A method for processing yolk peptides rich in glycerylphosphorylcholine

Abstract

The application discloses a processing method of yolk peptide rich in glycerophosphorylcholine, and comprises the following steps: S1, protease treatment: mixing yolk powder with water, adjusting pH to 7-10, adding protease, and carrying out enzymolysis at a temperature of 45-55 DEG C for 4-8 h to prepare yolk enzymolysis liquid 1; S2, phospholipase treatment: adjusting the pH of the yolk enzymolysis liquid 1 obtained in step S1 to 5.5-7.5, adding phospholipase B, and stirring and enzymolyzing at 50-60 DEG C for 2-4 h to obtain yolk enzymolysis liquid 2; S3, flash evaporation treatment: sequentially carrying out flash evaporation, separation, concentration, high-temperature sterilization and spray drying treatment on the yolk enzymolysis liquid 2 obtained in step S2 to prepare the yolk peptide rich in glycerophosphorylcholine. The application can solve the problems of insufficient comprehensive utilization rate of yolk resources and low product added value, and the method is simple in operation and easy to realize industrialization.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food biotechnology, in particular to a processing method of egg yolk peptide rich in glycerophosphorylcholine. BACKGROUND

[0002] Glycerophosphorylcholine is a chemical substance with important biological functions, which has a significant impact on the health of the brain and nervous system. It has been widely used in nutritional supplements and clinical treatment fields, and shows broad application prospects. The preparation methods of glycerophosphorylcholine mainly include phospholipid hydrolysis method and chemical synthesis method. The chemical synthesis method has complex steps, and the purification process will cause a large amount of wastewater, and toxic and harmful synthesis precursors are easily left. The traditional phospholipid hydrolysis method usually extracts crude glycerophosphorylcholine containing phosphatidylcholine from egg yolk or other phospholipid-rich raw materials, and realizes the preparation of glycerophosphorylcholine through enzyme catalysis. This method needs to extract phospholipids first, which is complex and not conducive to the industrial application of the method.

[0003] CN118203092A, a preparation method of glycerophosphorylcholine-rich deoiled egg yolk powder, discloses a method of using fresh poultry egg yolk dilution liquid centrifugal supernatant rich in phosphatidylcholine as raw material, and obtaining a reaction liquid rich in glycerophosphorylcholine through phospholipase complex enzymolysis, and then obtaining the target product through spray drying and supercritical CO2 extraction. The invention uses egg yolk dilution centrifugal supernatant as raw material, and obtains glycerophosphorylcholine-rich deoiled egg yolk powder through the synergistic treatment method of phospholipase and supercritical CO2, which has the advantage of high glycerophosphorylcholine content. However, there is still the problem of low solubility of deoiled egg yolk powder. In addition, only egg yolk dilution centrifugal supernatant is used as raw material, and a large amount of phosphatidylcholine in the precipitate cannot be effectively utilized, resulting in waste of egg yolk resources.

[0004] At present, deoiling of egg yolk is mainly achieved by organic solvent extraction and supercritical or subcritical fluid extraction, which requires large equipment investment, high cost and complex operation, and is not conducive to its industrial application. When using traditional protease-assisted aqueous enzymatic deoiling, a large amount of phospholipid components exist in the egg yolk, which has good emulsifying property, and it is difficult to effectively deoiling by centrifugal deoiling. Therefore, it is necessary to develop a processing method for high-value egg yolk products with good solubility, high egg yolk phospholipid conversion efficiency, good deoiling effect and low cost in view of the defects of glycerophosphorylcholine-rich egg yolk products, which has important economic value. SUMMARY

[0005] In view of the problems of insufficient comprehensive utilization of egg yolk resources and low product added value in the prior art, the present application provides a processing method of egg yolk peptide rich in glycerophosphorylcholine. The method of the present application is simple to operate and easy to realize industrialization.

