CN117448407A - Method for co-producing hydrolyzed egg yolk peptide and phospholipid DHA - Google Patents
Method for co-producing hydrolyzed egg yolk peptide and phospholipid DHA Download PDFInfo
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- CN117448407A CN117448407A CN202311273031.XA CN202311273031A CN117448407A CN 117448407 A CN117448407 A CN 117448407A CN 202311273031 A CN202311273031 A CN 202311273031A CN 117448407 A CN117448407 A CN 117448407A
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- 210000002969 egg yolk Anatomy 0.000 title claims abstract description 82
- 238000000034 method Methods 0.000 title claims abstract description 41
- 150000003904 phospholipids Chemical class 0.000 title claims abstract description 39
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- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 18
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 18
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- 239000000843 powder Substances 0.000 claims abstract description 64
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 24
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- 239000004365 Protease Substances 0.000 claims abstract description 21
- 230000007062 hydrolysis Effects 0.000 claims abstract description 17
- 108091005804 Peptidases Proteins 0.000 claims abstract description 15
- 239000000287 crude extract Substances 0.000 claims abstract description 14
- 238000010008 shearing Methods 0.000 claims abstract description 12
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 11
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000012752 auxiliary agent Substances 0.000 claims abstract description 8
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000009849 deactivation Effects 0.000 claims abstract description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 3
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- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract 5
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- 238000004108 freeze drying Methods 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 241000287828 Gallus gallus Species 0.000 claims description 6
- 108090000526 Papain Proteins 0.000 claims description 6
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- 102000004142 Trypsin Human genes 0.000 claims description 5
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- 239000012588 trypsin Substances 0.000 claims description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 4
- 235000011152 sodium sulphate Nutrition 0.000 claims description 4
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 3
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical group [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- WBHQBSYUUJJSRZ-UHFFFAOYSA-M sodium bisulfate Chemical compound [Na+].OS([O-])(=O)=O WBHQBSYUUJJSRZ-UHFFFAOYSA-M 0.000 claims description 3
- 229910000342 sodium bisulfate Inorganic materials 0.000 claims description 3
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 claims description 3
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical group [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims description 3
- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 3
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- 239000007788 liquid Substances 0.000 claims description 2
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- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 12
- 229920001184 polypeptide Polymers 0.000 abstract description 10
- 235000013305 food Nutrition 0.000 abstract description 6
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- 108010033276 Peptide Fragments Proteins 0.000 abstract description 3
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- 238000009835 boiling Methods 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 230000000415 inactivating effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 102000004895 Lipoproteins Human genes 0.000 description 4
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- 210000004907 gland Anatomy 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229940106134 krill oil Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
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- 210000001525 retina Anatomy 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000004289 sodium hydrogen sulphite Substances 0.000 description 1
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- 238000001228 spectrum Methods 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
- C07F9/103—Extraction or purification by physical or chemical treatment of natural phosphatides; Preparation of compositions containing phosphatides of unknown structure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
The invention discloses a method for co-producing hydrolyzed egg yolk peptide and phospholipid DHA, which comprises the following steps: mixing yolk powder and a phosphoric acid buffer solution, performing ultrasonic shearing treatment, adding a hydrolysis auxiliary agent, and adjusting the pH to 7-10; the hydrolysis auxiliary agent is one or more of sulfate, sulfite or thiosulfate; then adding protease, performing enzyme hydrolysis reaction and enzyme deactivation to obtain crude extract, separating by layers to obtain phospholipid DHA at the upper layer and hydrolyzed egg yolk peptide at the lower layer. The method can obtain hydrolyzed egg yolk peptide and phospholipid DHA in one step. The solubility of the obtained yolk peptide is 100%, the small molecular peptide accounts for more than 95%, the hydrolysis degree is more than 10%, the protein content is more than 30g/L, the polypeptide content is more than 9g/L, the extracted yolk oil rate is more than 75%, and the phospholipid DHA ratio is more than 10%. The method is simple, the prepared hydrolyzed yolk powder peptide fragment has reasonable molecular mass distribution, is suitable for human digestion and utilization, can reduce the harm and waste of organic reagent for extracting food oil substances, and is suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of biological enzymolysis, and particularly relates to a method for co-producing hydrolyzed egg yolk peptide and phospholipid DHA.
