Processing method of egg yolk peptide rich in glycerophosphorylcholine

Abstract

The processing method comprises the following steps: S1, protease treatment: uniformly mixing egg yolk powder with water, adjusting the pH value to 7-10, adding protease, and carrying out enzymolysis for 4-8 hours at the temperature of 45-55 DEG C, so as to obtain an egg yolk enzymatic hydrolysate 1; s2, phospholipase treatment: adjusting the pH value of the yolk enzymatic hydrolysate 1 obtained in the step S1 to 5.5-7.5, adding phospholipase B, and performing stirring enzymolysis at 50-60 DEG C for 2-4 hours to obtain yolk enzymatic hydrolysate 2; s3, flash evaporation treatment: sequentially carrying out flash evaporation, separation, concentration, high-temperature sterilization and spray drying treatment on the egg yolk enzymatic hydrolysate 2 obtained in the step S2 to obtain the egg yolk peptide rich in glycerophosphorylcholine. The method can solve the problems of insufficient comprehensive utilization rate of egg yolk resources and low added value of products, and is simple to operate and easy to industrialize.

Description

Technical Field

[0001] The invention relates to the field of food biotechnology, and in particular to a processing method of glycerolphosphorylcholine-rich egg yolk peptide. Background Art

[0002] Glycerolphosphorylcholine is a chemical substance with important biological functions and has a significant impact on the health of the brain and nervous system. It has been widely used in the fields of nutritional supplementation and clinical treatment, and shows broad application prospects. The preparation methods of glycerolphosphorylcholine are mainly divided into two methods: phospholipid hydrolysis method and chemical synthesis method. The chemical synthesis method has complicated steps, the purification process will produce a large amount of wastewater, and toxic and harmful synthetic precursors are easily left. The traditional phospholipid hydrolysis method is usually used to efficiently extract the crude product containing phosphatidylcholine from egg yolk or other phospholipid-rich raw materials, and then prepare glycerolphosphorylcholine through enzyme catalysis. This method requires the extraction of phospholipids first, the steps are complicated, and it is not conducive to the industrial application of this method.

[0003] CN118203092A, a method for preparing de-oiled egg yolk powder rich in glycerol-phosphorylcholine, discloses a method of using the supernatant of the centrifugation of fresh poultry egg yolk diluted liquid rich in phosphatidylcholine as raw material, obtaining a reaction liquid rich in glycerol-phosphorylcholine by phospholipase composite enzymolysis, and then obtaining the target product by spray drying and supercritical CO2 extraction. The invention uses the supernatant of the centrifugation of egg yolk diluted liquid as raw material, and the de-oiled egg yolk powder rich in glycerol-phosphorylcholine obtained by the synergistic treatment method of phospholipase and supercritical CO2 has the advantage of high glycerol-phosphorylcholine content, but still has the problem of low solubility of the de-oiled egg yolk powder. In addition, only the supernatant of the centrifugation of egg yolk diluted liquid is used as raw material, and a large amount of phosphatidylcholine in the precipitate cannot be effectively utilized, and there is a problem of waste of egg yolk resources.

[0004] At present, the deoiling of egg yolk mainly relies on organic solvent extraction and supercritical or subcritical fluid extraction deoiling, which requires large equipment investment, high cost and complicated operation, which is not conducive to its industrial application. When using the traditional protease-assisted aqueous enzymatic deoiling method, due to the large amount of phospholipid components in the egg yolk, it is difficult to effectively deoil by centrifugal deoiling due to the good emulsification of the phospholipid components. Therefore, it is necessary to develop a set of high-value-added egg yolk product processing methods with good solubility, high egg yolk phospholipid conversion efficiency, good deoiling effect and low cost to address the defects of glycerol phosphorylcholine-rich egg yolk products, which has important economic value. Summary of the invention

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

[0006] The technical solution of the present invention is as follows:

[0007] The object of the present invention is to provide a processing method for glycerolphosphorylcholine-rich egg yolk peptide, comprising the following steps:

[0008] S1. Protease treatment: mix the egg yolk powder and water, adjust the pH to 7-10, add protease, and perform enzymolysis at 45-55° C. for 4-8 hours to obtain egg yolk enzymolysis solution 1;

[0009] S2, phospholipase treatment: the pH of the egg yolk enzymolyzate 1 obtained in step S1 is adjusted to 5.5-7.5, phospholipase B is added, and the egg yolk enzymolyzate is stirred and hydrolyzed at 50-60° C. for 2-4 hours to obtain an egg yolk enzymolyzate 2;

[0010] S3, post-treatment: the egg yolk enzymolyzate 2 obtained in step S2 is sequentially subjected to flash evaporation, separation, concentration, high temperature sterilization and spray drying to obtain the glycerolphosphorylcholine-rich egg yolk peptide.