[0006] The technical scheme of the present application is as follows:

[0007] The application aims to provide a processing method of egg yolk peptide rich in glycerophosphorylcholine, comprising the following steps:

[0008] S1, protease treatment: mix egg yolk powder with water, adjust pH to 7-10, add protease, and enzymolysis at 45-55℃ for 4-8h to obtain egg yolk enzymolysis liquid 1;

[0009] S2, phospholipase treatment: adjust pH of the egg yolk enzymolysis liquid 1 obtained in step S1 to 5.5-7.5, add phospholipase B, and stir and enzymolyze at 50-60℃ for 2-4h to obtain egg yolk enzymolysis liquid 2;

[0010] S3, post-treatment: sequentially perform flash evaporation, separation, concentration, high-temperature sterilization and spray drying treatment on the egg yolk enzymolysis liquid 2 obtained in step S2 to obtain the egg yolk peptide rich in glycerophosphorylcholine.

[0011] In an embodiment of the application, in S1, the mass ratio of egg yolk powder to water is 1:1.5-3; and the dosage of protease relative to egg yolk powder is 3000000-6000000U / kg.

[0012] In an embodiment of the application, in S2, the dosage of phospholipase B relative to egg yolk powder is 400000-600000U / kg.

[0013] In an embodiment of the application, in S3, the flash evaporation is performed at 90-110℃ for 30-60s.

[0014] In an embodiment of the application, in S3, the separation is first performed by rotary membrane separation, and then centrifugal oil removal.

[0015] In an embodiment of the application, in S3, the specific method of separation is as follows:

[0016] (1) dilute the egg yolk enzymolysis liquid 2 with purified water according to a volume ratio of 1:2-4, and then perform slurry and residue separation by rotary ceramic membrane, with an inlet membrane pressure of 0.5-2bar, and collect the slurry;

[0017] (2) continuously centrifugal oil removal is performed on the collected slurry to separate oil phase and water phase.

[0018] In step (2), the centrifugal conditions are 9000-9500rpm, the flow rate is 1-3t / h, and the back pressure is 0.1-0.2MPa.

[0019] In step (2), the centrifugal is performed by a disc centrifuge, which is a continuous centrifugal equipment.

[0020] In one embodiment of the present application, in S3, the concentration is vacuum concentration of the obtained water phase after separation to a solid content of 30-50%, and the concentration conditions are: temperature 55-70 DEG C, vacuum degree -0.06 to -0.08 MPa, time 1-3 h.

[0021] In one embodiment of the present application, in S3, the high-temperature sterilization conditions are: 80-95 DEG C, 2-5 min.

[0022] In one embodiment of the present application, in S3, the spray drying conditions are: inlet temperature 180-195 DEG C, outlet temperature 75-85 DEG C.

[0023] In one embodiment of the present application, the phospholipase B is lysophospholipase.

[0024] The present application has the beneficial technical effects of:

[0025] The present application uses egg yolk powder as a starting material, adds water in a certain proportion, and mixes uniformly, and then is subjected to the steps of protease treatment, phospholipase treatment, rotary membrane separation, flash enzyme removal, centrifugal oil removal, vacuum concentration, high-temperature sterilization, and spray drying to obtain an egg yolk peptide product rich in glycerophosphorylcholine.

[0026] The present application uses the hydrolysis of protease to destroy the molecular structure of egg yolk phosphatidylcholine-rich lipoprotein, improve the solubility of egg yolk, and make it easier for phospholipase to hydrolyze phosphatidylcholine and improve the yield of glycerophosphorylcholine.

