CN110679953A - Preparation method of nano liposome embedded with egg white source active peptide - Google Patents
Preparation method of nano liposome embedded with egg white source active peptide Download PDFInfo
- Publication number
- CN110679953A CN110679953A CN201911009978.3A CN201911009978A CN110679953A CN 110679953 A CN110679953 A CN 110679953A CN 201911009978 A CN201911009978 A CN 201911009978A CN 110679953 A CN110679953 A CN 110679953A
- Authority
- CN
- China
- Prior art keywords
- egg white
- active peptide
- liposome
- source active
- embedded
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 92
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 92
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 82
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 title claims abstract description 73
- 235000014103 egg white Nutrition 0.000 title claims abstract description 73
- 210000000969 egg white Anatomy 0.000 title claims abstract description 73
- 239000002502 liposome Substances 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000002245 particle Substances 0.000 claims abstract description 21
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims abstract description 16
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 16
- 229940067606 lecithin Drugs 0.000 claims abstract description 16
- 235000010445 lecithin Nutrition 0.000 claims abstract description 16
- 239000000787 lecithin Substances 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims abstract description 11
- 238000000265 homogenisation Methods 0.000 claims abstract description 10
- 239000000843 powder Substances 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 238000000108 ultra-filtration Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 claims description 7
- 210000002969 egg yolk Anatomy 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
- 108091005658 Basic proteases Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 230000009849 deactivation Effects 0.000 claims description 5
- 238000004925 denaturation Methods 0.000 claims description 5
- 230000036425 denaturation Effects 0.000 claims description 5
- 238000009826 distribution Methods 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000011033 desalting Methods 0.000 claims description 2
- 238000009776 industrial production Methods 0.000 claims description 2
- 238000011031 large-scale manufacturing process Methods 0.000 claims description 2
- 231100000252 nontoxic Toxicity 0.000 claims description 2
- 230000003000 nontoxic effect Effects 0.000 claims description 2
- 238000001694 spray drying Methods 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 239000011162 core material Substances 0.000 claims 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims 2
- 230000003647 oxidation Effects 0.000 claims 2
- 238000007254 oxidation reaction Methods 0.000 claims 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- 230000007547 defect Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 claims 1
- 239000006185 dispersion Substances 0.000 claims 1
- 230000003301 hydrolyzing effect Effects 0.000 claims 1
- 238000011005 laboratory method Methods 0.000 claims 1
- 238000005057 refrigeration Methods 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 238000011161 development Methods 0.000 abstract description 6
- 238000010521 absorption reaction Methods 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 5
- 235000016709 nutrition Nutrition 0.000 abstract description 4
- 230000001737 promoting effect Effects 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 230000000968 intestinal effect Effects 0.000 abstract description 3
- 230000035764 nutrition Effects 0.000 abstract description 3
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 abstract 1
- 210000004051 gastric juice Anatomy 0.000 abstract 1
- 238000001728 nano-filtration Methods 0.000 description 12
- 238000001727 in vivo Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 5
- 235000013601 eggs Nutrition 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 102000038379 digestive enzymes Human genes 0.000 description 3
- 108091007734 digestive enzymes Proteins 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000002390 cell membrane structure Anatomy 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 229940040371 lecithin 10 mg Drugs 0.000 description 1
- 229940062519 lecithin 3 mg/ml Drugs 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000007830 nerve conduction Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/035—Organic compounds containing oxygen as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/05—Organic compounds containing phosphorus as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention provides a preparation method of a nano liposome embedded with egg white active peptide. Aims to solve the problem that the egg white active peptide is very easy to degrade in gastric juice and intestinal juice and can not be completely absorbed. The preparation method comprises the steps of dissolving film-forming materials such as lecithin, cholesterol and the like in ethanol, reducing the particle size through high-speed homogenization and high-pressure homogenization, and then dropping the solution into the aqueous solution of the dissolved egg white active peptide to form the egg white source active peptide-embedded nano liposome. The invention fills the blank in the aspect of protecting and promoting the biological absorption and utilization of the egg white source active peptide, and has important significance for the high-value utilization of protein and the development of short peptide nutrition industry.
