Process for preparing active polypeptide for promoting calcium absorption through multi-enzyme synergistic directional enzymolysis of egg yolk protein

Abstract

A process for preparing active polypeptide for promoting calcium absorption through multi-enzyme synergistic directional enzymolysis of egg yolk protein comprises the following steps: (1) raw material pretreatment: adopting degreased-lecithin-removed egg yolk powder as a raw material, and eliminating steric hindrance of lipid to enzymolysis; (2) double-enzyme synergistic enzymolysis: compounding neutral protease and trypsin according to a mass ratio of 2: (0.9-1.1), and carrying out synchronous enzymolysis for 2.5-3.5 hours under the conditions that the total enzyme amount is 2.25% (w / w), the temperature is 53-63 DEG C and the pH is 6.5-7.5; (3) separation: filtering the enzymatic hydrolysate through a plate frame, controlling the aperture to be 0.22-0.45 mu m, the gradient pressure to be 0.2-0.4 MPa and the temperature to be 55-60 DEG C, and then separating the filtrate through a 5kDa ultrafiltration membrane; and (4) concentrating and drying: concentrating the filtrate at low temperature until the solid content is 25-30%, and then performing spray drying to obtain yolk polypeptide powder with the polypeptide molecular weight being less than 5kDa and the proportion being more than 90%. The method has the technical effects of efficient directional enzymolysis, high product purity and strong calcium absorption promoting activity, and the egg yolk polypeptide powder with the polypeptide molecular weight being less than 5kDa and the proportion being more than 90% when the polypeptide molecular weight is more than 100Da is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food processing, and specifically relates to a process for preparing calcium absorption promoting active polypeptides by multi-enzyme synergistic complex directional enzymolysis of egg yolk protein, which is suitable for the development of functional food and calcium nutritional supplements. BACKGROUND

[0002] Traditional technologies generally use single protease to hydrolyze egg yolk protein to prepare calcium binding peptides, which has the problems of single enzyme cutting site, low peptide yield, product molecular weight dispersion and the like, which brings troubles such as insufficient functional activity and low industrialization efficiency to application.

[0003] In the existing published patent literature, patent application 1 with the application number 202410700733.X and the name of egg yolk peptide for promoting calcium absorption and its preparation method and application adopts the scheme of extracting egg yolk peptide from fertilized chicken eggs after hatching, but it has the following shortcomings or problems: first, although this scheme improves the utilization rate of egg yolk protein to a certain extent and prepares egg yolk peptides with calcium chelation activity, it relies on chicken embryo eggs at a specific hatching stage as raw materials, and the process is complex and has strict requirements on hatching days, storage conditions and the like, resulting in high cost; second, although the use of ethanol and ethyl acetate mixture and the step of ultrafiltration separation of small molecular components in the defatting and extraction process can effectively enrich the target peptide segment, the operation is complicated and time-consuming, especially in large-scale production scenarios, which may face the problems of high equipment investment and long operation cycle; finally, this method does not explicitly solve the problem of high loss rate of active fragments caused by secondary processing in the traditional enzymolysis process, and the content of bitter polypeptides in the product still cannot be effectively controlled. Therefore, this technical scheme may be limited by the balance problem between cost and efficiency in actual promotion.

[0004] Patent application 2 with application number 201710004653.0 and title of a kind of electron beam irradiation combined with enzyme method separates and purifies high affinity calcium of wheat germ polypeptide adopts electron beam irradiation modification combined with alkaline protease hydrolysis method, but it has the following shortcomings or problems: on the one hand, the protein structure change induced by electron beam irradiation can indeed improve the enzymolysis efficiency and generate a large number of functional peptides, but the electron beam irradiation technology itself needs expensive special equipment and strict radiation safety protection measures, which makes it difficult to realize low-cost large-scale commercial production; on the other hand, although the method uses various separation and purification means (such as ultrafiltration, anion exchange chromatography, gel filtration chromatography and reverse phase high performance liquid chromatography), high affinity calcium of wheat germ polypeptide is successfully obtained, but these complex separation steps not only increase the process difficulty, but also significantly prolong the overall processing time, further increasing the industrialization cost. In addition, due to the optimization of multiple gradient elution conditions in the separation process, batch consistency problems may occur in actual operation, thereby affecting the product quality stability. At the same time, the scheme is mainly aimed at wheat germ protein rather than egg yolk protein, the source of raw materials is narrow, which limits its applicability.

