CN112111546B - A kind of industrialized preparation method of phosphatin phosphopeptide and yolk polypeptide - Google Patents

Abstract

The invention discloses an industrialized preparation method of phosvitin phosphopeptide and yolk polypeptide, and belongs to the field of biotechnology. The invention uses defatted egg yolk powder, especially the defatted egg yolk powder with serious denaturation, loss of functionality and serious value reduction after industrial extraction of lecithin as raw materials, and separates egg yolk polypeptides with a ceramic membrane after the first enzymatic hydrolysis. and phosvitin, the second enzymatic hydrolysis is performed after the alkali treatment of phosvitin, thereby simultaneously preparing yolk polypeptide and phosvitin phosphopeptide. The method of the invention can make full use of the defatted egg yolk powder, especially can effectively improve the value of the low-value defatted egg yolk powder after industrial extraction of lecithin, and simultaneously realize the industrialized production of egg yolk polypeptide and egg yolk phosphopeptide.

Description

A kind of industrialized preparation method of phosphatin phosphopeptide and yolk polypeptide

technical field

The invention relates to an industrialized preparation method of phosphatin phosphopeptide and yolk polypeptide, and belongs to the field of biotechnology.

Background technique

Egg yolk has high nutritional value, and the main components are protein and lipid, among which, egg yolk lecithin is a functional component with important value. With the development of egg intensive processing, a large amount of defatted egg yolk powder by-products will be produced in the process of extracting high-quality lecithin from egg yolk. Due to the use of organic solvents such as alcohol in the extraction process, the protein in the defatted egg yolk powder is severely denatured and basically lost. The original functionality has reduced its value, so it is currently mostly used as animal feed, resulting in a great waste of protein resources. Since the 20th century, many scholars have isolated a variety of active peptides from egg yolks. At present, it has been found that the active peptides of egg yolk protein have various physiological functions such as antioxidant, lowering blood pressure, and promoting the absorption of mineral elements, and can be widely used in Food, health products and medicines and other related fields have great development prospects. Phosphosphatin phosphopeptide is an active peptide hydrolyzed by phosvitin, which is the protein with the highest degree of phosphorylation in nature. It consists of 216 amino acids, half of which are serine and 100 of which are serine. The left and right serines are phosphorylated. After being ingested by the human body, phosvitin cannot be hydrolyzed and utilized by the human body under the protection of a large number of phosphate groups. Therefore, it must be processed in vitro to prepare phosvitin phosphopeptide before it can be absorbed and utilized by the human body. The phosvitin phosphopeptide contains a large number of phosphoserine residues, which can complex with metal ions and have good metal ion chelation ability, and many studies have shown that the phosvitin phosphopeptide has better calcium holding capacity than A commercialized casein phosphopeptide that promotes calcium absorption. In addition, phosvitin phosphopeptide also has anti-inflammatory and immune-enhancing functions.

However, the existing research on egg yolk polypeptide mainly focuses on its functionality, while for the preparation process of egg yolk polypeptide, the research mainly focuses on the optimization of its enzymatic hydrolysis process, and the raw materials used in the research are mostly prepared by Soxhlet extraction. The defatted egg yolk powder has a relatively low degree of denaturation, and its properties are quite different from those of the industrially produced defatted egg yolk powder. The previous process for preparing phosvitin phosphopeptide is often to extract phosvitin from egg yolk, which leads to the inability of comprehensive utilization of egg yolk, and expensive separation and purification methods are used, which is not suitable for industrialized large-scale production.

SUMMARY OF THE INVENTION

In order to solve at least one of the above-mentioned problems, the present invention uses defatted egg yolk powder, especially the defatted egg yolk powder with severe denaturation, loss of functionality and serious reduction in value after industrial extraction of lecithin as a raw material, through two enzymatic hydrolysis, the first enzymatic hydrolysis Then, the yolk polypeptide and phosvitin are separated with a ceramic membrane, and the phosvitin is treated with alkali and then subjected to a second enzymatic hydrolysis, thereby simultaneously preparing the yolk polypeptide and the phosvitin phosphopeptide. The method of the invention can make full use of the defatted egg yolk powder, especially can effectively improve the value of the low-value defatted egg yolk powder after industrial extraction of lecithin, and simultaneously realize the industrialized production of egg yolk polypeptide and egg yolk phosphopeptide.

The first object of the present invention is to provide a method for industrialized simultaneous preparation of phosvitin phosphopeptide and yolk polypeptide, comprising the following steps:

(1) pretreatment: the defatted egg yolk powder is dissolved and then homogenized to obtain a homogenate of the defatted egg yolk powder;

(2) Enzymolysis for the first time: the defatted egg yolk powder homogenate of step (1) is subjected to enzymolysis to obtain an egg yolk polypeptide enzymolysis solution;

(3) clarification and separation: pH adjustment is performed on the yolk polypeptide enzymatic hydrolyzate solution obtained in step (2), and then clarification and separation are carried out with a ceramic membrane to obtain yolk polypeptide clarified solution and phosvitin precipitate;

(4) carrying out nanofiltration desalting concentration, refining and drying to the egg yolk polypeptide clarified liquid obtained in step (3) to obtain egg yolk polypeptide product;

(5) subjecting the phosvitin precipitate obtained in step (3) to alkali treatment for dephosphorization, second enzymatic hydrolysis, clarification, desalting concentration, purification and drying to obtain a phosvitin phosphopeptide product.