[0027] The present application uses the hydrolysis of phospholipase B to degrade the phospholipid components with good emulsification in egg yolk, and combines the denaturation of protein by flash treatment to greatly improve the oil removal capacity in the centrifugal oil removal step, so that the residual lipid in the egg yolk peptide rich in glycerophosphorylcholine is significantly reduced. DETAILED DESCRIPTION

[0028] Detection method:

[0029] 1. Determination of soluble nitrogen yield in sample:

[0030] According to GB 5009.5-2016, the protein content in the egg yolk powder and the protease enzyme hydrolysis supernatant is detected, and the calculation formula of the soluble nitrogen yield is as follows:

[0031]

[0032] In the formula:

[0033] V- volume of enzyme hydrolysis supernatant, mL;

[0034] X1- protein content in enzyme hydrolysis supernatant, g / mL;

[0035] M - mass of egg yolk powder, g;

[0036] X2 - protein content in egg yolk powder, g / 100g.

[0037] 2. Peptide content determination in sample

[0038] Refer to Appendix B of GB / T 22492-2008.

[0039] 3. Lipid content determination in sample

[0040] Refer to GB5009.6-2016.

[0041] 4. Phosphatidylcholine and glycerophosphocholine content in sample

[0042] Determined by high performance liquid chromatography-evaporative light scattering detection technology. Accurately weigh the sample and dissolve it in methanol, shake it uniformly, filter it through a 0.22 μm organic membrane, and use the filtrate. The detection conditions are as follows: chromatographic column, Waters HILIC Silica silica gel chromatographic column (250x4.6mm, 5μm); mobile phase, methanol water solution (volume ratio 95:5); flow rate 1.0mL / min; column temperature 35℃; injection volume 10μL; evaporative light detector drift tube temperature 70℃, carrier gas flow rate 2.0mL / min.

[0043] The calculation formula of GPC yield in phospholipase B hydrolysate is as follows:

[0044]

[0045] 5. Powder solubility determination

[0046] Accurately weigh 5g of sample in a beaker, add 10mL of deionized water, stir at 25℃ for 30min, fully dissolve, centrifuge at 4000r / min for 15min, pour the supernatant into an evaporating dish of known constant weight (m1), then evaporate to dryness in a 90℃ water bath, and then place it in a 105℃ oven to dry to constant weight (m2).

[0047] The solubility can be calculated according to the following formula:

[0048]

[0049] 6. Protein content determination

[0050] Refer to GB 5009.5-2016.

[0051] 7. Molecular weight distribution

[0052] Sample treatment: The sample solution was prepared at a concentration of 5 mg / mL, filtered through a water-based microporous filter (0.45 μm) and then injected. The liquid phase conditions were as follows: chromatographic column: TSKgel G2000SWxl liquid chromatographic analysis column; mobile phase: water / acetonitrile / trifluoroacetic acid (40:60:0.07); flow rate: 1 mL / min; column temperature: 25°C; detection wavelength: 214 nm.

[0053] The standard samples were cytochrome C (12384 Da), bovine insulin (5733.49 Da), bacitracin (1421.69 Da), reduced glutathione (307.32 Da), and glycine (75.07 Da).

[0054] Standard curve: y = -0.5409x + 7.218, R2= 0.9853.

[0055] Example 1

[0056] A processing method of a glycerophosphorylcholine-rich egg yolk peptide, comprising the following steps:

[0057] (1) Protease treatment: water was added according to a mass ratio of egg yolk powder to water of 1:1.5, mixed, the pH was adjusted to 10, 4000000 U / kg of alkaline protease was added, and hydrolysis was performed at a temperature of 55°C for 4 h;

[0058] (2) Phospholipase treatment: the egg yolk enzymatic hydrolysate obtained in step (1) was adjusted to a pH of 7.5, 400000 U / kg of phospholipase B was added, and enzymatic hydrolysis was performed at a temperature of 50°C for 4 h with stirring to obtain an egg yolk enzymatic hydrolysate;

[0059] (3) Flash treatment: the egg yolk enzymatic hydrolysate obtained in step (2) was subjected to flash treatment under the following conditions: 90°C, 60 s;

[0060] (4) Rotary membrane separation: the egg yolk enzymatic hydrolysate obtained in step (3) was further diluted with purified water at a material-to-water ratio of 1:2, and then slurry-separation was performed using a rotary ceramic membrane, with an inlet membrane pressure of 2.0 bar;