[0005] In summary, the above two existing technologies have not completely overcome the key defects in the traditional enzymolysis process, such as high loss rate of active fragments, prominent bitter taste of products, long process cycle and high industrialization cost. Therefore, developing a directional enzymolysis process that can not only ensure high retention rate of active peptide segments but also significantly reduce production cost is still a technical bottleneck that needs to be broken through in the field of functional foods. SUMMARY

[0006] The present application aims to provide a process for preparing calcium absorption promoting active polypeptide by multi-enzyme synergistic directional enzymolysis of egg yolk protein, which breaks through the bottleneck of low enzymolysis efficiency and instability of active peptides in traditional process through triple technology coupling of defatting pretreatment-double enzyme synchronous enzymolysis-separation, realizes directional enrichment of small molecule calcium absorption promoting peptides and long-term activity maintenance.

[0007] In order to achieve the above purpose, the following scheme is adopted: a process for preparing calcium absorption promoting active polypeptide by multi-enzyme synergistic directional enzymolysis of egg yolk protein, comprising the following steps: (1) raw material pretreatment: defatted-ovophospholipid egg yolk powder is used as raw material to eliminate the steric hindrance of lipids to enzymolysis; (2) double enzyme synergistic enzymolysis: neutral protease and trypsin are compounded according to mass ratio 2: (0.9-1.1), synchronous enzymolysis is carried out under the conditions of total enzyme amount 2-2.5% (w / w), temperature 53-63℃, pH 6.5-7.5 for 2.5-3.5 hours; (3) Separation: the enzymatic solution is filtered by a plate and frame filter, with a pore size of 0.22-0.45 μm and a gradient pressure of 0.2-0.4 MPa, and the temperature is maintained at 55-60°C, and then the filtrate is separated by a 5 kDa ultrafiltration membrane; (4) Concentration and drying: the separated filtrate is concentrated by a single-effect concentrator to a solid content of 25-30%, and then spray dried to obtain egg yolk polypeptide powder with a molecular weight of <5 kDa and a proportion of >90%.

[0008] As a preferred, in the raw material pretreatment, the low-temperature continuous phase change extraction method is used, the whole egg yolk powder is subjected to butane+ethanol continuous phase change extraction process, the oil and lecithin are rapidly separated at low temperature, and high-purity egg white is obtained, with a protein content of ≥80%, a fat content of <1%, and a lecithin content of <1%.

[0009] As a preferred, in the double-enzyme synergistic enzymolysis, the mass ratio of the neutral protease to the trypsin is 2:1.

[0010] As a preferred, in the double-enzyme synergistic enzymolysis, the enzymolysis temperature is 58°C, the pH value is 7, and the enzymolysis time is 3 hours.

[0011] As a preferred, in the separation, the filter cloth has a pore size range of 0.33 μm, and the pressure range during filtration is 0.13-0.38 MPa.

[0012] As a preferred, in the separation, the pressure during filtration is initially 0.13-0.15 MPa, in the main stage 0.23-0.25 MPa, and in the final stage 0.35-0.38 MPa, and the liquid temperature control range is 55-60°C.

[0013] As a preferred, in the concentration and drying, the polypeptide after ultrafiltration membrane separation has a molecular weight range of <5 kDa and >100 Da, and a proportion of >90% in the total protein.

[0014] As a preferred, in the concentration and drying, the liquid solid content after concentration by the single-effect concentrator is 27-28%.

[0015] As a preferred, in the concentration and drying, the inlet air temperature of spray drying is 180°C, and the outlet air temperature is 90-100°C.