In one embodiment of the present invention, the defatted egg yolk powder can be a commercially available defatted egg yolk powder, or a low-value defatted egg yolk powder after industrial extraction of lecithin, and the protein content is 60%-90%.

In an embodiment of the present invention, the homogenization treatment in step (1) is wet pulverization or homogenization treatment.

In an embodiment of the present invention, the pretreatment described in step (1) is as follows: firstly dissolving the defatted egg yolk powder, controlling the solid-liquid ratio (w/v) to be 1:8-1:12, and mixing evenly; After wet pulverization or homogenization, it is sieved to obtain a homogenate of defatted egg yolk powder.

In an embodiment of the present invention, the pretreatment described in step (1) is as follows: firstly dissolving the defatted egg yolk powder, controlling the solid-liquid ratio (w/v) to be 1:8-1:12, and mixing evenly; After 500-5000rpm rotary disc wet grinding or 10-100MPa high pressure homogenization, it is passed through a 100-300 mesh sieve to obtain a defatted egg yolk powder homogenate.

In an embodiment of the present invention, the first enzymatic hydrolysis described in step (2) is to adjust the homogenate of the defatted egg yolk powder obtained in step (1) to pH=6.5-7.5; The amount of neutral protease added is 1000-3000U/g, the enzymolysis temperature is 30-60°C, and the enzymolysis time is 2-4h.

In one embodiment of the present invention, the clarification and separation described in step (3) is to adjust the pH of the yolk polypeptide enzymatic hydrolyzate to 4.0-4.5, and stir at 30-100 rpm for 2-5 min, and then separate the yolk with a ceramic membrane to obtain the yolk Peptide and phosvitin precipitation.

In an embodiment of the present invention, the parameters of the ceramic membrane in step (3) are: the pore size is 50-500 nm.

In an embodiment of the present invention, the parameters of the clarification and separation of the ceramic membrane in step (3) are: the pressure is 0.1-1MPa, and the temperature is 20-65°C.

In one embodiment of the present invention, the nanofiltration desalting concentration described in step (4) is: desalting the egg yolk polypeptide clarified liquid through a nanofiltration membrane with a molecular weight of 300, adding 3 times the volume of pure water for desalting; obtaining egg yolk polypeptide Concentrate; the solid content of egg yolk polypeptide concentrate is 8-25%.

In an embodiment of the present invention, the refining described in step (4) is: adding 0.5-2% (w/v) activated carbon to the egg yolk polypeptide concentrate for decolorization, adjusting pH=4.0-4.5, at 30 Decolorize at -60°C for 30-90min, and sterilize after decolorization to obtain refined egg yolk polypeptide; the sterilization adopts microfiltration sterilization, specifically, 0.15-0.65 μm membrane microfiltration sterilization is used.

In one embodiment of the present invention, the drying described in step (4) is to dry the refined egg yolk polypeptide by spray drying, the air inlet temperature is 160-180°C, and the air outlet temperature is 70-90°C to obtain the egg yolk polypeptide product (powder).

In one embodiment of the present invention, the alkali treatment and dephosphorization in step (5) is as follows: the phosvitin precipitate obtained in step (3) is treated with 0.05-0.25M NaOH solution at 25-60° C. After stirring for 2-4 hours, dephosphorization is carried out to obtain partially dephosphorized phosvitin after alkali treatment.

In an embodiment of the present invention, the second enzymatic hydrolysis described in step (5) is: using alkaline protease to carry out the second enzymatic hydrolysis of the partially dephosphorized phosvitin after the alkali treatment, adding an enzyme The amount is 1000-3000U/g, pH=8.0-10.5, enzymatic hydrolysis is carried out at 30-60 DEG C for 2-4 hours; phosvitin phosphopeptide enzymatic hydrolysis solution is obtained.

In one embodiment of the present invention, the clarification described in step (5) is to use a ceramic membrane to process the phosvitin phosphopeptide enzymatic hydrolysis solution to obtain a phosvitin phosphopeptide clear solution; wherein the parameters of the ceramic membrane are The pore size is 50-500nm, and the parameters of the ceramic membrane clarification and separation are: the pressure is 0.1-1MPa, and the temperature is 20-65℃.