Background
The hydrolyzed yolk powder is a functional substance extracted from ovum gallus Domesticus flavus, and can be further extracted by proteolytic hydrolysis, ultrasonic salting, centrifugal separation, spray drying, etc. to obtain small peptide product. The hydrolyzed yolk powder is formally approved as a new resource food in 9/2008 and can be used in general foods. The protein and polypeptide in the hydrolyzed yolk powder can promote bone growth and health, and has anti-inflammatory and antioxidant bioactivity, and has important effect in preventing diseases. It also has the advantages of easy digestion and absorption, no generation of allergen or dermatitis, etc. Therefore, the hydrolyzed yolk powder can be widely applied to the related fields of foods, health-care products, medicines and the like, is also very wide in application population, can be added to bone health-care products for infants, teenagers and children, and has good industrial application prospect.
The phospholipid DHA has unique biological activity and function, has relatively high bioavailability, and has far higher human absorption rate than glyceride DHA and ethyl ester DHA. In addition, after the phospholipid is combined with DHA, the oxidation resistance of DHA can be improved. In addition, in some studies, phospholipid DHA exhibits a range of benefits, and phospholipid DHA can enhance the action of hydrophilic and hydrophobic compounds beneficial to health, such as cell differentiation agents, anticancer compounds, and anti-obesity compounds. The krill oil treatment can obviously reduce total cholesterol, low density lipoprotein and triglyceride and manage hyperlipidemia when being administered to patients with hyperlipidemia. Can also relieve menstrual pain, treat mental diseases, reduce dementia risk, maintain higher proportion of serum phospholipid DHA, be beneficial to reducing cardiovascular risk, improve cognitive function defect and the like. It also has effects of promoting infant retina development, reducing risk of nervous system diseases, and resisting tumor, and can be used as health product and food nutrition enhancer.
The hydrolyzed yolk powder in the market at present is mostly obtained by taking defatted yolk powder as a raw material and performing enzymolysis reaction. Although the grease is extracted more thoroughly, the grease is extracted by using an organic reagent, the time and the labor are wasted, the production cost is high, protein in the defatted yolk powder is denatured, caking is formed, the hydrolysis is difficult, and further damage and treatment are needed. Therefore, development of a production process with low production cost, short period and easy control is needed.
Disclosure of Invention
Aiming at the problems that in the existing method, the first step of extraction and oil extraction is needed for egg yolk powder before proteolysis, and the denaturation of lipid macroprotein embedded by an oil layer in the process is serious, so that the proteolysis of the later step is difficult, and the ratio of substances such as small peptide, amino acid and the like is too high due to excessive enzymatic hydrolysis after protein precipitation, the invention develops a method for co-producing hydrolyzed egg yolk peptide and phospholipid DHA, which can realize the one-step enzymolysis extraction of various active substances, is green and has low carbon pollution, can reduce the cost and the waste and pollution of organic reagents, and is suitable for industrial production.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a method for co-producing hydrolyzed egg yolk peptide and phospholipid DHA, comprising the following steps:
(1) Pretreatment: mixing yolk powder and a phosphoric acid buffer solution, performing ultrasonic post-shearing treatment, adding a hydrolysis auxiliary agent, and adjusting the pH to 7-10, preferably 8, so as to obtain a pretreated substrate; the hydrolysis auxiliary agent is one or more of sulfate, sulfite or thiosulfate;
(2) Hydrolysis: adding protease into the pretreated substrate, performing enzyme hydrolysis reaction and enzyme deactivation to obtain a crude extract, performing layering separation on the crude extract to obtain phospholipid DHA at the upper layer and hydrolyzed yolk peptide at the lower layer.
Preferably, the yolk powder in the step (1) is one or more of pure raw yolk powder, soybean yolk powder or phospholipid DHA yolk powder. More preferably, the phospholipid-type DHA egg yolk powder is derived from chicken eggs fed with DHA algae residues or algae powder.
Preferably, the phospholipid DHA egg yolk powder is obtained by the following method:
separating yolk of chicken eggs fed with DHA algae residue or algae powder, and spray drying to obtain phospholipid type DHA yolk powder.