[0016] Technical effects: 1) The present application realizes efficient preparation of high-activity, small-molecule calcium absorption promoting peptides by optimizing key technologies such as raw material pretreatment, double-enzyme synergistic enzymolysis, separation and spray drying, and has higher industrialization efficiency and economy. Compared with traditional single-enzyme hydrolysis process and electron beam irradiation combined with enzymolysis process, the production cost and process complexity are greatly reduced, not only the production efficiency is improved, but also the energy consumption and labor cost are reduced, further enhancing the industrialization feasibility of the present application.

[0017] 2) The present application solves the problem of high loss rate of target functional fragments in traditional process (loss rate of existing technology > 30%), and retains key active peptide segments through double-enzyme synergistic directional enzymolysis, while significantly improves the peptide yield to more than 90%, which is better than traditional single-enzyme hydrolysis process (peptide yield < 20%).

[0018] 3) The small-molecule polypeptide (<5kDa accounting for >90%) obtained by the present application has better calcium binding capacity and bioavailability, can effectively improve the calcium transport efficiency of Caco-2 cells, and meets the demand of high-efficiency calcium nutritional supplement, which makes up for the problem of molecular weight dispersion and insufficient function of traditional process products. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a calcium chelation activity comparison chart of the samples of examples 1-3 and comparative examples 1-5 of the present application; Figure 2 is a sensory evaluation score comparison chart of the samples of examples 1-3 and comparative examples 1-5 of the present application. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0021] Example 1: The present application provides a process for preparing calcium absorption promoting active polypeptide from egg yolk protein by multi-enzyme synergistic directional enzymolysis, comprising the following steps: (1) Raw material pretreatment: defatted-destripped lecithin egg yolk powder is used as raw material to eliminate the steric hindrance of lipids to enzymolysis; low-temperature continuous phase change extraction method is used, and full-fat egg yolk powder is subjected to butane+ethanol continuous phase change extraction process to realize low-temperature rapid separation of oil and lecithin, and high-purity egg yolk protein is obtained, with protein content ≥80%, fat content <1% and lecithin content <1%.

[0022] (2) Dual-enzyme synergistic enzymolysis: neutral protease and trypsin are compounded in a mass ratio of 2:0.9, and synchronous enzymolysis is carried out under the conditions of a total enzyme amount of 2.5% (w / w), a temperature of 53°C, and pH 6.5 for 2.5 hours; the solid-liquid ratio is 1:10.

[0023] (3) Separation: the enzymolysis liquid is subjected to plate and frame filtration, the pore size is controlled to be 0.22 μm, the pressure range during filtration is 0.1 MPa initially→0.2 MPa in the main stage→0.35 MPa in the end stage, the liquid temperature is maintained at 55°C, and then the filtrate is separated by a 5 kDa ultrafiltration membrane.

[0024] (4) Concentration and drying: low-temperature concentration is carried out by using a single-effect concentrator until the solid content is 25%, spray drying (the inlet air temperature is 180°C, and the outlet air temperature is 90°C) is carried out, and egg yolk polypeptide powder with a polypeptide molecular weight of <5 kDa and >100 Da accounting for >90% is obtained.

[0025] Example 2 The application provides a process for preparing calcium absorption promoting active polypeptides by multi-enzyme synergistic directional enzymolysis of egg yolk protein, which comprises the following steps: (1) Raw material pretreatment: defatted-ovophospholipid egg yolk powder is used as the raw material to eliminate the steric hindrance of lipids to enzymolysis; low-temperature continuous phase change extraction is used, full-fat egg yolk powder is subjected to butane+ethanol continuous phase change extraction process, and low-temperature rapid separation of oil and ovophospholipids is realized, so that high-purity egg yolk protein is obtained, the protein content of which is ≥80%, the fat content is <1%, and the ovophospholipid content is <1%.

[0026] (2) Dual-enzyme synergistic enzymolysis: neutral protease and trypsin are compounded in a mass ratio of 2:1.0, and synchronous enzymolysis is carried out under the conditions of a total enzyme amount of 2.25% (w / w), a temperature of 58°C, and pH 7.0 for 3.0 hours; the solid-liquid ratio is 1:9.5.