[0061] (5) Centrifugal deoiling: the egg yolk enzymatic hydrolysate obtained in step (4) was subjected to continuous centrifugal deoiling to obtain an oil phase and an aqueous phase, with a centrifugal condition of 9000 rpm, a flow rate of 3 t / h, and a back pressure of 0.2 MPa;

[0062] (6) Vacuum concentration: the aqueous phase obtained in step (5) was subjected to vacuum concentration to a solid content of 50%, with a concentration condition of a temperature of 70°C and a vacuum degree of -0.06 MPa;

[0063] (7) High-temperature sterilization: the concentrate obtained in step (6) was subjected to high-temperature sterilization, with a sterilization condition of a temperature of 95°C and a time of 2 min;

[0064] (8) Spray drying: the sterilized concentrate obtained in step (7) is subjected to spray drying to obtain the egg yolk peptide rich in glycerophosphorylcholine, and the drying conditions are: inlet temperature 195°C, outlet temperature 85°C.

[0065] The product indicators obtained in this example are shown in Table 1 below.

[0066] Table 1

[0067] Index Example 1 Solubility (g / 100 mL) 98.5±1.4 Protein content (g / 100 g) 68.95±1.5 Peptide content (g / 100 g) 61±0.7 Lipid content (g / 100 g) 6.1±0.6 GPC content (g / 100 g) 7.2±0.4 MW > 1000 Da 0 500 < MW < 1000 Da 5.36 200 < MW < 500 Da 45.55 MW < 200 Da 49.09

[0068] Example 2

[0069] A method for processing an egg yolk peptide rich in glycerophosphorylcholine, comprising the following steps:

[0070] (1) Protease treatment: water is added according to an egg yolk powder to water mass ratio of 1:2.5, mixed, the pH is adjusted to 8.5, 6000000 U / kg of alkaline protease is added, and hydrolysis is carried out at a temperature of 50°C for 6h;

[0071] (2) Phospholipase treatment: the egg yolk enzymatic hydrolysate obtained in step (1) is adjusted to a pH of 6.5, 500000 U / kg of phospholipase B is added, and enzymatic hydrolysis is carried out at a temperature of 55°C for 3h to obtain an egg yolk enzymatic hydrolysate;

[0072] (3) Flash treatment: the egg yolk enzymatic hydrolysate obtained in step (2) is subjected to flash enzyme removal, and the conditions are: 100°C, 45s;

[0073] (4) Rotary membrane separation: the egg yolk enzymatic hydrolysate obtained in step (3) is further diluted with purified water according to a material to water ratio of 1:3, and then slurry and residue separation is carried out using a rotary ceramic membrane, and the inlet membrane pressure is 1.5 bar;

[0074] (5) Centrifugal oil removal: the filtered egg yolk enzymatic hydrolysate obtained in step (4) is subjected to continuous centrifugal oil removal to obtain an oil phase and an aqueous phase, and the centrifugal conditions are: 9200 rpm, flow rate 2t / h, back pressure 0.15 MPa;

[0075] (6) Vacuum concentration: the aqueous phase obtained in step (5) is subjected to vacuum concentration to a solid content of 35%, and the concentration conditions are: temperature 60°C, vacuum degree -0.07 MPa;

[0076] (7) High-temperature sterilization: the concentrate obtained in step (6) is subjected to high-temperature sterilization, and the sterilization conditions are: temperature 85°C, time 3 min;

[0077] (8) Spray drying: the sterilized concentrate obtained in step (7) is subjected to spray drying to obtain the egg yolk peptide rich in glycerophosphorylcholine, and the drying conditions are: inlet temperature 185°C, outlet temperature 80°C.