In one embodiment of the present invention, the desalting concentration described in step (5) is: desalting and concentrating the phosvitin phosphopeptide clarified solution, using a nanofiltration membrane with a molecular weight of 300, adding three times the volume of pure water , to obtain a phosvitin phosphopeptide concentrate, wherein the solid content of the phosvitin phosphopeptide concentrate is 8-25%.

In an embodiment of the present invention, the refining described in step (5) is: performing clarification and microfiltration sterilization on the phosvitin phosphopeptide concentrate to obtain a purified phosvitin phosphopeptide concentrate ; The clarification treatment is carried out by using ceramic membrane, the specific parameters are using 50-500nm ceramic membrane, the filtration pressure is 0.1-1MPa, and the temperature is 20-65°C. The microfiltration sterilization is specifically the use of 0.15-0.65 μm membrane microfiltration sterilization.

In one embodiment of the present invention, the drying described in step (5) is to spray-dry the purified phosvitin phosphopeptide concentrate after purification, the air inlet temperature is 170-190° C., and the air outlet temperature is 70° C. -90°C to obtain phosvitin phosphopeptide product (powder).

The second object of the present invention is the phosvitin phosphopeptide product and the yolk polypeptide product prepared by the method of the present invention.

The third object of the present invention is to provide a method for separating yolk polypeptide and phosvitin, comprising the steps of:

The mixture containing yolk protein and phosvitin is decomposed into a yolk polypeptide enzymatic hydrolysis solution by enzymatic hydrolysis; then the pH is adjusted to precipitate the unenzymatic phosvitin protein, and the yolk polypeptide solution is obtained by clarification and separation through a ceramic membrane and phosvitin precipitation.

In one embodiment of the present invention, the enzymatic hydrolysis is carried out with neutral protease, the amount of neutral protease added is 1000-3000 U/g, the enzymatic hydrolysis temperature is 30-60 °C, and the enzymatic hydrolysis time is 2-4 h .

In one embodiment of the present invention, the pH is adjusted to 4.0-4.5.

In an embodiment of the present invention, the pore size of the ceramic membrane is 50-500 nm, and the parameters for the clarification and separation of the ceramic membrane are: the pressure is 0.1-1 MPa, and the temperature is 20-65°C.

In one embodiment of the present invention, the described method for separating yolk polypeptide and phosvitin comprises the following steps:

(1) pretreatment: dissolving the defatted egg yolk powder and then carrying out homogenization to obtain a homogenate of the defatted egg yolk powder;

(2) Enzymolysis for the first time: the defatted egg yolk powder homogenate of step (1) is subjected to enzymolysis to obtain an egg yolk polypeptide enzymolysis solution;

(3) Clarification and separation: the pH of the yolk polypeptide enzymatic hydrolyzate solution obtained in step (2) is adjusted, and then clarified and separated with a ceramic membrane to obtain the yolk polypeptide clarified solution and the yolk phosphoprotein precipitate.

In one embodiment of the present invention, the defatted egg yolk powder may be a commercially available defatted egg yolk powder, or a low-value defatted egg yolk powder after industrial extraction of lecithin.

In an embodiment of the present invention, the homogenization treatment in step (1) is wet pulverization or homogenization treatment.

In an embodiment of the present invention, the pretreatment described in step (1) is as follows: firstly dissolving the defatted egg yolk powder, controlling the solid-liquid ratio (w/v) to be 1:8-1:12, and mixing evenly; After wet pulverization or homogenization, it is sieved to obtain a homogenate of defatted egg yolk powder.

In an embodiment of the present invention, the pretreatment described in step (1) is as follows: firstly dissolving the defatted egg yolk powder, controlling the solid-liquid ratio (w/v) to be 1:8-1:12, and mixing evenly; After 500-5000rpm rotary disc wet grinding or 10-100MPa high pressure homogenization, it is passed through a 100-300 mesh sieve to obtain a defatted egg yolk powder homogenate.

In an embodiment of the present invention, the first enzymatic hydrolysis described in step (2) is to adjust the homogenate of the defatted egg yolk powder obtained in step (1) to pH=6.5-7.5; The amount of neutral protease added is 1000-3000U/g, the enzymolysis temperature is 30-60°C, and the enzymolysis time is 2-4h.

In one embodiment of the present invention, the clarification and separation described in step (3) is to adjust the pH of the yolk polypeptide enzymatic hydrolyzate to 4.0-4.5, and stir at 30-100 rpm for 2-5 min, and then separate the yolk with a ceramic membrane to obtain the yolk Peptide and phosvitin precipitation.

In an embodiment of the present invention, the parameters of the ceramic membrane in step (3) are: the pore size is 50-500 nm.

In an embodiment of the present invention, the parameters of the clarification and separation of the ceramic membrane in step (3) are: the pressure is 0.1-1MPa, and the temperature is 20-65°C.

The fourth object of the present invention is the application of the phosvitin phosphopeptide product and the yolk polypeptide product of the present invention in the preparation of medicines, foods and health care products.