[0027] (3) Separation: the enzymolysis liquid is subjected to plate and frame filtration, the pore size is controlled to be 0.33 μm, the pressure range during filtration is 0.13 MPa initially→0.23 MPa in the main stage→0.38 MPa in the end stage, the liquid temperature is maintained at 58°C, and then the filtrate is separated by a 5 kDa ultrafiltration membrane.

[0028] (4) Concentration and drying: after the filtrate is separated by the 5 kDa ultrafiltration membrane, low-temperature concentration is carried out by using a single-effect concentrator until the solid content is 27.5%, spray drying (the inlet air temperature is 180°C, and the outlet air temperature is 95°C) is carried out, and egg yolk polypeptide powder with a polypeptide molecular weight of <5 kDa and >100 Da accounting for >90% is obtained.

[0029] Example 3 The application provides a process for preparing calcium absorption promoting active polypeptides by multi-enzyme synergistic directional enzymolysis of egg yolk protein, which comprises the following steps: (1) Raw material pretreatment: Defatted-De-egg lecithin yolk powder is used as raw material to eliminate the steric hindrance of lipids to enzymolysis; low-temperature continuous phase change extraction method is used, full-fat yolk powder is subjected to butane+ethanol continuous variable phase extraction process to realize low-temperature rapid separation of oil and lecithin, and high-purity egg yolk protein is obtained, the protein content of which is ≥80%, the fat content is <1%, and the lecithin content is <1%.

[0030] (2) Double-enzyme synergistic enzymolysis: neutral protease and trypsin are compounded according to a mass ratio of 2:1.1, and are synchronously enzymolyzed under the conditions of a total enzyme amount of 2.1% (w / w), a temperature of 63°C, a pH of 7.5, and a material-liquid ratio of 1:11 for 3.5 hours.

[0031] (3) Separation: the enzymolysis liquid is subjected to plate and frame filtration, the pore size is controlled to be 0.45 μm, the pressure range during filtration is 0.15 MPa initially→0.25 MPa in the main stage→0.4 MPa at the end, and the liquid temperature is maintained at 60°C, and then the filtrate is separated by a 5 kDa ultrafiltration membrane.

[0032] (4) Concentration and drying: after the filtrate is separated by the 5 kDa ultrafiltration membrane, low-temperature concentration is carried out by using a single-effect concentrator to a solid content of 30%, spray drying (the inlet air temperature is 180°C, and the outlet air temperature is 100°C) is carried out, and egg yolk polypeptide powder with a polypeptide molecular weight of <5 kDa and >100 Da accounting for >90% is obtained.

[0033] Comparative example: Comparative example 1: pepsin: pH 1.5; temperature 58°C; enzyme-substrate ratio 1%; time 3h The raw material is defatted-de-egg lecithin yolk powder (the protein content is ≥80%, the fat content is <1%, and the lecithin content is <1%); pepsin is added for enzymolysis, the enzyme amount is 1% (w / w), the temperature is 58°C, the pH is 1.5, and synchronous enzymolysis is carried out for 3h; after the filtrate is separated by a 5 kDa ultrafiltration membrane, the concentration and drying process is carried out according to the method of step 4 in the embodiment 1.

[0034] Comparative example 2: trypsin: pH 7.5; temperature 58°C; enzyme-substrate ratio 1%; time 3h The raw material is defatted-de-egg lecithin yolk powder (the protein content is ≥80%, the fat content is <1%, and the lecithin content is <1%); trypsin is added for enzymolysis, the enzyme amount is 1% (w / w), the temperature is 58°C, the pH is 7.5, and synchronous enzymolysis is carried out for 3h; after the filtrate is separated by a 5 kDa ultrafiltration membrane, egg yolk polypeptide powder is obtained by freeze-drying.