[0078] Example 3

[0079] A processing method of egg yolk peptide rich in glycerophosphorylcholine, comprising the following steps:

[0080] (1) Protease treatment: water is added according to the mass ratio of egg yolk powder to water 1:3, mixed, the pH is adjusted to 7.5, 8000000 U / kg of neutral protease is added, and hydrolysis is carried out at a temperature of 55℃ for 4h;

[0081] (2) Phospholipase treatment: the egg yolk liquid obtained in step (1) is adjusted to pH 7.5, 600000 U / kg of phospholipase B is added, and enzyme hydrolysis is carried out at a temperature of 60℃ for 2h to obtain an egg yolk enzyme hydrolysate;

[0082] (3) Flash treatment: the egg yolk enzyme hydrolysate obtained in step (2) is subjected to flash treatment under the following conditions: 110℃, 30s;

[0083] (4) Rotary membrane separation: the egg yolk enzyme hydrolysate obtained in step (3) is further diluted with purified water at a material to water ratio of 1:4, and then slurry and residue are separated by rotary ceramic membrane, and the inlet membrane pressure is 0.5bar;

[0084] (5) Centrifugal deoiling: the filtered egg yolk enzyme hydrolysate obtained in step (4) is subjected to continuous centrifugal deoiling to obtain an oil phase and an aqueous phase, and the centrifugal conditions are as follows: 9000rpm, flow rate 1t / h, back pressure 0.1MPa;

[0085] (6) Vacuum concentration: the aqueous phase obtained in step (5) is vacuum concentrated to a solid content of 30%, and the concentration conditions are as follows: temperature 55℃, vacuum degree-0.08MPa;

[0086] (7) High-temperature sterilization: the concentrate obtained in step (6) is subjected to high-temperature sterilization, and the sterilization conditions are as follows: 80℃, 5min;

[0087] (8) Spray drying: the sterilized concentrate obtained in step (7) is subjected to spray drying to obtain egg yolk peptide rich in glycerophosphorylcholine, and the drying conditions are as follows: inlet temperature 180℃, outlet temperature 75℃.

[0088] Comparative Example 1

[0089] Referring to Example 1, the difference is only that the protease treatment of step (1) is omitted, and the other steps and parameters remain unchanged.

[0090] Comparative Example 2

[0091] Referring to Example 1, the difference is only that the phospholipase B treatment of step (2) is omitted, and the other steps and parameters remain unchanged.

[0092] Comparative Example 3

[0093] Reference to Example 1, the only difference is that the flash treatment of step (4) is omitted, other steps and parameters remain unchanged.

[0094] Comparative Example 4

[0095] Reference to Example 1, the only difference is that the phospholipase B in step (2) is replaced by phospholipase A1, other steps and parameters remain unchanged.

[0096] Comparative Example 5

[0097] Reference to Example 1, the only difference is that the order of step (1) and step (2) is exchanged, other steps and parameters remain unchanged.

[0098] The performance test results of the products obtained in Examples 1-3 and Comparative Examples 1-5 are shown in Table 2 below.

[0099] Table 2

[0100]

[0101]

[0102] Performance comparison:

[0103] From Table 2, it can be seen that the soluble nitrogen yield, glycerophosphorylcholine yield and peptide content in Examples 1-3 are at a relatively high level, and the lipid content is relatively low.

[0104] Comparing Comparative Example 1 with Example 1, it can be seen that without protease treatment, the soluble nitrogen yield, glycerophosphorylcholine yield and peptide content are significantly reduced, and the lipid content in the product is also significantly increased, indicating that protease treatment can significantly improve the hydrolysis of phospholipase B and the efficiency of centrifugal oil removal.

[0105] Comparing Comparative Example 2 with Example 1, it can be seen that without phospholipase B treatment, the glycerophosphorylcholine is below the detection limit, and the lipid content is significantly increased, indicating that phospholipase B hydrolysis can greatly improve the efficiency of centrifugal oil removal.