Description
Technical Field
The invention relates to the technical field of encapsulation methods of physiologically active substances, in particular to a nano liposome with sustained release characteristic and embedded egg white source active peptide and a preparation method thereof.
Background
The eggs are rich in nutrient components and can quickly supplement various nutrient substances, the heat of the eggs is only half cup of milk, but other nutrient components are much higher than that of the milk. The eggs are rich in lipid substances and are concentrated in yolk parts, wherein the yolk lecithin accounts for about 10-15%. The yolk lecithin has the functions of eliminating free radicals, delaying body aging, promoting lipid metabolism, participating in nerve conduction, assisting in improving memory, enhancing human immunity and the like. The egg white contains a large amount of egg white protein with high biological value, the egg white contains more protein content and contains various essential amino acids for human bodies, and the egg white is a good protein resource for obtaining bioactive peptides.
The egg white protein is subjected to enzymolysis to obtain a plurality of peptide segments consisting of amino acids, namely the egg white source active peptide, wherein the egg white source active peptide has the advantages of unique in vivo or in vitro biological activity, high nutritional value, various biological activities, almost no toxic or side effect and the like, but can not be absorbed in a complete form because the egg white source active peptide is very easily degraded in a gastrointestinal tract digestive system in vivo, the egg white source active peptide must be degraded by complex digestive enzymes of the gastrointestinal tract to play the biological activity in vivo, then penetrates through a small intestinal epithelium absorption and permeation barrier in a complete form, is transported to an action target point through blood circulation and the like after being completely absorbed by an organism, and the function of regulating the physiological function of the organism can be realized finally only after the egg white protein is accumulated to a certain. Therefore, the development of a technical means for effectively protecting the egg white source active peptide to improve the stability and the absorption and utilization rate in vivo is urgent.
The liposome (1iposome), also called lipid globule, refers to a closed vesicle formed by embedding a drug in an amphiphilic lipid molecule and having a phospholipid bilayer structure, which is similar to a cell membrane structure and mainly comprises phospholipid and cholesterol, and the liposome can be used for embedding a water-soluble drug or a fat-soluble drug. The liposome is used as a drug carrier, has the characteristics of easy degradation in organisms, no immunity, no toxicity, drug release control, drug toxicity reduction and the like, is emphasized and widely applied. The liposome is used as a nano wall material, and has important significance for embedding and protecting the egg white source active peptide.
The research on the physiological activity of the short peptide at home and abroad is widely carried out, but the practical application research is obviously insufficient. After passing through a gastrointestinal tract digestive enzyme system and a high-acidity environment, the short peptide is seriously degraded and is degraded by peptidase after entering small intestinal cells, so that the possibility that the short peptide can exert physiological activity in an intact state is very low, and most researches cannot be practically applied. Therefore, the analysis of the protein modification mechanism and the complete transport process of the short peptide provides theoretical reference for the in vivo delivery of the short peptide, and has important significance for the high-valued utilization of protein and the development of the short peptide nutrition industry.
Disclosure of Invention
Step one, liposome preparation:
dissolving a certain amount of egg yolk lecithin and cholesterol in 95% hot ethanol to ensure that the concentration of the lecithin is 0.5-20 mg/ml and the concentration of the cholesterol is 0.1-2 mg/ml, homogenizing for 5min at 19,000 rpm by using a high-speed homogenizer, carrying out primary homogenization, and then carrying out high-pressure homogenization by using a high-pressure homogenizer at the homogenization pressure of 10-60 mpa for 1-11 min. The blank liposome with the particle size less than 150nm, the potential less than-25 mV and good stability is obtained.