[0035] Comparative example 3: neutral protease: pH 7.0; temperature 58°C; enzyme-substrate ratio 1%; time 3h Raw material selection defatted-destripped lecithin egg yolk powder (protein content ≥80%, fat content <1%, lecithin content <1%); adding neutral protease enzyme hydrolysis, under the conditions of enzyme amount 1% (w / w), temperature 58°C, pH 7.0, synchronous enzyme hydrolysis for 3h; the filtrate was separated by 5kDa ultrafiltration membrane, and the concentration and drying process was according to the method of step 4 in example 2.

[0036] Comparative example 4: alkaline protease: pH 8.0; temperature 58°C; enzyme substrate ratio 1%; time 3h Raw material selection defatted-destripped lecithin egg yolk powder (protein content ≥80%, fat content <1%, lecithin content <1%); adding alkaline protease enzyme hydrolysis, under the conditions of enzyme amount 1% (w / w), temperature 58°C, pH 8.0, synchronous enzyme hydrolysis for 3h; the filtrate was separated by 5kDa ultrafiltration membrane, and the concentration and drying process was according to the method of step 4 in example 3.

[0037] Comparative example 5: papain: pH 6.5; temperature 58°C; enzyme substrate ratio 1%; time 3h Raw material selection defatted-destripped lecithin egg yolk powder (protein content ≥80%, fat content <1%, lecithin content <1%); adding papain enzyme hydrolysis, under the conditions of enzyme amount 1% (w / w), temperature 58°C, pH 6.5, synchronous enzyme hydrolysis for 3h; the filtrate was separated by 5kDa ultrafiltration membrane, and the freeze-drying obtained egg yolk polypeptide powder.

[0038] Comparative example 6: neutral protease + papain, complex enzyme hydrolysis; Raw material selection defatted-destripped lecithin egg yolk powder (protein content ≥80%, fat content <1%, lecithin content <1%); adding neutral protease + papain enzyme hydrolysis, control enzyme hydrolysis time 3h, neutral protease addition amount 1.5%, papain addition amount 0.75%, enzyme hydrolysis temperature 58°C, solid-liquid ratio 1:10, pH 7.0; the filtrate was separated by 5kDa ultrafiltration membrane, and the freeze-drying obtained egg yolk polypeptide powder.

[0039] Comparative example 7: neutral protease + pepsin; twice enzyme hydrolysis (separately added); Raw material selection defatted-destripped lecithin egg yolk powder (protein content ≥80%, fat content <1%, lecithin content <1%); due to the different optimum pH of enzyme hydrolysis, neutral protease and pepsin were used for enzyme hydrolysis respectively, once enzyme hydrolysis control enzyme hydrolysis time 2.5h, neutral protease addition amount 1.5%, enzyme hydrolysis temperature 58°C, solid-liquid ratio 1:10, pH 7.0; twice enzyme hydrolysis control enzyme hydrolysis time 2.5h, pepsin addition amount 0.75%, enzyme hydrolysis temperature 58°C, solid-liquid ratio 1:10, pH control at 1.2; the filtrate was separated by 5kDa ultrafiltration membrane, and the concentration and drying process was according to the method of step 4 in example 1.

[0040] Comparative Example 8: Neutral protease + pepsin, complex enzymatic hydrolysis Raw material selection defatted-destripped lecithin egg yolk powder (protein content ≥80%, fat content <1%, lecithin content <1%); while adding neutral protease + pepsin enzymatic hydrolysis, control enzymatic hydrolysis time 3 h, neutral protease addition amount 1.5%, pepsin addition amount 0.75%, enzymatic hydrolysis temperature 58°C, solid-liquid ratio 1:10, pH 7.0; the filtrate was separated by 5kDa ultrafiltration membrane, and the concentration and drying process was according to the method of step 4 described in Example 3.

[0041] Comparative Example 9: Trypsin + pepsin, complex enzymatic hydrolysis Raw material selection defatted-destripped lecithin egg yolk powder (protein content ≥80%, fat content <1%, lecithin content <1%); while adding trypsin + pepsin enzymatic hydrolysis, control enzymatic hydrolysis time 3 h, trypsin addition amount 1.5%, pepsin addition amount 0.75%, enzymatic hydrolysis temperature 58°C, solid-liquid ratio 1:10, pH 7.0; the filtrate was separated by 5kDa ultrafiltration membrane, and the concentration and drying process was according to the method of step 4 described in Example 2.