Beneficial effects of the present invention:

(1) The present invention provides a kind of defatted egg yolk powder, especially the defatted egg yolk powder with severe denaturation, loss of functionality and reduced value after industrial extraction of lecithin as raw material, and simultaneously producing egg yolk polypeptide and phosphatin phosphopeptide method The method can realize industrialized large-scale production, realize the deep processing of low-value defatted egg yolk powder, increase the added value of the product, and extend the production line for deep processing of egg products.

(2) The process of separating egg yolk polypeptide clarified liquid and phosvitin precipitation with ceramic membrane according to the present invention can greatly improve the separation efficiency.

(3) The present invention utilizes low-value defatted egg yolk powder to produce yolk polypeptide and yolk phosphopeptide, thereby improving the utilization value of defatted egg yolk powder.

(4) The molecular weight of the egg yolk polypeptide produced by the present invention is mainly distributed in 180-500 Da, which is the most suitable peptide segment for the human body to absorb, which can effectively promote the human body to absorb and give full play to its biological activity.

(5) The yield of phosvitin phosphopeptide produced by the present invention is obviously higher than that of other preparation methods in the laboratory; the yield of yolk polypeptide by the method of the present invention reaches more than 69.8%, and the yield of phosvitin phosphopeptide reaches 69.8% or more. More than 8%, the N/P of phosvitin phosphopeptide is lower than 7.2.

Description of drawings

Fig. 1 is the production process flow chart of the present invention.

FIG. 2 is a graph showing the molecular weight of egg yolk polypeptide in the egg yolk polypeptide product in Example 2. FIG.

FIG. 3 is a map of the molecular weight of the phosvitin phosphopeptide in the phosvitin phosphopeptide product in Example 2. FIG.

Detailed ways

The preferred embodiments of the present invention will be described below, and it should be understood that the embodiments are used to better explain the present invention and are not intended to limit the present invention.

testing method:

Determination method of egg yolk polypeptide yield: Kjeldahl method was used for determination, the protein content in defatted egg yolk powder and the protein content in polypeptide liquid were detected, and the protein content in polypeptide liquid was used to characterize polypeptide yield.

In the formula: V: the volume of the polypeptide liquid; X ₁ : the protein content in the polypeptide liquid; M: the mass of the defatted protein powder; X ₂ : the protein content of the defatted protein powder.

N/P determination method: N/P is an index used to characterize phosvitin phosphopeptide. The lower the N/P, the higher the purity of phosvitin phosphopeptide; the number of moles of N in the product is determined by Kjeldahl The mole number of P was detected by molybdenum blue spectrophotometry.

Molecular weight test of peptides: High performance liquid chromatography was used, and the chromatographic conditions were as follows: Waters 600 high performance liquid chromatograph (with 2487 UV detector and M 32 workstation), TSKgelG2000 SW×L 300mm×7.8mm, mobile phase was acetonitrile/water /trifluoroacetic acid, 45/55/0.1 (V/V/V), detection wavelength 220nm, flow rate 0.5mL/min, column temperature 30°C.

Example 1

An industrialized method for simultaneously preparing phosvitin phosphopeptide and yolk polypeptide, comprising the following steps:

(1) Dissolve defatted egg yolk powder (containing 85% protein), and control the ratio of material to liquid to be 1:8 (w/v);

(2) adopt 3000rpm rotary disc wet pulverization, then pass through 100 mesh sieves to obtain defatted egg yolk powder homogenate;

(3) The obtained defatted egg yolk powder was homogenized to adjust pH=7.0, and neutral protease was used for enzymolysis, the addition amount was 2000U/g, the stirring time for enzymolysis was 2.5h, the pH was adjusted to 4.2 after enzymolysis, and the stirring was carried out at 30rpm for 2min. Filter and separate with 100nm ceramic membrane at 0.1MPa and 30℃ to obtain yolk polypeptide clear liquid and phosvitin precipitation;

(4) Desalting the egg yolk polypeptide clear liquid obtained in step (3) by nanofiltration membrane with a molecular weight of 300, adding 3 times the volume of pure water for desalting, to obtain egg yolk polypeptide concentrate (solid content is 10%);

(5) Add 1.0% (v/w) 200-mesh powdered activated carbon to the egg yolk polypeptide concentrate, decolorize at pH 4.0 and temperature 50°C for 30 min, and remove the activated carbon by plate-and-frame circular filtration; Microfiltration sterilization to obtain refined egg yolk polypeptide;

(6) spray drying the obtained refined yolk polypeptide, the air inlet temperature is 180°C, and the air outlet temperature is 80°C to obtain the yolk polypeptide powder product;

(7) stirring the phosvitin precipitation obtained in step (3) in a 0.1M NaOH solution at 30° C. at 50 rpm for 2 h; obtaining partially dephosphorized phosvitin after alkali treatment;