Step two, preparing the egg white source active peptide:
taking a certain amount of egg white protein powder, and mixing the egg white protein powder with the feed liquid according to the mass ratio of 1: 5-1: 20, preparing an egg white protein powder raw material liquid, heating to 80-100 ℃, continuing for 5-10 min, performing denaturation treatment, then cooling to 50-60 ℃, adjusting the pH value to 8-11, adding alkaline protease, continuing to hydrolyze for 2-3 h, performing enzyme deactivation in a boiling water bath, finally centrifuging for 9000r/min for 10min, taking supernatant, performing ultrafiltration and nanofiltration by using an ultrafiltration and nanofiltration device with the molecular weight of less than 1kDa, concentrating filtrate, and concentrating to obtain the egg white source active peptide.
Step three, preparation of liposome of embedded egg white source active peptide
Dissolving the egg white source active peptide prepared in the first step, wherein the concentration of the egg white source active peptide is 0.5-20 mg/ml which is the same as the concentration of lecithin, and taking the volume ratio of liposome to peptide as 1: 5-5: 1, dropwise adding the liposome into a peptide solution under a stirring state to obtain the egg white peptide-embedded nano liposome, carrying out ultrasonic treatment on the nano liposome after stirring for 0-200 w of ultrasonic power and 0-250 s of ultrasonic time, and measuring the particle size and potential distribution condition of the nano liposome to obtain the egg white peptide-embedded nano liposome solution with the particle size of less than 300nm, the potential of less than-35 mv and good stability.
In the first step, the concentration of the liposome lecithin is 0.5 mg/ml-20 mg/ml.
Step one, the concentration of cholesterol is 0.1 mg/ml-2 mg/ml.
In the first step, the homogenizing pressure is 10-60 mpa, and the homogenizing time is 1-11 min.
And in the second step, the ultrafiltration membrane is a nanofiltration membrane with the molecular weight of 30KDa, 10KDa, 3KDa and 1KDa, the nanofiltration membrane is 200Da, the supernatant sequentially passes through the nanofiltration membrane with the molecular weight of 30KDa, 10KDa, 3KDa and 1KDa, and the nanofiltration membrane with the molecular weight of 200Da is used for desalting treatment to obtain the egg white source active peptide with the molecular weight of 200 Da-1 KDa.
In the second step, the content of soluble solid matters in the enzymatic hydrolysate is 8 to 15 percent after rotary evaporation,
in the second step, the inlet temperature of the spraying parameters is 150-200 ℃, and the outlet temperature is 50-80 ℃.
The volume ratio of the liposome to the peptide in the step three is 1: 5-5: 1.
in the third step, the ultrasonic treatment ultrasonic power is 0-200 w, and the ultrasonic time is 0-250 s.
The invention has the advantages of
(1) The invention adopts a mode of combining high-pressure homogenization and ethanol injection, takes egg yolk lecithin and cholesterol as nano packing materials, embeds 200 Da-1 KDa of egg white source active peptide obtained by enzymolysis, prepares the egg white peptide nano liposome with better stability and functionality, and fills the blank in the aspect of protecting and promoting the biological absorption and utilization of the egg white source active peptide.
(2) The invention belongs to the deep processing technology of egg product resources, and the egg white source active peptide liposome prepared by adopting the egg yolk lecithin with higher content in egg yolk as the raw material of the nanometer wall material and the egg white source active peptide extracted from the egg white has greater reference significance for the development and utilization of the egg white source active peptide liposome in the fields of beverages, health products and the like, can promote the comprehensive development and utilization of high value-added components in the egg product industry, and improve the economic value
(3) The method provided by the invention uses a mode of combining high-pressure homogenization and ethanol injection, is simple to operate, mild in parameter conditions, capable of realizing large-scale production, short in production period, long in storage period and easy to apply to industrial production.