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

[0012] In one embodiment of the present invention, in S2, the amount of phospholipase B relative to egg yolk powder is 400,000 to 600,000 U / kg.

[0013] In one embodiment of the present invention, in S3, the flash evaporation conditions are: flash evaporation at 90-110° C. for 30-60 seconds.

[0014] In one embodiment of the present invention, in S3, separation is first performed by rotary membrane separation and then by centrifugal deoiling.

[0015] In one embodiment of the present invention, in S3, the specific method of separation is:

[0016] (1) diluting the egg yolk enzymatic hydrolysate 2 with purified water in a volume ratio of 1:2 to 4, and then using a rotating ceramic membrane to separate the slurry from the residue at a membrane inlet pressure of 0.5 to 2 bar, and collecting the slurry;

[0017] (2) The collected slurry is continuously centrifuged to deoil to separate the oil phase and the water phase.

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

[0019] In one embodiment of the present invention, in step (2), centrifugation is performed using a disc centrifuge, which is a continuous centrifugation device.

[0020] In one embodiment of the present invention, in S3, the concentration is to vacuum concentrate the separated aqueous phase to a solid content of 30-50%, and the concentration conditions are: temperature 55-70°C, vacuum degree -0.06 to -0.08 MPa, and time 1 to 3 hours.

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

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

[0023] In one embodiment of the invention, the phospholipase B is a lysophospholipase.

[0024] The beneficial technical effects of the present invention are:

[0025] The invention uses egg yolk powder as a starting material, adds water in a certain proportion and mixes evenly, and then undergoes protease treatment, phospholipase treatment, rotary membrane separation, flash evaporation enzyme inactivation, centrifugal deoiling, vacuum concentration, high-temperature sterilization and spray drying steps to obtain an egg yolk peptide product rich in glycerolphosphorylcholine. The method of the invention is simple to operate and easy to realize industrialization.

[0026] The invention utilizes the hydrolysis of protease to destroy the molecular structure of egg yolk phosphatidylcholine-enriched lipoprotein, improves the solubility of egg yolk, and makes it easier for phospholipase to hydrolyze phosphatidylcholine to increase the yield of phosphatidylcholine to glycerolphosphatidylcholine.

[0027] The present invention utilizes the hydrolysis effect of phospholipase B to degrade the phospholipid components with good emulsification in the egg yolk, and combines the denaturation effect of flash evaporation on the protein to greatly improve the deoiling capacity of the centrifugal deoiling 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 samples:

[0030] GB 5009.5-2016 was used to detect the protein content in egg yolk powder and protease hydrolysis supernatant, and the calculation formula for soluble nitrogen yield was as follows:

[0031]

[0032] Where:

[0033] V-volume of enzymatic supernatant, mL;

[0034] X1-protein content in the enzymatic supernatant, g / mL;

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

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

[0037] 2. Determination of Peptide Content in Samples

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

[0039] 3. Determination of lipid content in samples

[0040] Refer to GB5009.6-2016.

[0041] 4. Phosphatidylcholine and glycerophosphatidylcholine content in samples

[0042] The determination was carried out by high performance liquid chromatography-evaporative light scattering detection coupling technology. The sample was accurately weighed and dissolved in methanol. After shaking evenly, it was filtered through a 0.22 μm organic membrane and the filtrate was set aside. The detection conditions were as follows: chromatographic column, Waters HILICSilica silica gel chromatographic column (250×4.6mm, 5μm); the mobile phase was 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 hydrolyzate is as follows:

[0044]

[0045] 5. Powder Solubility Determination

[0046] Accurately weigh 5 g of sample into a beaker, add 10 mL of deionized water, stir at 25 °C for 30 min to fully dissolve, centrifuge at 4000 r / min for 15 min, pour the supernatant into an evaporating dish with a known constant weight (m1), then evaporate to dryness in a 90 °C water bath, and then dry in a 105 °C oven 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: Prepare a sample solution with a concentration of 5 mg / mL, filter it through a water-based microporous filter membrane (0.45 μm) and inject it. The liquid phase conditions are as follows: chromatographic column: TSKgel G2000SWxl liquid chromatography 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 standards 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 for egg yolk peptide rich in glycerolphosphorylcholine comprises the following steps:

[0057] (1) Protease treatment: add water according to the mass ratio of egg yolk powder to water of 1:1.5, mix well, adjust the pH to 10, add 4000000 U / kg of alkaline protease, and hydrolyze at 55°C for 4 h;

[0058] (2) Phospholipase treatment: the pH of the egg yolk hydrolysate obtained in step (1) was adjusted to 7.5, 400,000 U / kg of phospholipase B was added, and the mixture was stirred at 50° C. for 4 h to obtain an egg yolk hydrolysate;