(8) Adjust the pH=10 of the partially dephosphorized phosvitin after alkali treatment, add alkaline protease in an amount of 1000U/g, stir at 30rpm for 2h, and use a 100nm ceramic membrane at 0.5MPa, 35°C Lower filtration to obtain phosvitin phosphopeptide clarified solution;

(9) desalting and concentrating the phosvitin phosphopeptide clarified solution with a 300 molecular weight nanofiltration membrane, adding 3 times the volume of pure water for desalting; obtaining a phosvitin phosphopeptide concentrate (solid content is 15%);

(10) carrying out clarification treatment and microfiltration sterilization with the phosvitin phosphopeptide concentrate to obtain the purified phosvitin phosphopeptide concentrate; wherein the clarification treatment is carried out by using a ceramic membrane, and the specific parameters are: The ceramic membrane with a pore size of 100nm is filtered and clarified at 0.6MPa and 50°C; the microfiltration sterilization is specifically the use of a 0.22μm membrane for microfiltration sterilization.

(11) spray-drying the obtained purified phosvitin phosphopeptide concentrate with an inlet air temperature of 180° C. and an outlet air temperature of 80° C to obtain a phosvitin phosphopeptide powder product.

Example 2

An industrialized method for simultaneously preparing phosvitin phosphopeptide and yolk polypeptide, comprising the following steps:

(1) Dissolve the defatted egg yolk powder (containing 65% protein), and control the solid-liquid ratio to be 1:10 (w/v);

(2) adopting 3000rpm rotary disc wet method to pulverize 300 mesh sieves to obtain degreasing egg yolk powder homogenate;

(3) The obtained defatted egg yolk powder was homogenized to adjust pH=7.0, and neutral protease was used for enzymolysis, the addition amount was 3000U/g, and the enzymolysis time was stirred for 3h. The ceramic membrane was filtered and clarified at 0.3MPa and 50 °C, and the clarified and separated yolk polypeptide clarified liquid and phosvitin precipitate were obtained;

(4) Desalting the egg yolk polypeptide clear liquid obtained in step (3) by nanofiltration membrane with a molecular weight of 300, adding 3 times the volume of pure water for desalting, to obtain egg yolk polypeptide concentrate (solid content is 12%);

(5) Add 1.0% (v/w) 200-mesh powder activated carbon to the egg yolk polypeptide concentrate, decolorize at pH 4.0 and temperature 50°C for 30 minutes, and filter the activated carbon by plate and frame circulation; Membrane microfiltration sterilization to obtain refined egg yolk polypeptide;

(6) spray drying the obtained refined yolk polypeptide, the air inlet temperature is 180°C, and the air outlet temperature is 80°C to obtain the yolk polypeptide powder product;

(7) The phosvitin precipitation obtained in step (3) was stirred in a 0.1 M NaOH solution at 30° C. and 40 rpm for 2 h; the partially dephosphorized phosvitin after alkali treatment was obtained;

(8) Adjust the pH=10 of the partially dephosphorized phosvitin after alkali treatment, add alkaline protease in an amount of 3000U/g, stir for 3h at 30rpm, and use a 500nm ceramic membrane at 0.4MPa, 50 Filter and clarify at ℃ to obtain phosvitin phosphopeptide clarified solution;

(9) desalting and concentrating the phosvitin phosphopeptide clarified solution with a 300 molecular weight nanofiltration membrane, adding 3 times the volume of pure water for desalting; obtaining a phosvitin phosphopeptide concentrate (solid content is 15%);

(10) carrying out clarification treatment and microfiltration sterilization of the phosvitin phosphopeptide concentrate to obtain a purified phosvitin phosphopeptide concentrate; wherein the clarification treatment is carried out by using a ceramic membrane, using a 500nm ceramic membrane The membrane was filtered and clarified at 0.3MPa and 50°C, and microfiltration sterilization was specifically performed by using a 0.45 μm membrane microfiltration sterilization.

(11) spray-drying the obtained purified phosvitin phosphopeptide concentrate, with an inlet air temperature of 180°C and an outlet air temperature of 80°C, to obtain a phosvitin phosphopeptide powder product.

The obtained egg yolk product is subjected to molecular weight detection, and the detection results are shown in Table 1 and Figure 2: it can be seen from Table 1 and Figure 2 that the molecular weight distribution of the main peptide segments of the egg yolk polypeptide is concentrated in the 180-500Da range, indicating that the egg yolk polypeptide product The highest content is the small peptides from dipeptide to pentapeptide. These small peptides are most easily absorbed by the human body, indicating that egg yolk polypeptide products have good absorption performance and nutritional value in vivo.