(4) The yolk lecithin, the cholesterol and the egg white powder are extracted from the food, and the solvent is ethanol, so that the yolk lecithin, the cholesterol and the egg white powder are safe, non-toxic and low in use amount and have no harm to human bodies.
(5) The nanoliposome embedded with the egg white source active peptide is uniform and stable, has small particle size, is a nano-grade product, can be well absorbed by a human body, and plays a corresponding role.
(6) The experiment makes up for the limitation of the utilization of the egg white source active peptide to a certain extent. After passing through a gastrointestinal tract digestive enzyme system and a high-acidity environment, the short peptide is seriously degraded and is degraded by peptidase after entering small intestinal cells, so that the possibility that the short peptide can exert physiological activity in an intact state is very low, and most researches cannot be practically applied. Therefore, the liposome is used as a wall material to embed and deliver active peptide so as to improve the drug effect, and meanwhile, the sustained release of the polypeptide in vivo has important significance.
The invention is expected to promote the systematic research on the aspects of protecting and promoting the biological absorption and utilization of the egg white source active peptide and provide a new idea for the high-valued utilization of protein and the development of short peptide nutrition industry.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
step one, liposome preparation:
dissolving egg yolk lecithin and cholesterol in hot ethanol to make the concentration of lecithin 1mg/ml and cholesterol 0.2mg/ml, homogenizing at 19,000 rpm for 5min with a high speed homogenizer, primarily homogenizing, and homogenizing at high pressure of 40mpa for 7 min. The blank liposome with the particle size of 95 +/-10 nm, the potential of-30 +/-4 mV and good stability is obtained.
Step two, preparing the egg white source active peptide:
taking a certain amount of egg white protein powder, and mixing the egg white protein powder with the feed liquid according to the mass ratio of 1: 5 preparing an egg white protein powder raw material liquid, heating to 80 ℃, continuing for 8min, carrying out denaturation treatment, then cooling to 50 ℃, adjusting the pH value to 10.5, adding alkaline protease, carrying out continuous hydrolysis for 2h, carrying out enzyme deactivation in a boiling water bath, finally carrying out centrifugation treatment for 10min at 9000r/min, taking supernatant, carrying out ultrafiltration and nanofiltration by using an ultrafiltration and nanofiltration device with the molecular weight of less than 1000Da, concentrating the filtrate until the content of soluble solid matters is 15%, and concentrating to obtain the egg white source active peptide.
Step three, preparation of liposome of embedded egg white source active peptide
Dissolving the egg white source active peptide prepared in the first step, wherein the concentration of the egg white source active peptide is 1mg/ml which is the same as the concentration of lecithin, and taking the volume ratio of liposome to peptide as 1: 1, dripping the liposome into a peptide solution under the stirring state to obtain the egg white peptide-embedded nano liposome, carrying out ultrasonic treatment on the nano liposome with the ultrasonic power of 100w and the ultrasonic time of 100s after stirring, and measuring the particle size and the potential distribution condition of the nano liposome to obtain the egg white peptide-embedded nano liposome solution with the particle size of 240 +/-20 nm, the potential of-40 +/-4 mv and the embedding rate of 30 +/-5 percent and good stability.
Example 2:
step one, liposome preparation:
dissolving egg yolk lecithin and cholesterol in 95% hot ethanol to make the concentration of lecithin 3mg/ml and cholesterol 0.066mg/ml, homogenizing at 19,000 rpm for 5min with a high speed homogenizer, homogenizing at 40mpa for 7 min. The blank liposome with the particle size of 85 plus or minus 7nm, the potential of-28 plus or minus 6mV and good stability is obtained.
