition rate.

[0027] Table 4 Alpha-glucosidase inhibition rate determination results of different samples From the above table, it can be seen that: (1) The DPPH free radical scavenging rate of Examples 1-4 is ≥90.8%, which is increased by more than 15% compared with Comparative Example 2 and the commercially available product, because the high-activity peptide segment is retained intact; (2) The alpha-glucosidase inhibition rate of Examples 1-4 is ≥71.2%, which is significantly higher than that of Comparative Example 2 and the commercially available product, verifying the targeted functional role of the 2-3 kDa high-activity peptide.

[0028] 5. Process efficiency test (1) Enzymolysis time determination During the enzyme hydrolysis process, 2 mL of sample was taken every 15 min, 1 mL of 10% trichloroacetic acid solution was added to terminate the reaction, centrifuged at 4000 rpm for 10 min, and the supernatant was taken to determine the soluble nitrogen content by the Kjeldahl method; when the degree of hydrolysis reached 18%, the enzyme hydrolysis time at this time was recorded, which was the actual enzyme hydrolysis time.

[0029] (2) Retention recovery rate determination According to the "peptide molecular weight distribution determination" method, the mass concentration of 2-5 kDa peptides in the enzyme hydrolysate was detected, and the total mass was calculated combined with the volume of the enzyme hydrolysate; the mass concentration of 2-5 kDa peptides in the nanofiltration retentate was detected by the same method, and the total mass and the retention recovery rate were calculated combined with the volume of the retentate.

[0030] Table 5 Enzymolysis time and recovery rate data table of different samples From the above table, it can be seen that: (1) The enzymolysis time of Example 1 is only 1.5 h, which is shortened by 25-31% compared with Comparative Examples 1 and 2, proving that the "raw material grading screening + composite enzyme system" can improve the enzymolysis rate; (2) Total time consumption and recovery rate: the total time consumption of Examples 1-4 is ≤4.9 h, and the nanofiltration retention recovery rate is ≥93.5%, which reduces the loss of target peptides while ensuring efficiency, which is better than the "low efficiency and time consumption + peptide chain damage" problem of the traditional process.

[0031] 6. Storage stability test After crushing each peptide product, pass through an 80-mesh sieve, accurately weigh 5 g and place in a sealed aluminum foil bag, 3 parallel samples for each sample; store in a constant temperature and humidity chamber at 25°C and 60% relative humidity for 30 days; take samples at 0 days, 15 days and 30 days, respectively, and measure the water content (vacuum drying method, 105°C drying to constant weight) and DPPH radical scavenging rate, and calculate the 30-day DPPH retention rate.

[0032] Table 6 Water content and 30-day DPPH retention rate data table of different samples The 30-day water content change of Examples 1-4 is ≤0.4%, and the DPPH retention rate is ≥89.2%, while the activity retention rate of Comparative Example 2 and the commercial product is 10-12% lower, indicating that "dual-effect separation + freeze-drying" can reduce the oxidative degradation of peptide segments and improve storage stability.

[0033] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for extracting a bioactive peptide for a low-sugar food, characterized by, The key components in the extraction process are used in the following mass fraction ratio: 100 parts of low-sugar high-protein raw material, 400-600 parts of deionized water, 0.8-1.2 parts of composite directional enzyme system; the composite directional enzyme system is composed of neutral protease, saccharifying enzyme and phytase in a mass ratio of 3:2:1-4:3:2; the addition amount of 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution for adjusting pH during enzymolysis is 0.5-1.0% of the mass of the raw material slurry.

2. A method for extracting bioactive peptides specifically for low-sugar foods, the extraction method as described in claim 1, characterized in that, The method steps are as follows: S1, the low-sugar high-protein raw material is crushed to 80-100 mesh, and mixed with water in a mass ratio of 1:4-1:6 to obtain a raw material slurry; the low-sugar high-protein raw material is defatted low-sugar soybean protein isolate or low-lactose whey protein concentrate, wherein the protein content of the defatted low-sugar soybean protein isolate is 88-92%, and the sugar content is 0.5-1.0%, and the protein content of the low-lactose whey protein concentrate is 83-87%, and the sugar content is 1.5-2.0%; S2, add the composite directional enzyme system to the raw material slurry, and enzymolysis at a temperature of 35-40℃ and a pH of 6.5-7.0 for 1.0-2.0 h to obtain an enzymolysis liquid; the addition amount of the composite directional enzyme system is 0.8-1.2% of the mass of the low-sugar high-protein raw material; S3, sequentially separating the enzymatic hydrolysate through a 0.1-micron microfiltration membrane and a 1-kDa charge-modified nanofiltration membrane, and collecting the retentate of the 1-kDa charge-modified nanofiltration membrane; wherein the operating temperature of the 0.1-micron microfiltration membrane is 20-25 DEG C, and the operating pressure is 0.1-0.2 MPa; the 1-kDa charge-modified nanofiltration membrane is a polyethersulfone nanofiltration membrane with sulfonic acid groups grafted on the surface, and the sulfonic acid group loading is 0.8-2.0 micromol / cm 2 , the operating temperature is 20-25 DEG C, and the operating pressure is 0.3-0.5 MPa; S4, freeze-drying the cut-off liquid under the conditions of-45 to-50℃ and a vacuum degree of 0.08-0.10 MPa to obtain a low-sugar food special biological active peptide; the sugar content of the low-sugar food special biological active peptide is 0.2-0.3 g / 100 g, the proportion of biological active peptide with a molecular weight of 2-5 kDa is 90-95%, and the high-activity peptide with a molecular weight of 2-3 kDa accounts for 80-85% of the biological active peptide with a molecular weight of 2-5 kDa.

3. The method of claim 2, wherein, The low-sugar high-protein raw material is selected from one or more of defatted low-sugar soybean protein isolate, low-lactose whey protein concentrate and pea protein.

4. The method of claim 2, wherein, The crushed raw material is passed through a standard sieve, and the undersize is used to prepare the raw material slurry; when the raw material is mixed with water, mechanical stirring is used at a stirring rate of 200-300 rpm, and the stirring time is 15-20 min.

5. The method of claim 2, wherein, The enzymolysis process uses a constant-temperature water bath shaking device with a shaking rate of 150-200 rpm; during the enzymolysis process, the pH is maintained stable by adding 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution, and the pH fluctuation range is controlled within ±0.

2.

6. The method of claim 2, wherein, The 0.1 μm microfiltration membrane is a hydrophilic polyvinylidene fluoride microfiltration membrane, which is soaked in 0.1 mol / L sodium hydroxide solution for 30 min before use, and then washed with deionized water until neutral; The 1 kDa charge-modified nanofiltration membrane is soaked in deionized water at the operating pressure for 20-30 min before use to remove the residual protective agent on the surface of the membrane.

7. The method of claim 2, wherein, The cut-off liquid is placed at 4 DEG C for 1-2 h before freeze-drying, and the surface scum is removed; during the freeze-drying process, the cut-off liquid is cooled to-45 DEG C to-50 DEG C and frozen for 2-3 h, and then vacuum drying is carried out, and the drying time is 12-15 h, so as to ensure that the moisture content of the finished product is 1-3%.

8. The method of claim 2, wherein, The enzyme activity of the neutral protease is 50000-80000 U / g, the enzyme activity of the glucoamylase is 100000-150000 U / g, and the enzyme activity of the phytase is 5000-10000 U / g.

9. The method of claim 2, wherein, The DPPH free radical scavenging rate of the low-sugar food special biological active peptide is 85-95%, and the alpha-glucosidase inhibition rate is 65-75%.

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