[0042] Comparative Example 10: Trypsin + pepsin, two-step enzymatic hydrolysis (separately added) Raw material selection defatted-destripped lecithin egg yolk powder (protein content ≥80%, fat content <1%, lecithin content <1%); due to the different optimum pH of enzymatic hydrolysis, neutral protease and pepsin were used for enzymatic hydrolysis respectively, one-step enzymatic hydrolysis control enzymatic hydrolysis time 2.5h, trypsin addition amount 1.5%, enzymatic hydrolysis temperature 58°C, solid-liquid ratio 1:10, pH 7.8; two-step enzymatic hydrolysis control enzymatic hydrolysis time 2.5h, pepsin addition amount 0.75%, enzymatic hydrolysis temperature 58°C, solid-liquid ratio 1:10, pH control at 1.2; the filtrate was separated by 5kDa ultrafiltration membrane, and the concentration and drying process was according to the method of step 4 described in Example 1.

[0043] Test Example: (1) Experimental group: Examples 1, 2, 3; Control group: Comparative Examples 1-5, Comparative Examples 6-11; other reagents are all analytical pure.

[0044] (2) Experimental instruments Magnetic stirring water bath: SN-HWS-2DJ; pH meter: PHS-3C; centrifuge: 3-5N; drying oven: XMTA-7000-L; electronic balance scale: ME204; freeze dryer; multifunctional enzyme marker: model 2300, Perkin Elmer (3) Detection method 3.1 Determination of molecular weight of hydrolyzed egg yolk powder Sample pretreatment: 50 mg sample was weighed and dissolved in distilled water to 2 mg / ml, and filtered using a 0.22 μm polyether sulfone filter. Liquid phase conditions: mobile phase: water, 45% acetonitrile, 0.01% TFA; wavelength: 220 nm; detector: SPD-M400; column type: TSKgel G2000SWxL column (7.8 mm l.D. x 30 cm, 5 μm); column temperature: 25 °C; procedure: gradient elution, 40 min; injection volume: 20 μL. Standard curve was drawn; the protein standard selected for drawing the standard curve was: cytochrome c, aprotinin, bacitracin, ethionine-ethionine-tyrosine-arginine, ethionine-ethionine-ethionine. The standard curve obtained was: Y = -0.2292x + 6.809 (R2= 0.9971), where x is the running time and y is the log10 (relative molecular mass).

[0045] 3.2 Evaluation of in vitro activity - calcium chelating activity Take 1 mL of 200 mg / mL enzyme hydrolysate supernatant, 1 mL of 100 mg / mL CaCl2and 2 mL of 0.1 M phosphate buffer solution (pH = 7.8) in a 10 mL centrifuge tube, react in a 37 °C water bath for 10 min, centrifuge at 4000 r / min for 10 min. Take the supernatant and dilute 100 times, take 50 μL to a 96-well plate, then add 150 μL of calcium color developing solution and react, then measure at 575 nm. Measure the calcium content of the supernatant after reaction, which is the chelated calcium content of the hydrolysate. The higher the calcium content, the better the calcium chelating activity of the hydrolysate.

[0046] 3.3 Determination of the degree of hydrolysis of egg yolk powder OPA reaction was used to determine the degree of hydrolysis. Each peptide bond hydrolyzed releases a free amine group. The free amine group reacts with OPA (o-phthaldehyde) to form a yellow complex. The absorbance at 340 nm can be measured using a spectrophotometer. 0.100 g of enzyme hydrolysate was dissolved in 100 mL, filtered, 500 μL of the filtrate was taken, 3 mL of OPA reagent was added, and the reaction was allowed to proceed for 2 min, using distilled water as the reference to measure A340 nm. The determination was repeated 5 times under the same conditions.