Step two, preparing the egg white source active peptide:
taking a certain amount of egg white protein powder, and mixing the egg white protein powder with the feed liquid according to the mass ratio of 1: 10 preparing an egg white protein powder raw material liquid, heating to 80 ℃, continuing for 8min, carrying out denaturation treatment, then cooling to 60 ℃, adjusting the pH value to 11, adding alkaline protease, carrying out continuous hydrolysis for 3h, carrying out enzyme deactivation in a boiling water bath, finally carrying out centrifugation treatment for 10min at 9000r/min, taking supernatant, carrying out ultrafiltration and nanofiltration by using an ultrafiltration and nanofiltration device with the molecular weight of less than 1kDa, concentrating the filtrate until the content of soluble solid matters is 12%, and carrying out spray drying after concentration to obtain the egg white source active peptide. Step three, preparation of liposome of embedded egg white source active peptide
Dissolving the egg white source active peptide prepared in the first step, wherein the concentration of the egg white source active peptide is 3mg/ml which is the same as the concentration of lecithin, and taking the volume ratio of liposome to peptide as 1: 1, dripping the liposome into a peptide solution under the stirring state to obtain the egg white peptide-embedded nano liposome, carrying out ultrasonic treatment on the nano liposome with the ultrasonic power of 100w and the ultrasonic time of 100s after stirring, and measuring the particle size and the potential distribution condition of the nano liposome to obtain the egg white peptide-embedded nano liposome solution with good stability, the particle size of 200 +/-10 nm, the potential of-40 +/-5 mv and the embedding rate of 35 +/-5%.
Example 3:
step one, liposome preparation:
dissolving egg yolk lecithin in 95% hot ethanol to make the concentration of lecithin 10mg/ml and cholesterol 2mg/ml, homogenizing at 19,000 rpm for 5min with a high speed homogenizer, homogenizing at high pressure of 40mpa for 7 min. The blank liposome with the particle size of 95 +/-8 nm, the potential of more than 31 +/-6 mV and good stability is obtained.
Step two, preparing the egg white source active peptide:
taking a certain amount of egg white protein powder, and mixing the egg white protein powder with the feed liquid according to the mass ratio of 1: 8 preparing an egg white protein powder raw material liquid, heating to 80 ℃, continuing for 10min, carrying out denaturation treatment, then cooling to 60 ℃, adjusting the pH value to 11, adding alkaline protease, carrying out boiling water bath enzyme deactivation after continuing hydrolysis for 3h, finally carrying out centrifugation treatment at 9000r/min for 10min, taking supernatant, carrying out ultrafiltration and nanofiltration by using an ultrafiltration and nanofiltration device with the molecular weight of less than 1kDa, concentrating filtrate, and concentrating to obtain the egg white source active peptide.
Step three, preparation of liposome of embedded egg white source active peptide
Dissolving the egg white source active peptide prepared in the first step, wherein the concentration of the egg white source active peptide is 10mg/ml which is the same as the concentration of lecithin, and taking the volume ratio of liposome to peptide as 5: and 3, dropwise adding the liposome into the peptide solution under the stirring state to obtain the egg white peptide-embedded nano liposome, carrying out ultrasonic treatment on the nano liposome with ultrasonic power of 200w and ultrasonic time of 120s after stirring, and measuring the particle size and potential distribution condition of the nano liposome to obtain the egg white peptide-embedded nano liposome solution with good stability, wherein the particle size is 240 +/-20 nm, the potential is-40 +/-4 mv, and the embedding rate is 50 +/-5%.