[0059] (3) Flash evaporation: flash evaporation the egg yolk enzymatic hydrolysate obtained in step (2) at 90° C. for 60 seconds;

[0060] (4) Rotating membrane separation: the egg yolk enzymatic hydrolysate obtained in step (3) is further diluted with purified water at a feed-water ratio of 1:2, and then the pulp-residue separation is performed using a rotating ceramic membrane at an inlet pressure of 2.0 bar;

[0061] (5) Centrifugal deoiling: The egg yolk enzymatic hydrolysate obtained in step (4) is continuously deoiled by centrifugation to obtain an oil phase and an aqueous phase. The centrifugal conditions are 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) is vacuum concentrated to a solid content of 50% under the following conditions: temperature 70° C., vacuum degree -0.06 MPa;

[0063] (7) High temperature sterilization: The concentrate obtained in step (6) is subjected to high temperature sterilization under the following conditions: temperature 95° C., time 2 min;

[0064] (8) Spray drying: spray drying the sterilized concentrate obtained in step (7) to obtain egg yolk peptide rich in glycerophosphorylcholine, the drying conditions being: 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 / 100mL) 98.5±1.4 Protein content (g / 100g) 68.95±1.5 Peptide content (g / 100g) 61±0.7 Lipid content (g / 100g) 6.1±0.6 GPC content (g / 100g) 7.2±0.4 MW>1000Da 0 500<MW≤1000Da 5.36 200<MW≤500Da 45.55 MW≤200Da 49.09

[0068] Example 2

[0069] A processing method for egg yolk peptide rich in glycerolphosphorylcholine comprises the following steps:

[0070] (1) Protease treatment: add water according to the mass ratio of egg yolk powder to water of 1:2.5, mix well, adjust the pH to 8.5, add 6000000 U / kg of alkaline protease, and hydrolyze at 50°C for 6 h;

[0071] (2) Phospholipase treatment: the egg yolk hydrolysate obtained in step (1) was adjusted to pH 6.5, 500,000 U / kg of phospholipase B was added, and the mixture was stirred at 55° C. for 3 h to obtain an egg yolk hydrolysate;

[0072] (3) Flash evaporation: flash evaporation of the egg yolk enzymatic hydrolysate obtained in step (2) to inactivate enzymes at 100° C. for 45 seconds;

[0073] (4) Rotating membrane separation: the egg yolk enzymatic hydrolysate obtained in step (3) is further diluted with purified water at a feed-water ratio of 1:3, and then the pulp-residue separation is performed using a rotating ceramic membrane at an inlet pressure of 1.5 bar;

[0074] (5) Centrifugal deoiling: The filtered egg yolk enzymatic hydrolysate obtained in step (4) is continuously deoiled by centrifugation to obtain an oil phase and an aqueous phase. The centrifugal conditions are 9200 rpm, a flow rate of 2 t / h, and a back pressure of 0.15 MPa;

[0075] (6) Vacuum concentration: The aqueous phase obtained in step (5) is vacuum concentrated to a solid content of 35% under the following conditions: 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 under the following conditions: temperature 85° C., time 3 min;

[0077] (8) Spray drying: spray drying the sterilized concentrate obtained in step (7) to obtain 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 for egg yolk peptide rich in glycerolphosphorylcholine comprises the following steps:

[0080] (1) Protease treatment: add water according to the mass ratio of egg yolk powder to water of 1:3, mix well, adjust the pH to 7.5, add 8000000 U / kg of neutral protease, and hydrolyze at 55°C for 4 h;

[0081] (2) Phospholipase treatment: the pH of the egg yolk liquid obtained in step (1) was adjusted to 7.5, 600,000 U / kg of phospholipase B was added, and the mixture was stirred for enzymatic hydrolysis at 60° C. for 2 h to obtain an egg yolk enzymolyzate;

[0082] (3) Flash evaporation: The egg yolk enzymatic hydrolysate obtained in step (2) is flash-evaporated at 110° C. for 30 seconds.

[0083] (4) Rotary membrane separation: the egg yolk enzymatic hydrolysate obtained in step (3) is further diluted with purified water at a feed-water ratio of 1:4, and then the pulp-residue separation is performed using a rotary ceramic membrane with an inlet pressure of 0.5 bar;

[0084] (5) Centrifugal deoiling: The filtered egg yolk enzymatic hydrolysate obtained in step (4) is continuously centrifuged to deoil to obtain an oil phase and an aqueous phase. The centrifugal conditions are 9000 rpm, a flow rate of 1 t / h, and a back pressure of 0.1 MPa;

[0085] (6) Vacuum concentration: The aqueous phase obtained in step (5) is vacuum concentrated to a solid content of 30% under the following conditions: temperature 55° C., vacuum degree -0.08 MPa;

[0086] (7) High temperature sterilization: The concentrate obtained in step (6) is subjected to high temperature sterilization at 80° C. for 5 min;

[0087] (8) Spray drying: spray drying the sterilized concentrate obtained in step (7) to obtain egg yolk peptide rich in glycerophosphorylcholine, the drying conditions being: inlet temperature 180° C., outlet temperature 75° C.