[0047] Where h is the number of hydrolyzed peptide bonds, mmol / g; htot is the total number of peptide bonds, mmol / g, egg white protein is 8.2 mmol / g; Cserine is the serine amino millimolar in the sample solution, mmol / L; V is the sample dissolution constant volume, L; N is the dilution multiple of the sample solution; m is the sample mass, g; ω is the protein content in the sample, %; β is the constant, 0.4 for egg white protein; α is the constant, 1 for egg white protein.

[0048] 3.4 Yield calculation The yield of the enzymatic hydrolysate prepared from the de-ovoleithin egg yolk powder was calculated according to the following formula: In the formula, m1 is the mass of the enzymatic hydrolysate supernatant; m2 is the mass of the de-ovoleithin egg yolk powder; A is the solid content of the supernatant; and B is the protein content. , In the formula, m1 is the mass of the enzymatic hydrolysate supernatant; m2 is the mass of the de-ovoleithin egg yolk powder; A is the solid content of the supernatant; and B is the protein content.

[0049] Table 1 Performance indicators of Examples 1-3 and Comparative Examples 1-5

[0050] Proteases have specificity and specificity. One protease can only recognize and cut one kind of peptide bond with a specific amino acid sequence, thereby affecting the properties of the enzymatic hydrolysate.

[0051] According to the data in Table 1 and Table 2, the calcium chelating activity of the enzymatic hydrolysates of Examples 1-3 was significantly higher than that of Comparative Examples 1-5, and the calcium chelating activity was ranked as follows: Example 2 > Example 3 > Example 1 > neutral protease (Comparative Example 3) > trypsin (Comparative Example 2) > alkaline protease (Comparative Example 4) > papain (Comparative Example 5) > pepsin (Comparative Example 1). Figure 1 According to the data in Table 1 and Table 2, the calcium chelating activity of the enzymatic hydrolysates of Examples 1-3 was significantly higher than that of Comparative Examples 1-5, and the calcium chelating activity was ranked as follows: Example 2 > Example 3 > Example 1 > neutral protease (Comparative Example 3) > trypsin (Comparative Example 2) > alkaline protease (Comparative Example 4) > papain (Comparative Example 5) > pepsin (Comparative Example 1).

[0052] Figure 2 According to the data in Table 1 and Table 2, the calcium chelating activity of the enzymatic hydrolysates of Examples 1-3 was significantly higher than that of Comparative Examples 1-5, and the calcium chelating activity was ranked as follows: Example 2 > Example 3 > Example 1 > neutral protease (Comparative Example 3) > trypsin (Comparative Example 2) > alkaline protease (Comparative Example 4) > papain (Comparative Example 5) > pepsin (Comparative Example 1).

[0053] In the yield, enzymatic hydrolysate molecular weight of 100-5000 Da relative content indicators, still in the effect of example 1, example 2, example 3 is good.

[0054] Table 2 Performance indicators of Comparative Examples 6-10

[0055] ​It can be further known from Table 2 that the indexes of Examples 1, 2 and 3 are all higher than those of Comparative Examples 6-10. The analysis reason is that the double-enzyme synergistic enzymolysis process breaks through the bottleneck of limited single enzymolysis enzyme cutting site, significantly improves the degree of hydrolysis and the yield of peptides, at the same time avoids the damage of secondary enzymolysis to active peptide segments, reduces the risk of bitter polypeptide exposure; compared with the multi-stage separation process, the simplified low-pressure membrane separation and ultrafiltration separation technology significantly shortens the process cycle, reduces the equipment investment and operation cost, and is more suitable for large-scale production; under the action of the composite enzyme and the primary enzymolysis process of the application, the application realizes the precise control of the molecular weight of polypeptides (the proportion of <5000 Da is >90%), ensures the high calcium binding capacity and the calcium absorption promoting effect of the product, and the spatial steric hindrance of the short peptide chain is smaller, so there are more calcium binding sites exposed, which is easier to coordinate with calcium ions, so as to chelate more calcium ions. It is significantly better than the performance of the product with dispersed molecular weight (most >3000 Da) in the traditional process. Moreover, it is found in the experiment that the process efficiency of the application is very high, and the process cycle of the prior art can be shortened to 3-4 hours.