Table 1 Molecular weight distribution of egg yolk polypeptides in egg yolk polypeptide products

Molecular weight/Da keep time Mn Mw MP area %area 10000 11.871 13685 13750 13915 2112 0.00 10000-5000 13.683 5708 5784 5000 65875 0.07 5000-3000 14.588 3569 3634 2999 609917 0.65 3000-2000 15.305 2346 2377 2001 1742566 1.87 2000-1000 16.502 1325 1377 1018 11069623 11.88 1000-500 17.540 655 679 566 25145478 26.98 500-180 18.802 287 309 278 43251229 46.40 <180 19.568 99 128 180 11319078 12.14

The obtained phosvitin phosphopeptide powder product is subjected to molecular weight detection, and the test results are shown in Table 2 and Figure 3: From Table 2 and Figure 3, it can be seen that the molecular weight distribution of the main peptide segments of phosvitin phosphopeptide is concentrated. In the range of 180-2000Da, it shows that the phosvitin phosphopeptide product has a small molecular weight and is easily absorbed by the human body, and its metal ion chelation ability is good, indicating that the phosvitin phosphopeptide product has good absorption performance and metal ions in the body. Ion chelating ability.

Table 2 Molecular weight distribution of phosvitin phosphopeptides in phosvitin phosphopeptide products

Molecular weight/Da keep time Mn Mw MP area %area 10000 12.457 12097 12500 9995 287105 0.52 10000-5000 13.683 6351 6563 5000 2868022 5.23 5000-3000 14.588 3758 3839 2999 5154956 9.40 3000-2000 15.305 2416 2449 2001 5689853 10.38 2000-1000 15.912 1384 1439 1420 11305016 20.62 1000-500 17.133 698 725 713 11988263 21.86 500-180 18.613 301 323 309 14105637 25.72 <180 19.568 45 112 180 3435543 6.27

Example 3

An industrialized method for simultaneously preparing phosvitin phosphopeptide and yolk polypeptide, comprising the following steps:

(1) Dissolve defatted egg yolk powder (containing 75% protein), and control the ratio of material to liquid to be 1:9 (w/v);

(2) adopt 3000rpm rotary disc wet pulverization to pass through 200 mesh sieves, obtain defatted egg yolk powder homogenate;

(3) The obtained defatted egg yolk powder was homogenized to adjust pH=7.0, and neutral protease was used for enzymolysis. The 200nm ceramic membrane was filtered and clarified at 0.2MPa and 60°C to clarify the separated yolk polypeptide clarified liquid and phosvitin precipitation;

(4) Desalting the egg yolk polypeptide clarified solution obtained in step (3) through a nanofiltration membrane with a molecular weight of 300, adding 3 times the volume of pure water for desalting to obtain egg yolk polypeptide concentrate (solid content is 13%);

(5) Add 2.0% (v/w) 200-mesh powder activated carbon to the egg yolk polypeptide concentrate, decolorize at pH 4.0 and temperature 30°C for 30 min, and filter the activated carbon by plate and frame circulation; Microfiltration sterilization to obtain refined egg yolk polypeptide;

(6) spray-drying the obtained refined yolk polypeptide, the air inlet temperature is 180°C, and the air outlet temperature is 80°C to obtain a yolk polypeptide powder product;

(7) stirring the phosvitin precipitation obtained in step (3) in a 0.1M NaOH solution at 50° C. at 40 rpm for 2 h; obtaining partially dephosphorized phosvitin after alkali treatment;

(8) Adjust the pH=10 of the partially dephosphorized phosvitin after alkali treatment, add alkaline protease in an amount of 1000U/g, stir for 2h at 50rpm, and use a 200nm ceramic membrane at 0.2MPa, 35 Filter and clarify at ℃ to obtain phosvitin phosphopeptide clarified solution;

(9) desalting and concentrating the phosvitin phosphopeptide clarified liquid with a 300 molecular weight nanofiltration membrane, adding 3 times the volume of pure water for desalting; obtaining a phosvitin phosphopeptide concentrate (solid content is 14%);

(10) carrying out clarification treatment and microfiltration sterilization of the phosvitin phosphopeptide concentrate to obtain a purified phosvitin phosphopeptide concentrate; wherein the clarification treatment is carried out by using a ceramic membrane, using a 200nm ceramic membrane The membrane was filtered and clarified at 0.3MPa and 50°C, and microfiltration sterilization was specifically performed by using a 0.5 μm membrane microfiltration sterilization.

(11) spray-drying the obtained purified phosvitin phosphopeptide concentrate with an inlet air temperature of 180° C. and an outlet air temperature of 80° C to obtain a phosvitin phosphopeptide powder product.