Preferably, the mass ratio of the yolk powder to the phosphate buffer solution in the step (1) is 1:5-10.
Preferably, the concentration of the phosphate buffer in the step (1) is 10 to 50mmol/L.
Preferably, the sulfate in the step (1) is sodium sulfate or sodium bisulfate, the sulfite is sodium bisulfite or sodium sulfite, and the thiosulfate is sodium thiosulfate.
Preferably, the mass ratio of the yolk powder to the hydrolysis auxiliary agent in the step (1) is 100:2-5.
Preferably, the power of the ultrasonic wave in the step (1) is 65-325W, the time is 10-30 min, and the shearing is performed by a shearing machine, and the time is 0.5-5 min.
Preferably, the protease in the step (2) is one or more of neutral protease, alkaline protease, flavourzyme, papain or trypsin. More preferably, the protease is a mixture of neutral protease, alkaline protease and flavourzyme, and the mass ratio of the neutral protease, the alkaline protease and the flavourzyme is 2:1:0.5. The hydrolyzed product under the condition has the best comprehensive polypeptide content and product taste, and is more suitable for food application.
Preferably, the amount of protease used in step (2) is 2% (w/w) to 6% (w/w) of the total mass of the pretreated substrate.
Preferably, the crude extract is obtained through centrifugal treatment after enzyme deactivation in the step (2), wherein the centrifugal rotation speed is 8000-12000 r/min, and the time is 10-30 min.
Preferably, the method for inactivating enzyme in the step (2) comprises the following steps: boiling.
Preferably, the method for obtaining the hydrolyzed egg yolk peptide in the step (2) comprises the following steps: and (5) decoloring the lower layer liquid, and freeze-drying.
Preferably, the decoloring treatment method is one or more of activated carbon decoloring, macroporous resin decoloring or sephadex decoloring.
Preferably, the freeze drying is performed by a gland type freeze dryer under the conditions that the cold trap temperature is between minus 60 ℃ and minus 80 ℃ and the vacuum degree is between 0 Pa and 2 Pa.
Preferably, the method for obtaining the phospholipid DHA in the step (2) comprises the following steps: and (3) rotary evaporating the supernatant, wherein the temperature of the rotary evaporation is 60-80 ℃ and the time is 5-20 min.
The invention has the beneficial effects that:
(1) The invention provides a method for directly carrying out enzymolysis on yolk powder to obtain phospholipid DHA and yolk peptide by a one-step method, which does not need organic solvent to extract oil, has mild process and is green and safe; the enzyme can be reused, so that waste and pollution are avoided, and the method accords with the low-carbon environment-friendly concept.
(2) The invention further provides a method for preparing the phospholipid DHA, which comprises the steps of feeding chickens with DHA algae residues and algae powder, obtaining eggs containing the phospholipid DHA through in-vivo enrichment, spray-drying the eggs into the phospholipid DHA yolk powder, and then extracting the phospholipid DHA from the yolk powder. The method for extracting the phospholipid DHA is not an organic solvent extraction method, and no organic solvent is left;
(3) The invention provides a processing method combining physical and chemical pretreatment and enzymolysis treatment, which is used for efficiently obtaining yolk polypeptide, wherein the peptide with the relative molecular weight of less than 10000Da accounts for more than 95%, the peptide with the relative molecular weight of less than 2000Da accounts for more than 80%, the processing method reaches the national primary standard of peptides, the hydrolysis degree is more than 10%, the protein content is more than 30g/L, the polypeptide content is more than 9g/L, the extracted yolk oil rate is more than 75%, and the proportion of phospholipid DHA is more than 10%.
Since the change of the disulfide bond content in the protein has a significant effect on the proteolytic properties and the functional activity of the enzymatic product thereof, the sulfate, sulfite or thiosulfate, which can be used as a food additive, can break the disulfide bonds of the protein. The salts have a binding effect with proteins, the main forces of the binding of the salts are hydrogen bonds and van der Waals forces, and the salts change the conformation of the proteins by reducing disulfide bonds in the proteins to form sulfonates. Ultrasonic shearing and sulfate, sulfite or thiosulfate can open disulfide bonds of proteins through a physical method and a chemical method respectively, so that the content of free sulfhydryl groups and the surface hydrophobicity are increased, and certain complementarity exists. The ultrasonic shearing adopted in the method can cause cavitation to influence the change of protein conformation, so that more proteins and polypeptides are adsorbed on the interface, and the solubility of the proteins is improved; the added sulfate, sulfite or thiosulfate can break disulfide bonds of the protein, so that the mechanism of the protein is looser, hydrophobic groups are better exposed, the protein and grease are separated out, the solubility of the protein is increased, and enzymatic reaction is facilitated. The method provided by the invention is scientific and effective, and the prepared product meets the requirements.