Claims

1. A process for the preparation of a calcium absorption promoting active polypeptide by multi-enzyme synergistic directed enzymatic hydrolysis of egg yolk protein characterized by: Comprise the following steps: (1) raw material pretreatment: using defatted-eliminating lecithin egg yolk powder as raw material, eliminating the steric hindrance of lipid to enzymolysis; (2) double enzyme synergistic enzymolysis: neutral protease and trypsin are compounded according to the mass ratio of 2: (0.9-1.1), and the synchronous enzymolysis is carried out under the conditions of total enzyme amount 2-2.5% (w / w), temperature 53-63 DEG C, pH 6.5-7.5 for 2.5-3.5 hours; (3) separation: the enzymolysis liquid is filtered by plate frame, the aperture is controlled to be 0.22-0.45 μm, the gradient pressure is 0.2-0.4 MPa, and the temperature is maintained at 55-60 DEG C, then the filtrate is separated by 5kDa ultrafiltration membrane; (4) concentration and drying: the filtrate after separation is concentrated by a single-effect concentrator to a solid content of 25-30% at low temperature, and then spray dried to obtain egg yolk polypeptide powder with a polypeptide molecular weight of <5kDa accounting for >90%.

2. The process for the preparation of calcium absorption promoting active polypeptides by multi-enzyme synergistic directed zymolysis of egg yolk proteins according to claim 1, characterized in that: In the raw material pretreatment, the low-temperature continuous phase change extraction method is used, the whole fat egg yolk powder is subjected to butane+ethanol continuous phase change extraction process, the oil and lecithin are rapidly separated at low temperature, and the high-purity egg yolk protein is obtained, the protein content is ≥80%, the fat content is <1%, and the lecithin content is <1%.

3. The process for the preparation of calcium absorption promoting active polypeptides by multi-enzyme synergistic directed zymolysis of egg yolk proteins according to claim 1, characterized in that: In the double enzyme synergistic enzymolysis, the mass ratio of neutral protease to trypsin is 2:

1.

4. The process for the preparation of calcium absorption promoting active polypeptides by multi-enzyme synergistic directed zymolysis of egg yolk proteins according to claim 1, characterized in that: In the double enzyme synergistic enzymolysis, the enzymolysis temperature is 58 DEG C, the pH value is 7, and the enzymolysis time is 3 hours.

5. The process for the preparation of calcium absorption promoting active polypeptides by synergistically directed zymolysis of egg yolk proteins according to claim 1, characterized in that: In the separation, the filter cloth has a pore size of 0.33 μm, and the pressure during filtration is 0.13-0.38 MPa.

6. The process for the preparation of calcium absorption promoting active polypeptides by multi-enzyme synergistic directed zymolysis of egg yolk proteins as claimed in claim 5, wherein: In the separation, the pressure during filtration is initially 0.13-0.15 MPa, in the main stage 0.23-0.25 MPa, in the end stage 0.35-0.38 MPa, and the liquid temperature is 55-60 DEG C.

7. The process for the preparation of calcium absorption promoting active polypeptides by synergistically directed zymolysis of egg yolk proteins according to claim 1, characterized in that: In the concentration and drying, the polypeptide after ultrafiltration membrane separation has a molecular weight of <5kDa and >100Da, and the proportion in total protein is >90%.

8. The process for the preparation of calcium absorption promoting active polypeptides by synergistically directed zymolysis of egg yolk proteins according to claim 1, characterized in that: In the concentration and drying, the liquid solid content after single-effect concentrator concentration is 27-28%.

9. The process for the preparation of calcium absorption promoting active polypeptides by synergistically directed zymolysis of egg yolk proteins according to claim 1, characterized in that: In the concentration and drying, the inlet air temperature of spray drying is 180 DEG C, and the outlet air temperature is 90-100 DEG C.

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