Example 4

An industrialized method for simultaneously preparing phosvitin phosphopeptide and yolk polypeptide, comprising the following steps:

(1) Dissolve defatted egg yolk powder (containing 90% protein), and control the solid-liquid ratio to be 1:10 (w/v);

(2) wet pulverizing through a 300-mesh sieve to obtain a homogenate of defatted egg yolk powder;

(3) The obtained defatted egg yolk powder was homogenized to adjust pH=7.0, and neutral protease was used for enzymolysis. The 100nm ceramic membrane was filtered and clarified at 0.8MPa and 30°C, and the yolk polypeptide clarified liquid and phosvitin precipitate were obtained by separation;

(4) Desalting the egg yolk polypeptide clarified solution obtained in step (3) through a nanofiltration membrane with a molecular weight of 300, adding 3 times the volume of pure water for desalting, to obtain an egg yolk polypeptide concentrate (solid content is 18%);

(5) Add 1.0% (v/w) 200-mesh powder activated carbon to the egg yolk polypeptide concentrate, decolorize at pH 4.0 and temperature 50°C for 30 min, and filter out the activated carbon by circular filtration; Microfiltration sterilization to obtain refined egg yolk polypeptide;

(6) spray-drying the obtained refined yolk polypeptide, the air inlet temperature is 180°C, and the air outlet temperature is 80°C to obtain a yolk polypeptide powder product;

(7) stirring the phosvitin precipitation obtained in step (3) in a 0.2M NaOH solution at 30° C. at 30 rpm for 3 hours; obtaining partially dephosphorized phosvitin after alkali treatment;

(8) Adjust pH=10 of the partially dephosphorized phosvitin after alkali treatment, add alkaline protease in an amount of 3000U/g, stir for 3h at 30rpm, and use a 100nm ceramic membrane at 0.3MPa, 50 Filter and clarify at ℃ to obtain phosvitin phosphopeptide clarified solution;

(9) desalting and concentrating the phosvitin phosphopeptide clarified liquid with a 300 molecular weight nanofiltration membrane, adding 3 times the volume of pure water for desalting; obtaining a phosvitin phosphopeptide concentrate (solid content is 11%);

(10) carrying out clarification treatment and microfiltration sterilization to the phosvitin phosphopeptide concentrate to obtain the purified phosvitin phosphopeptide concentrate; wherein the clarification treatment is performed by using a ceramic membrane, using a 100 nm ceramic membrane The membrane is filtered and clarified at 0.6MPa and 30°C, and microfiltration sterilization is specifically performed by using a membrane of 0.22-0.45 μm for microfiltration sterilization.

(11) spray-drying the obtained purified phosvitin phosphopeptide concentrate with an inlet air temperature of 180° C. and an outlet air temperature of 80° C to obtain a phosvitin phosphopeptide powder product.

The yolk polypeptide powder products and phosvitin phosphopeptide powder products obtained in Examples 1-4 were tested for performance, and the test results are shown in Table 3.

The test result of table 3 embodiment 1-4

Comparative Example 1

Step (2) in Example 2 was adjusted to heat the defatted egg yolk powder solution of Step (1) at 90° C. for 30 min; otherwise, the same as in Example 2, yolk polypeptide powder products and phosvitin phosphopeptide powder products were obtained.

Comparative Example 2

Step (2) of Example 2 was adjusted to treat the defatted egg yolk powder solution of Step (1) with 300W ultrasonic wave for 10 min; otherwise, the same as in Example 2, yolk polypeptide powder products and phosvitin phosphopeptide powder products were obtained.

Comparative Example 3

Step (2) of Example 2 was omitted, and the others were the same as those of Example 2, to obtain a yolk polypeptide powder product and a phosvitin phosphopeptide powder product.

The yolk polypeptide powder products and phosvitin phosphopeptide powder products obtained in Comparative Examples 1-3 were tested for performance, and the test results are shown in Table 4.

Table 4 Test results of comparative examples 1-3

Example 5

The pore size of the ceramic membrane in step (3) in Example 2 was adjusted as shown in Table 5, and the others were the same as those in Example 2 to obtain yolk polypeptide powder products and phosvitin phosphopeptide powder products.

The obtained yolk polypeptide powder product and phosvitin phosphopeptide powder product were tested for performance, and the test results are shown in Table 5.

Table 5 Test results of ceramic membranes with different pore sizes

Comparative Example 4

In step (3) in the adjustment example 2, the clarification by ceramic membrane filtration is centrifugation (centrifugal force 4000g, centrifugation time is 30min) and the others are consistent with embodiment 2, and severe emulsification occurs during the separation process, which cannot be effectively separated. The egg yolk polypeptide obtained and phosvitin phosphopeptides of lower purity.

Comparative Example 5

In step (3) in the adjustment example 2, the clarification of ceramic membrane filtration is plate and frame filtration (filtration pressure 6bar, and the filter aid is diatomaceous earth), and the others are consistent with embodiment 2. Effective separation, and the loss is relatively large, the yield of the obtained yolk polypeptide and phosvitin phosphopeptide is low.

The yolk polypeptide powder products and phosvitin phosphopeptide powder products obtained in Comparative Examples 4 and 5 were tested for performance, and the test results are shown in Table 6.

The test results of table 6 comparative examples 4 and 5

Example 6

The dosage of neutral protease in step (3) in Example 2 was adjusted as shown in Table 7, and the others were the same as those in Example 2 to obtain yolk polypeptide powder products and phosvitin phosphopeptide powder products.