Drawings
FIG. 1 is a flow chart of the method of example 1.
FIG. 2 shows a spectrum of phospholipid DHA analyzed by GC-MS in example 1.
Example 1
Comparative example, no physical and chemical pretreatment steps:
(1) And (3) feeding chickens with DHA algae residues or algae powder to obtain DHA-enriched eggs. Washing and drying egg, separating to obtain yolk by egg separator, and spray drying to obtain yolk powder.
Pretreatment: mixing yolk powder and 0.01M phosphate buffer solution according to a mass ratio of 1:5, and directly adjusting PH=8 without pretreatment to obtain a pretreated sample.
(2) Adding neutral protease into the pretreated sample obtained in the step (1), wherein the enzyme addition amount is 2% (w/w) of the total mass of the pretreated sample, mixing to carry out hydrolysis reaction, boiling in water bath, inactivating enzyme at high temperature for 20min, centrifuging for 10min at 12000r/min, and obtaining the dissolved hydrolyzed yolk powder and phospholipid DHA crude extract.
(3) And (3) layering and separating the crude extract obtained in the step (2), extracting the phospholipid DHA from the upper layer by rotary evaporation at 75 ℃ for 10min, decoloring the supernatant by macroporous resin for 20min, freeze-drying, crushing and sieving to obtain hydrolyzed yolk powder.
The protein is detected by BCA method, the content of the polypeptide is detected by trichloroacetic acid method, the Degree of Hydrolysis (DH), the molecular weight of the polypeptide is detected by SDS-PAGE and HPLC, and phospholipid DHA is detected by GC-MS method.
Example 2
Comparative example, no physical, chemical pretreatment step and no protease added:
the experimental conditions were the same as in example 1 except that no protease was added to the resulting pretreated sample in step (2) as a blank.
Example 3
In the comparative example, no physical and chemical pretreatment steps are carried out, and complex protease is added:
the experimental conditions were the same as in example 1 except that neutral protease, alkaline protease and flavourzyme were added to the pretreated sample in step (2) in the amounts of 2%, 1% and 0.5% (w/w) of the total mass of the pretreated sample, respectively.
Example 4
Comparative example, no physical pretreatment step, with chemical pretreatment step:
the experimental conditions were the same as in example 1, except that sodium sulfite was added to the sample mixed in step (1) in an amount of 5% by mass of the yolk powder.
Example 5
In the comparative example, there is a physical pretreatment step, no chemical pretreatment step, and a complex protease is added:
the experimental conditions were the same as in example 1, except that the samples mixed in step (1) were subjected to physical ultrasonic treatment at a power of 65W for 30min and sheared for 3min to aid in enzymolysis, and neutral protease, alkaline protease and flavourzyme were added to the pretreated samples in step (2) in an amount of 2%, 1% and 0.5% (W/W) of the total mass of the pretreated samples, respectively.
Example 6
Comparative example, with physical pretreatment step, without chemical pretreatment step:
the experimental conditions were the same as in example 1, except that the samples mixed in step (1) were subjected to physical ultrasonic treatment at a power of 65W for 30min and sheared for 3min to aid in enzymolysis.
Example 7
Comparative example, without physical, chemical pretreatment steps, will be treated in UP aqueous environment:
the experimental conditions were the same as in example 1 except that the yolk powder and UP water were mixed in the mass ratio of 1:5 in step (1).
Example 8
The experimental conditions were the same as in example 1, except that the samples mixed in step (1) were subjected to physical ultrasonic treatment at a power of 65W for 30min and sheared for 3min, and then sodium sulfite was added in an amount of 5% of the mass of the yolk powder to aid enzymolysis.