[0106] Comparing Comparative Example 3 with Example 1, it can be seen that without flash treatment, the peptide content in the product will be reduced to a certain extent, which is related to the denaturation effect of flash high-temperature treatment on heat-sensitive soluble proteins. Correspondingly, the lipid content is also increased to a certain extent, indicating that flash treatment can destroy the emulsifying activity of the egg yolk hydrolysate to a certain extent, and improve the efficiency of centrifugal oil removal.

[0107] Comparing Comparative Example 4 with Example 1, it can be seen that replacing phospholipase B with phospholipase A1 has no significant effect on the soluble nitrogen yield and peptide content, but the glycerophosphorylcholine yield will be significantly reduced, and the lipid content in the product will be increased. Correspondingly, indicating that phospholipase B has stronger hydrolysis ability on phosphatidylcholine than phospholipase A1.

[0108] From comparative example 1 and comparative example 5, it can be seen that the order of the enzymatic steps has no significant effect on the yield of soluble nitrogen and the content of peptides, but the yield of glycerophosphorylcholine is significantly reduced because the phospholipase hydrolysis is not preceded by protease treatment.

[0109] The above examples are not intended to limit the scope of the present application, nor are the described steps intended to limit the order of their execution. Those skilled in the art will make obvious modifications to the present application in light of the prior art knowledge, which also fall within the scope of protection defined by the claims of the present application.

Claims

1. A processing method for egg yolk peptides rich in glycerophosphorylcholine, characterized in that, Includes the following steps: S1. Protease treatment: Mix egg yolk powder with water, adjust the pH to 7-10, add protease, and hydrolyze at 45-55℃ for 4-8 hours to obtain egg yolk hydrolysate 1. S2, Phospholipase treatment: Adjust the pH of the egg yolk hydrolysate 1 obtained in step S1 to 5.5-7.5, add phospholipase B, and stir at 50-60℃ for 2-4 hours to obtain egg yolk hydrolysate 2. S3. Post-processing: The egg yolk hydrolysate 2 obtained in step S2 is subjected to flash evaporation, separation, concentration, high-temperature sterilization and spray drying to obtain the egg yolk peptide rich in glycerophosphorylcholine.

2. The processing method according to claim 1, characterized in that, In S1, the mass ratio of egg yolk powder to water is 1:1.5 to 3; the amount of protease relative to egg yolk powder is 3,000,000 to 6,000,000 U / kg.

3. The processing method according to claim 1, characterized in that, In S2, the relative amount of phospholipase B to egg yolk powder is 400,000–600,000 U / kg.

4. The processing method according to claim 1, characterized in that, In S3, the flash evaporation conditions are: flash evaporation treatment at 90-110℃ for 30-60 seconds.

5. The processing method according to claim 1, characterized in that, In S3, the separation process involves first using a rotating membrane for separation, followed by centrifugation to remove oil.

6. The processing method according to claim 1, characterized in that, In S3, the specific method for separation is as follows: (1) Dilute egg yolk hydrolysate 2 with purified water at a volume ratio of 1:2 to 4, then use a rotating ceramic membrane to separate the slurry and residue, with a membrane pressure of 0.5 to 2 bar, and collect the slurry; (2) The collected slurry is continuously centrifuged to remove oil, and the oil phase and water phase are separated.

7. The processing method according to claim 6, characterized in that, In step (2), the centrifugation conditions are 9000-9500 rpm, the flow rate is 1-3 t / h, and the back pressure is 0.1-0.2 MPa.

8. The processing method according to claim 1, characterized in that, In S3, concentration involves vacuum concentrating the separated aqueous phase to a solid content of 30-50%. The concentration conditions are: temperature 55-70℃, vacuum degree -0.06 to -0.08 MPa.

9. The processing method according to claim 1, characterized in that, In S3, the high-temperature sterilization conditions are: 80-95℃, 2-5 min.

10. The processing method according to claim 1, characterized in that, In S3, the spray drying conditions are: inlet temperature 180-195℃, outlet temperature 75-85℃.

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