Claims (5)
1. A preparation method of a nano liposome embedded with albumen-source active peptide is characterized by comprising the following steps:
step one, liposome preparation:
dissolving a certain amount of egg yolk lecithin and cholesterol in hot ethanol at the temperature of 50-70 ℃ to ensure that the concentration of the lecithin is 0.5-20 mg/ml and the concentration of the cholesterol is 0.1-2 mg/ml, homogenizing for 5min at the rotating speed of 19,000 rpm by using a high-speed homogenizer, carrying out primary homogenization, and then carrying out high-pressure homogenization by using a high-pressure homogenizer at the homogenizing pressure of 10-60 mpa for 1-11 min; obtaining blank liposome with particle size less than 150nm, potential less than-25 mV and good stability;
step two, preparing the egg white source active peptide:
taking a certain amount of egg white protein powder, and mixing the egg white protein powder with the feed liquid according to the mass ratio of 1: 5-1: 20 preparing an egg white protein powder raw material liquid, heating to 80-100 ℃, continuing for 5-10 min, performing denaturation treatment, then cooling to 50-60 ℃, adjusting the pH value to 7-11, adding alkaline protease, continuously hydrolyzing for 2-3 h, performing enzyme deactivation in a boiling water bath, finally centrifuging for 9000r/min for 10min, sequentially passing the supernatant through ultrafiltration membranes with molecular weights of 30KDa, 10KDa, 3KDa and 1KDa, and then performing desalting treatment by using a sodium filter membrane with a molecular weight of 200Da to obtain an egg white source active peptide with a molecular weight of 200Da to 1 KDa; concentrating the filtrate, and spray drying to obtain egg white source active peptide;
step three, preparing the liposome of the embedded egg white source active peptide:
dissolving the egg white source active peptide prepared in the first step, wherein the concentration of the egg white source active peptide is 0.5-20 mg/ml which is the same as the concentration of lecithin, and taking the volume ratio of liposome to peptide as 1: 5-5: 1, dropwise adding the liposome into a peptide solution under a stirring state to obtain the egg white peptide-embedded nano liposome, carrying out ultrasonic treatment on the nano liposome after stirring for 0-200 w of ultrasonic power and 0-250 s of ultrasonic time, and measuring the particle size and potential distribution condition of the nano liposome to obtain the egg white peptide-embedded nano liposome solution with the particle size of less than 300nm, the potential of less than 35mv and good stability.
2. The method for preparing the nano liposome embedded with the egg white source active peptide according to claim 1, which is characterized in that: the yolk lecithin and the cholesterol are dissolved in hot ethanol at the temperature of 50-70 ℃ to ensure that the concentration of the lecithin is 0.5-20 mg/ml, and the ethanol replaces the traditional chloroform for preparing the liposome, so the preparation method is safe, non-toxic and harmless to human bodies.
3. The method for preparing the nano liposome embedded with the egg white source active peptide according to claim 1, which is characterized in that: the embedded core material is egg white source active peptide with the molecular weight of 200 Da-1 KDa, the oxidation resistance of the embedded core material is 3 times that of the egg white source active peptide without separating components through detection, and the embedded core material has good oxidation resistance and good functionality.
4. The method for preparing the nano liposome embedded with the egg white source active peptide according to claim 1, which is characterized in that: the product has small particle size, the particle size of blank liposome is less than 150nm, the particle size of liposome embedded with egg white peptide is less than 300nm, the storage stability is good, the particle size potential has no obvious change when the product is stored for one month under the conditions of normal temperature and refrigeration, and the product can be stored for a long time.