Example 9
The experimental conditions were the same as in example 1, except that the mixed sample in step (1) was subjected to physical ultrasonic treatment at a power of 65W for 30min and then sheared for 3min, and then sodium sulfite was added in an amount of 5% of the mass of the yolk powder to aid enzymolysis. Then in the step (2), neutral protease, alkaline protease and flavourzyme are added into the pretreated sample, wherein the added enzyme amounts are respectively 2%, 1% and 0.5% (w/w) of the total mass of the pretreated sample.
Example 10
The experimental conditions were the same as in example 1, except that the samples mixed in step (1) were subjected to physical ultrasonic treatment at a power of 65W for 30min and then sheared for 3min, and then sodium sulfite was added in an amount of 5% by mass of the yolk powder to aid in enzymolysis, and neutral protease and alkaline protease were added to the pretreated samples in step (2) in an amount of 2% and 1% (W/W) of the total mass of the pretreated samples.
Example 11
The experimental conditions were the same as in example 1, except that the mixed sample in step (1) was subjected to physical ultrasonic treatment at a power of 65W for 30min and then sheared for 3min, and then sodium sulfite was added in an amount of 5% of the mass of the yolk powder to aid enzymolysis. And (2) adding neutral protease and flavourzyme into the pretreated sample in the step (2), wherein the added enzyme amount is 2% and 1% (w/w) of the total mass of the pretreated sample.
Example 12
The experimental conditions were the same as in example 1, except that the mixed sample in step (1) was subjected to physical ultrasonic treatment at a power of 65W for 30min and then sheared for 3min, and then sodium sulfite was added in an amount of 5% of the mass of the yolk powder to aid enzymolysis. And (2) adding neutral protease and papain into the pretreated sample in the step (2), wherein the enzyme adding amount is 2% and 1% (w/w) of the total mass of the pretreated sample.
Example 13
(1) Yolk powder was obtained in the same manner as in (1) in example 1.
Pretreatment: mixing yolk powder and 0.05M phosphate buffer solution according to a mass ratio of 1:5, performing physical ultrasonic treatment, performing power of 325W for 10min, shearing for 5min, adding sodium bisulfate accounting for 5% of the mass of the yolk powder to assist enzymolysis, and adjusting PH=7 to obtain a pretreated sample.
(2) Adding neutral protease and alkaline protease into the pretreated sample obtained in the step (1), mixing the mixture with the enzyme amount which is 2 percent and 1 percent (w/w) of the total mass of the pretreated sample, carrying out hydrolysis reaction, boiling in water bath, inactivating enzyme at high temperature for 20min, centrifuging for 30min at 8000r/min, and obtaining the dissolved hydrolyzed yolk powder and phospholipid DHA crude extract.
(3) And (3) layering and separating the crude extract obtained in the step (2), extracting the phospholipid DHA from the upper layer by rotary evaporation at 80 ℃ for 5min, decoloring the supernatant by using active carbon for 20min, freeze-drying, crushing and sieving to obtain hydrolyzed yolk powder.
Example 14
(1) Yolk powder was obtained in the same manner as in (1) in example 1.
Pretreatment: mixing yolk powder and 0.01M phosphate buffer solution according to a mass ratio of 1:10, performing physical ultrasonic treatment, performing power 260W for 15min, shearing for 30s, adding sodium thiosulfate accounting for 2% of the mass of the yolk powder to assist enzymolysis, and adjusting pH=7 to obtain a pretreated sample.
(2) Adding neutral protease, alkaline protease and flavourzyme into the pretreated sample obtained in the step (1), wherein the enzyme addition amounts are respectively 2%, 1% and 0.5% (w/w) of the total mass of the pretreated sample, mixing to carry out hydrolysis reaction, boiling in water bath, inactivating enzyme at high temperature for 20min, and centrifuging for 15min at 10000r/min to obtain the dissolved hydrolyzed yolk powder and phospholipid DHA crude extract.
(3) And (3) layering and separating the crude extract obtained in the step (2), extracting the phospholipid DHA from the upper layer by rotary evaporation at 60 ℃ for 20min, decoloring the supernatant by using sephadex for 20min, freeze-drying, crushing and sieving to obtain the hydrolyzed yolk powder.
Example 15
(1) Yolk powder was obtained in the same manner as in (1) in example 1.