The obtained yolk polypeptide powder product and phosvitin phosphopeptide powder product were tested for performance, and the test results are shown in Table 7.

The test result of the dosage of table 7 different neutral protease

Example 7

The dosage of alkaline protease in step (8) in Example 2 was adjusted as shown in Table 8, and the others were consistent with Example 2 to obtain yolk polypeptide powder products and phosvitin phosphopeptide powder products.

The obtained yolk polypeptide powder product and phosvitin phosphopeptide powder product were tested for performance, and the test results are shown in Table 8.

The test result of the consumption of table 8 different alkaline protease

Comparative Example 6

A method for preparing phosvitin phosphopeptide in a laboratory, comprising the following steps:

(1) Dissolve defatted egg yolk powder (containing 65% protein) with 12% sodium chloride solution, and control the ratio of material to liquid to be 1:10 (w/v);

(2) Centrifuge at 8000g for 30min, and take the supernatant;

(3) heating the supernatant to 90°C for 30min;

(4) suction filtration, removes impurity protein, obtains phosvitin solution;

(5) stirring the phosvitin solution obtained in step (4) with a 0.1M NaOH solution at 50° C. at 40 rpm for 2 h; obtaining partially dephosphorized phosvitin after alkali treatment;

(6) adjusting the pH=10 of the partially dephosphorized phosvitin after the alkali treatment, adding alkaline protease, adding an amount of 3000U/g, and stirring the enzymolysis time at 30rpm for 3h to obtain an enzymolysis solution;

(7) Centrifuge the enzymatic hydrolyzate obtained in step (6) at 8000 g for 30 min to obtain a phosvitin phosphopeptide supernatant.

(8) freeze-drying the phosvitin phosphopeptide supernatant to obtain a phosvitin phosphopeptide powder product.

The above is the extraction method commonly used in the laboratory. Only one phosvitin phosphopeptide product is obtained by this method, and the yield is only 2.3%, and the N/P is 5.6. A huge waste of resources.

Comparative Example 7

A method for preparing egg yolk polypeptide in a laboratory, comprising the following steps:

(1) Dissolve defatted egg yolk powder (containing 65% protein) with 0.1M sodium hydroxide solution, control the material-liquid ratio to be 1:10 (w/v), and stir at 30rpm for 3h at 37°C;

(2) Adjust the pH of the system after (1) treatment to 10, add neutral protease, the amount of addition is 3000U/g, and the enzymolysis time is 3h with stirring at 50rpm to obtain an enzymolysis solution;

(3) Centrifuge the enzymatic hydrolyzate solution obtained in (2) at 8000 g for 30 min to obtain egg yolk polypeptide supernatant.

(4) freeze-drying the yolk polypeptide supernatant to obtain a yolk polypeptide powder product.

The above is the extraction method commonly used in the laboratory. Only one yolk polypeptide product was obtained by this method. The yield was 68.9% and the N/P was 65.6. It is effectively separated, resulting in lower added value of the product.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the technology and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (2)

1. a method for industrialized preparation of phosvitin phosphopeptide and yolk polypeptide simultaneously, is characterized in that, comprises the steps: (1) Preprocessing: First dissolve the defatted egg yolk powder, control the material-liquid ratio to be 1:8-1:12, and mix evenly; after that, after wet pulverization or homogenization, sieve to obtain the defatted egg yolk powder homogenate; (2) The first enzymatic hydrolysis: the defatted egg yolk powder homogenate in step (1) is subjected to enzymatic hydrolysis to obtain an enzymatic hydrolysis solution of egg yolk polypeptide; wherein, neutral protease is used for enzymatic hydrolysis; The addition amount of neutral protease is 1000-3000U/g, the enzymolysis temperature is 30-60℃, and the enzymolysis time is 2-4h; (3) Clarification and separation: adjust the pH of the yolk polypeptide enzymatic hydrolysate obtained in step (2), and then use a ceramic membrane for clarification and separation to obtain yolk polypeptide clarified solution and phosvitin precipitate; wherein, the pore size of the ceramic membrane is: 200-500nm; (4) performing nanofiltration, desalting, concentration, refining and drying on the egg yolk polypeptide clarified liquid obtained in step (3) to obtain egg yolk polypeptide products; (5) Alkaline treatment, dephosphorization, second enzymatic hydrolysis, clarification, desalting concentration, refining and drying are performed on the phosvitin precipitate obtained in step (3) to obtain a phosvitin phosphopeptide product; Alkaline protease is used for enzymatic hydrolysis; the amount of enzyme added in the second enzymatic hydrolysis is 1000-3000 U/g, pH=8.0-10.5, and the enzymatic hydrolysis is carried out at 30-60° C. for 2-4 hours. 2 . The method according to claim 1 , wherein the pH is adjusted to 4.0-4.5 in step (3). 3 .

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