5. The method for preparing the nano liposome embedded with the egg white source active peptide according to claim 1, which is characterized in that: the preparation method combining high-pressure homogenization and ethanol injection is applied, the defect that the liposome prepared by a laboratory method film dispersion method is limited by the size of a rotary evaporator and cannot be produced in an enlarged manner is overcome, the operation is simple, the parameter condition is mild, the large-scale production can be realized, the production capacity can reach more than 5 liters each time, the production period is short, and the method is easy to apply to industrial production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911009978.3A CN110679953A (en) | 2019-10-23 | 2019-10-23 | Preparation method of nano liposome embedded with egg white source active peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911009978.3A CN110679953A (en) | 2019-10-23 | 2019-10-23 | Preparation method of nano liposome embedded with egg white source active peptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110679953A true CN110679953A (en) | 2020-01-14 |
Family
ID=69113858
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911009978.3A Pending CN110679953A (en) | 2019-10-23 | 2019-10-23 | Preparation method of nano liposome embedded with egg white source active peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110679953A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111888469A (en) * | 2020-09-11 | 2020-11-06 | 苏州瑞微生物科技有限公司 | IgY liposome spray and preparation method thereof |
| CN112293635A (en) * | 2020-11-07 | 2021-02-02 | 润科生物工程（福建）有限公司 | Spray drying process of instant chlorella protein peptide powder and application of instant chlorella protein peptide powder in solid beverage |
| CN113142588A (en) * | 2021-02-10 | 2021-07-23 | 大连工业大学 | Nano liposome base material powder for improving processing stability of egg white peptide chelated calcium and preparation method thereof |
| CN113208116A (en) * | 2021-02-10 | 2021-08-06 | 大连工业大学 | Nano liposome embedded with egg white peptide calcium compound and preparation method thereof |
| CN114081963A (en) * | 2021-11-16 | 2022-02-25 | 上海理工大学 | Nano-carrier for improving bioavailability of active peptide and preparation and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080213346A1 (en) * | 2005-03-28 | 2008-09-04 | Regeron, Inc. | Preparation of Nanoliposome-Encapsulating Proteins and Protein-Encapsulated Nanoliposome |
| CN101502326A (en) * | 2009-02-23 | 2009-08-12 | 江南大学 | Method for preparing vitamin E nano liposome |
| CN101804028A (en) * | 2010-04-20 | 2010-08-18 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for preparing traditional Chinese medicine liposome |
| CN103052717A (en) * | 2011-05-17 | 2013-04-17 | 中国食品发酵工业研究院 | Industrial production method for producing antihypertensive bioactive peptide |
| CN103479578A (en) * | 2012-06-14 | 2014-01-01 | 沈阳药科大学 | Pixantrone maleate liposome preparation and preparation process thereof |
| CN103637989A (en) * | 2013-11-07 | 2014-03-19 | 南昌大学 | Preparation method of tea polyphenol nano-liposomes by ethanol injection-dynamic high-pressure microfluidization-enzymolysis |
| CN107459547A (en) * | 2017-07-13 | 2017-12-12 | 浙江省农业科学院 | The coproduction separation method of various bioactivators in yolk |
| CN108432982A (en) * | 2018-03-07 | 2018-08-24 | 吉林大学 | A kind of fruit drink and preparation method thereof containing anti-oxidant egg white peptide nano liposomes |
-
2019
- 2019-10-23 CN CN201911009978.3A patent/CN110679953A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080213346A1 (en) * | 2005-03-28 | 2008-09-04 | Regeron, Inc. | Preparation of Nanoliposome-Encapsulating Proteins and Protein-Encapsulated Nanoliposome |
| CN101502326A (en) * | 2009-02-23 | 2009-08-12 | 江南大学 | Method for preparing vitamin E nano liposome |
| CN101804028A (en) * | 2010-04-20 | 2010-08-18 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for preparing traditional Chinese medicine liposome |
| CN103052717A (en) * | 2011-05-17 | 2013-04-17 | 中国食品发酵工业研究院 | Industrial production method for producing antihypertensive bioactive peptide |
| CN103479578A (en) * | 2012-06-14 | 2014-01-01 | 沈阳药科大学 | Pixantrone maleate liposome preparation and preparation process thereof |