Pretreatment: mixing yolk powder and 0.01M phosphate buffer solution according to a mass ratio of 1:7, performing physical ultrasonic treatment, performing power of 125W for 15min, shearing for 30s, adding sodium bisulphite accounting for 3% of the mass of the yolk powder to assist enzymolysis, and adjusting PH=10 to obtain a pretreated sample.
(2) Adding neutral protease, alkaline protease, flavourzyme and trypsin into the pretreated sample obtained in the step (1), wherein the enzyme addition amounts are respectively 2%, 1% and 2% (w/w) of the total mass of the pretreated sample, mixing to carry out hydrolysis reaction, boiling in water bath, inactivating enzyme at high temperature for 20min, and centrifuging for 15min at 10000r/min to obtain the dissolved hydrolyzed yolk powder and phospholipid DHA crude extract.
(3) And (3) layering and separating the crude extract obtained in the step (2), extracting the phospholipid DHA from the upper layer by rotary evaporation at 60 ℃ for 20min, decoloring the supernatant by macroporous resin for 20min, freeze-drying, crushing and sieving to obtain hydrolyzed yolk powder.
Example 16
The experimental conditions were the same as in example 1, except that the samples mixed in step (1) were subjected to physical ultrasonic treatment at a power of 65W for 30min and then sheared for 3min, and then 5% sodium sulfate by mass of the yolk powder was added to aid in enzymolysis. Then, neutral protease, flavourzyme, alkaline protease and papain are added to the pretreated sample in the step (2), wherein the added enzyme amounts are respectively 2%, 1% and 0.5% (w/w) of the total mass of the pretreated sample.
Example 17
The experimental conditions were the same as in example 1, except that the samples mixed in step (1) were subjected to physical ultrasonic treatment at a power of 65W for 30min and then sheared for 3min, and then 5% sodium sulfate by mass of the yolk powder was added to aid in enzymolysis. Then adding neutral protease, flavourzyme, alkaline protease, trypsin and papain to the pretreated sample in the step (2), wherein the addition amounts of the neutral protease, the flavourzyme, the alkaline protease, the trypsin and the papain are respectively 2%, 1% and 0.5% (w/w) of the total mass of the pretreated sample.
Example 18
The extraction effects in examples 1 to 17 are shown in Table 1, and the molecular weight distribution before and after the enzymolysis is shown in Table 2:
TABLE 1 oil extraction ratio of upper egg yolk grease, lower protein and polypeptide content and bottom sediment mass ratio in examples 1 to 17
TABLE 2 HPLC analysis of the molecular weight ratio before and after enzymatic hydrolysis
| Examples | <1000 | 1000-2000 | 2000-10000 | >10000 | Degree of hydrolysis% |
| 1 | 62.59％ | 20.59％ | 9.87％ | 6.95％ | 10.64 |
| 2 | 44.67％ | 15.66％ | 2.02％ | 37.57％ | 2.44 |
| 3 | 65.82％ | 18.46％ | 15.58％ | 0.17％ | 10.77 |
| 4 | 55.62％ | 14.49％ | 27.04％ | 2.85％ | 8.02 |
| 5 | 58.42％ | 11.41％ | 16.33％ | 13.84％ | 9.09 |
| 6 | 50.72％ | 17.21％ | 5.23％ | 26.74％ | 6.34 |
| 7 | 60.91％ | 21.71％ | 10.36％ | 7.02％ | 9.07 |
| 8 | 59.56％ | 21.76％ | 16.93％ | 1.75％ | 10.22 |
| 9 | 57.18％ | 25.52％ | 8.43％ | 8.87％ | 11.15 |
| 10 | 58.69％ | 25.43％ | 9.86％ | 6.02％ | 10.87 |
| 11 | 65.56％ | 15.74％ | 12.57％ | 6.12％ | 9.56 |
| 12 | 61.72％ | 16.51％ | 13.68％ | 8.07％ | 8.31 |
| 13 | 64.48％ | 18.04％ | 12.42％ | 5.06％ | 7.18 |
| 14 | 61.01％ | 22.19％ | 8.72％ | 8.08％ | 9.47 |
| 15 | 70.75％ | 19.24％ | 9.98％ | 0.03％ | 11.39 |
| 16 | 67.54％ | 20.39％ | 12.07％ | 0.07％ | 11.67 |
| 17 | 71.65％ | 18.18％ | 9.39％ | 0.17％ | 12.22 |
As can be seen from table 1, compared with the non-enzymatic hydrolysis, the single enzymatic hydrolysis and the compound enzymatic hydrolysis, the physical ultrasound and the chemical addition of the sulfate can increase the extraction rate of the upper egg yolk oil, increase the content of the lower protein and polypeptide and reduce the ratio of the bottom sediment quality, the effect of adding the sulfate is most remarkable, and the combination effect of the physical and chemical coupling auxiliary different proteases is better.