[0088] Comparative Example 1

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

[0090] Comparative Example 2

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

[0092] Comparative Example 3

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

[0094] Comparative Example 4

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

[0096] Comparative Example 5

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

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

[0099] Table 2

[0100]

[0101]

[0102] Performance comparison:

[0103] As can be seen from Table 2, the soluble nitrogen yield, glycerolphosphorylcholine yield and peptide content in Examples 1-3 are all at a relatively high level, and the lipid content is relatively low.

[0104] By comparing Example 1 and Comparative Example 1, it can be seen that without protease treatment, the soluble nitrogen yield, glycerolphosphorylcholine 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 effect of phospholipase B and the centrifugal deoiling efficiency.

[0105] By comparing Example 1 and Comparative Example 2, it can be seen that without phospholipase B treatment, glycerolphosphorylcholine is below the detection limit and the lipid content is significantly increased, indicating that phospholipase B hydrolysis can greatly improve the efficiency of centrifugal deoiling.

[0106] By comparing Example 1 and Comparative Example 3, it can be seen that without flash evaporation treatment, the peptide content in the product will be reduced to a certain extent. This is related to the denaturing effect of flash evaporation high temperature treatment on heat-sensitive soluble proteins. The corresponding lipid content is also increased to a certain extent, indicating that flash evaporation treatment can destroy the emulsifying activity of egg yolk hydrolysate to a certain extent and improve the efficiency of centrifugal deoiling.

[0107] By comparing Example 1 and Comparative Example 4, it can be seen that after replacing phospholipid B with phospholipase A1, there is no significant effect on the soluble nitrogen yield and peptide content, but the yield of glycerolphosphocholine will be significantly reduced, and the lipid content in the corresponding product will also be increased, indicating that phospholipase B has a stronger hydrolysis ability for phosphatidylcholine than phospholipase A1.

[0108] Comparison of Example 1 and Comparative Example 5 shows that after the enzymatic hydrolysis steps are switched, there is no significant effect on the soluble nitrogen yield and peptide content, but because no protease treatment is performed before phospholipase hydrolysis, the yield of glycerolphosphorylcholine is significantly reduced.

[0109] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for processing glycerolphosphorylcholine-rich egg yolk peptide, characterized in that: The steps include: S1. Protease treatment: mix the egg yolk powder and water, adjust the pH to 7-10, add protease, and perform enzymolysis at 45-55° C. for 4-8 hours to obtain egg yolk enzymolysis solution 1; S2, phospholipase treatment: the pH of the egg yolk enzymolyzate 1 obtained in step S1 is adjusted to 5.5-7.5, phospholipase B is added, and the egg yolk enzymolyzate is stirred and hydrolyzed at 50-60° C. for 2-4 hours to obtain an egg yolk enzymolyzate 2; S3, post-treatment: the egg yolk enzymolyzate 2 obtained in step S2 is sequentially subjected to flash evaporation, separation, concentration, high temperature sterilization and spray drying to obtain the glycerolphosphorylcholine-rich egg yolk peptide.

2. The processing method according to claim 1, characterized in that: In S1, the mass ratio of egg yolk powder to water is 1:1.5-3; the amount of protease relative to egg yolk powder is 3000000-6000000 U / kg.

3. The processing method according to claim 1, characterized in that: In S2, the amount of phospholipase B relative to egg yolk powder is 400,000 to 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° C. for 30-60 seconds.

5. The processing method according to claim 1, characterized in that: In S3, separation is first performed by rotary membrane separation and then by centrifugal deoiling.

6. The processing method according to claim 1, characterized in that: In S3, the specific method of separation is: (1) diluting the egg yolk enzymatic hydrolysate 2 with purified water in a volume ratio of 1:2 to 4, and then using a rotating ceramic membrane to separate the slurry from the residue at a membrane inlet pressure of 0.5 to 2 bar, and collecting the slurry; (2) The collected slurry is continuously centrifuged to deoil to separate the oil phase and the water phase.

7. The processing method according to claim 6, characterized in that: In step (2), the centrifugal 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, the concentration is to vacuum concentrate the separated aqueous phase to a solid content of 30-50%, and the concentration conditions are: temperature 55-70°C, vacuum degree -0.06 to -0.08 MPa.

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

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

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