| CN103637989A (en) * | 2013-11-07 | 2014-03-19 | 南昌大学 | Preparation method of tea polyphenol nano-liposomes by ethanol injection-dynamic high-pressure microfluidization-enzymolysis |
| CN107459547A (en) * | 2017-07-13 | 2017-12-12 | 浙江省农业科学院 | The coproduction separation method of various bioactivators in yolk |
| CN108432982A (en) * | 2018-03-07 | 2018-08-24 | 吉林大学 | A kind of fruit drink and preparation method thereof containing anti-oxidant egg white peptide nano liposomes |
Non-Patent Citations (1)
| Title |
|---|
| 吴琦: ""抗氧化蛋清肽脂质体的制备及其产品的研发"", 《中国优秀博硕士学位论文全文数据库（硕士） 工程科技Ⅰ辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111888469A (en) * | 2020-09-11 | 2020-11-06 | 苏州瑞微生物科技有限公司 | IgY liposome spray and preparation method thereof |
| CN112293635A (en) * | 2020-11-07 | 2021-02-02 | 润科生物工程（福建）有限公司 | Spray drying process of instant chlorella protein peptide powder and application of instant chlorella protein peptide powder in solid beverage |
| CN113142588A (en) * | 2021-02-10 | 2021-07-23 | 大连工业大学 | Nano liposome base material powder for improving processing stability of egg white peptide chelated calcium and preparation method thereof |
| CN113208116A (en) * | 2021-02-10 | 2021-08-06 | 大连工业大学 | Nano liposome embedded with egg white peptide calcium compound and preparation method thereof |
| CN114081963A (en) * | 2021-11-16 | 2022-02-25 | 上海理工大学 | Nano-carrier for improving bioavailability of active peptide and preparation and application thereof |
| CN114081963B (en) * | 2021-11-16 | 2023-09-26 | 上海理工大学 | Nanometer carrier for improving bioavailability of active peptide and preparation and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110679953A (en) | Preparation method of nano liposome embedded with egg white source active peptide | |
| CN102940214B (en) | Fruit and vegetable fiber nutrition powder production and production method and application thereof | |
| CN110089753A (en) | A kind of preparation method for the nanoparticle improving curcumin bioavailability | |
| CN100569795C (en) | A kind of oxidation resistance zymolyte that is rich in pollen active peptide | |
| CN105054073B (en) | A kind of water miscible vitamine D3 nano particle and preparation method thereof | |
| CN109588721B (en) | Carotenoid-protein particle and preparation method and application thereof | |
| Mosquera et al. | Incorporation of liposomes containing squid tunic ACE‐inhibitory peptides into fish gelatin | |
| CN102228125B (en) | Preparation method of algal active peptide | |
| CN106916871A (en) | A kind of bionic enzymatic prepares the production method of freshwater mussel meat small peptide | |
| CN106333359A (en) | Oyster polypeptide and spirulina nutritional tablet and preparation method thereof | |
| CN104095243A (en) | Preparation technology for enzymolysis bioactive peptide nanometer selenium | |
| CN106978462A (en) | A kind of bionic enzymatic prepares the production method of corn Gly-His-Lys | |
| CN111004829A (en) | Preparation method of black fungus active peptide | |
| JP2012232963A (en) | Absorption-enhancing composition containing composite of basic physiologically active protein and sophorolipid | |
| Sun et al. | Development of zein-based nutraceutical delivery systems: A systematic overview based on recent researches | |
| CN113397175B (en) | Apostichopus japonicus oligopeptide cross-linked microcapsule and preparation method and application thereof | |
| CN101589823B (en) | Production process of sea cucumber health milk | |
| CN107048417B (en) | Preparation method of peach kernel polypeptide oral liquid with high bioavailability | |
| CN106011209A (en) | Preparation method of collagen peptide | |
| CN104096231A (en) | Targeting nano sono-sensitizer and preparation method thereof | |
| CN111329988A (en) | Donkey-hide gelatin peptide buccal tablet rich in free arginine and preparation method thereof | |
| CN115644453A (en) | Method for stabilizing DHA emulsion by soybean polypeptide-polyphenol antioxidant particles | |
| CN106265412B (en) | A method of hydrolyzed pearl solution is prepared using probiotics fermention | |
| CN106858614A (en) | Compound amino acid selenium yeast oral liquid | |
| CN107114555A (en) | A kind of bionic enzymatic prepares the production method of wheat Gly-His-Lys |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200114 |
|
| WD01 | Invention patent application deemed withdrawn after publication |