As can be seen from Table 2, the enzymatic hydrolysis of the single and complex enzymatic proteins, although having a high degree of hydrolysis, is only a part of the soluble proteins, and has a limited enzymatic hydrolysis effect on the insoluble macromolecular lipoproteins. Therefore, pretreatment is needed, fat-soluble proteins are dissolved and increased through a physical ultrasonic method, proteins with molecular weight of more than 10000Da after compound enzymolysis are subjected to enzymolysis to reduce the molecular weight, chemical addition of sulfate can damage the structure of lipoprotein, so that lipoprotein precipitation promotes oil extraction rate, simultaneously, lipoproteins are dissolved to increase peptide fragments with molecular weight of 2000-10000Da, and the peptide fragments after enzymolysis gradually reduce the molecular weight along with the combination of physicochemical coupling auxiliary multiple proteases, and hydrolyzed yolk peptides can be obtained through a purification step.
Claims (10)
1. The method for co-producing the hydrolyzed egg yolk peptide and the phospholipid DHA is characterized by comprising the following steps of:
(1) Pretreatment: mixing yolk powder and a phosphoric acid buffer solution, performing ultrasonic post-shearing treatment, adding a hydrolysis auxiliary agent, and adjusting the pH to 7-10, preferably 8, so as to obtain a pretreated substrate; the hydrolysis auxiliary agent is one or more of sulfate, sulfite or thiosulfate;
(2) Hydrolysis: adding protease into the pretreated substrate, performing enzyme hydrolysis reaction and enzyme deactivation to obtain a crude extract, performing layering separation on the crude extract to obtain phospholipid DHA at the upper layer and hydrolyzed yolk peptide at the lower layer.
2. The method of claim 1, wherein the yolk powder in step (1) is one or more of pure raw yolk powder, soybean yolk powder, and phospholipid-type DHA yolk powder; preferably, the phospholipid type DHA egg yolk powder is derived from chicken eggs fed with DHA algae residues or algae powder.
3. The method of claim 1, wherein the mass ratio of the yolk powder to the phosphate buffer solution in the step (1) is 1:5-10.
4. The method of claim 1, wherein the sulfate in step (1) is sodium sulfate or sodium bisulfate, the sulfite is sodium bisulfite or sodium sulfite, and the thiosulfate is sodium thiosulfate.
5. The method according to claim 1, wherein the mass ratio of the yolk powder to the hydrolysis auxiliary agent in the step (1) is 100:2-5.
6. The method of claim 1, wherein the power of the ultrasound in step (1) is 65-325 w for 10-30 min, and the shearing is performed by a shearing machine for 0.5-5 min.
7. The method of claim 1, wherein the protease in step (2) is one or more of neutral protease, alkaline protease, flavourzyme, papain or trypsin; preferably, the protease is a mixture of neutral protease, alkaline protease and flavourzyme, and the mass ratio of the neutral protease, the alkaline protease and the flavourzyme is 2:1:0.5.
8. The method according to claim 1, wherein the amount of protease used in step (2) is 2% (w/w) to 6% (w/w) of the total mass of the pretreated substrate.
9. The method according to claim 1, wherein the hydrolyzed egg yolk peptide obtained in step (2) is obtained by: decoloring the lower layer liquid and freeze-drying; preferably, the decoloring treatment method is one or more of activated carbon decoloring, macroporous resin decoloring or sephadex decoloring.
10. The method according to claim 1, wherein the phospholipid-type DHA in step (2) is obtained by: and (3) rotary evaporating the supernatant, wherein the temperature of the rotary evaporation is 60-80 ℃ and the time is 5-20 min.
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