CN108456707A - A kind of preparation method of pupa albumen anti-oxidation peptide and the application of pupa albumen anti-oxidation peptide - Google Patents
A kind of preparation method of pupa albumen anti-oxidation peptide and the application of pupa albumen anti-oxidation peptide Download PDFInfo
- Publication number
- CN108456707A CN108456707A CN201810464907.1A CN201810464907A CN108456707A CN 108456707 A CN108456707 A CN 108456707A CN 201810464907 A CN201810464907 A CN 201810464907A CN 108456707 A CN108456707 A CN 108456707A
- Authority
- CN
- China
- Prior art keywords
- silkworm chrysalis
- preparation
- pupa albumen
- pupa
- ultrafiltration
- 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
- 241000382353 Pupa Species 0.000 title claims abstract description 91
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 230000003064 anti-oxidating effect Effects 0.000 title claims abstract description 17
- 241000255789 Bombyx mori Species 0.000 claims abstract description 69
- 102000004190 Enzymes Human genes 0.000 claims abstract description 57
- 108090000790 Enzymes Proteins 0.000 claims abstract description 57
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 57
- 235000012054 meals Nutrition 0.000 claims abstract description 38
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 36
- 230000009849 deactivation Effects 0.000 claims abstract description 36
- 239000012528 membrane Substances 0.000 claims abstract description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000000843 powder Substances 0.000 claims abstract description 28
- 239000000126 substance Substances 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 239000000706 filtrate Substances 0.000 claims abstract description 21
- 239000003208 petroleum Substances 0.000 claims abstract description 18
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- 235000013305 food Nutrition 0.000 claims abstract description 8
- 239000012466 permeate Substances 0.000 claims abstract description 8
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 238000000926 separation method Methods 0.000 claims abstract description 6
- 238000005238 degreasing Methods 0.000 claims description 20
- 108090000604 Hydrolases Proteins 0.000 claims description 16
- 102000004157 Hydrolases Human genes 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 239000007790 solid phase Substances 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 101800000068 Antioxidant peptide Proteins 0.000 claims description 3
- 108091005804 Peptidases Proteins 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 102000035195 Peptidases Human genes 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
- 230000002000 scavenging effect Effects 0.000 abstract description 17
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 abstract description 12
- 235000020776 essential amino acid Nutrition 0.000 abstract description 11
- 239000003797 essential amino acid Substances 0.000 abstract description 11
- 230000003078 antioxidant effect Effects 0.000 abstract description 8
- 238000002604 ultrasonography Methods 0.000 abstract description 8
- 239000003963 antioxidant agent Substances 0.000 abstract description 5
- 238000005374 membrane filtration Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000012545 processing Methods 0.000 abstract description 3
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 54
- 239000000243 solution Substances 0.000 description 36
- 150000001413 amino acids Chemical class 0.000 description 32
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- 230000031700 light absorption Effects 0.000 description 15
- 239000000413 hydrolysate Substances 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- 239000011521 glass Substances 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 230000008569 process Effects 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 239000013065 commercial product Substances 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 235000019580 granularity Nutrition 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- 238000009777 vacuum freeze-drying Methods 0.000 description 7
- 238000009835 boiling Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- -1 aromatic amino acid Chemical class 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000004365 Protease Substances 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 4
- 235000019634 flavors Nutrition 0.000 description 4
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 4
- 210000001541 thymus gland Anatomy 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N EtOH Substances CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 0.000 description 3
- 150000005693 branched-chain amino acids Chemical class 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000001471 micro-filtration Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 229940079877 pyrogallol Drugs 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 244000189799 Asimina triloba Species 0.000 description 1
- 235000006264 Asimina triloba Nutrition 0.000 description 1
- 241000255783 Bombycidae Species 0.000 description 1
- 241000255791 Bombyx Species 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 229910015400 FeC13 Inorganic materials 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 239000002826 coolant Substances 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 229960004279 formaldehyde Drugs 0.000 description 1
- 235000019256 formaldehyde Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical compound O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- LQRJAEQXMSMEDP-XCHBZYMASA-N peptide a Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(=O)NCCCC[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)C(\NC(=O)[C@@H](CCCCN)NC(=O)CNC(C)=O)=C/C=1C=CC=CC=1)C(N)=O)C(=O)C(\NC(=O)[C@@H](CCCCN)NC(=O)CNC(C)=O)=C\C1=CC=CC=C1 LQRJAEQXMSMEDP-XCHBZYMASA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- KKCBUQHMOMHUOY-UHFFFAOYSA-N sodium oxide Chemical compound [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 description 1
- 229910001948 sodium oxide Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 230000035922 thirst Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- 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
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of preparation methods of pupa albumen anti-oxidation peptide, belong to food processing technology field.The preparation method includes：1) dried silkworm chrysalis meal is mixed with petroleum ether, ultrasound is separated by solid-liquid separation；2) degreased pupa powder, water and pupa albumen hydrolase are mixed and is digested；3) by dried silkworm chrysalis meal enzymolysis liquid enzyme deactivation, obtained supernatant is used 0.40~0.50 μm of membrane filtration, collects filtrate by centrifugation；4) the ultrafiltration membrane ultrafiltration that filtrate is used to 3000Da, collects permeate, obtains ultrafiltrate；5) the ultrafiltration membrane ultrafiltration that ultrafiltrate is used to 200Da, collects trapped substance.Essential amino acids content reaches 43% or so in the pupa albumen peptide that the present invention is prepared, total antioxidant capacity, to superoxide anion (O2) Scavenging activity, the Scavenging activity to OH Scavenging activities and to DPPH it is relatively strong.
Description
Technical field
The invention belongs to food processing technology fields, and in particular to a kind of preparation method of pupa albumen anti-oxidation peptide and silkworm
The application of pupa protein antioxidant peptide.
Background technology
About 600,000 tons of China's amount of cocoon production at present, account for 80% of the world or so.Silkworm chrysalis (Silkwormpupa) is Bombycidae elder brother
The pupa of worm bombyx mori (Bombyx moilL.), silkworm chrysalis is traditional Chinese medicine,《Compendium of Materia Medica》、《General Records of Holy Universal Relief》、《Peaceful holy benevolent prescription》、《East
The precious mirror of doctor》Equal ancient medicines classical works are all documented silkworm chrysalis function, it is believed that silkworm chrysalis have promote the production of body fluid to quench thirst, help digestion reason
Gas, relieving convulsion, establishing-Yang, key taste, wind-damp dispelling, long flesh are brought down a fever and other effects.
Protein content is up to 45%~55%, mostly globulin in silkworm chrysalis, 18 kinds of amino acid is rich in, wherein needed by human
8 kinds of amino acid contents be more than the 40% of total amount, it is necessary to the ratio between amino acid and nonessential amino acid are defended higher than the world more than 0.6
The reference protein pattern that raw tissue and FAO (Food and Agriculture Organization of the United Nation) (WHO/FAO) propose, is a kind of high-quality protein of full price, is had latent
Edible value.
Although pupa albumen is high-quality protein, since its big water solubility of molecular weight is poor, not only influences them and eating
Application in product industry also influences human body and is digested and assimilated to it.There is also the macromoleculars of about 30kDa or more for pupa albumen
Albumen may cause groups of people allergic reaction occur, these all hinder application of the pupa albumen in food, also give silkworm chrysalis egg
White direct utilize brings risk.
Moreover, because the condition of silkworm chrysalis filature generally use is high temperature highly basic, therefore the silkworm chrysalis after filature not only has and is difficult
The fishy smell eliminated, and protein is denaturalized, and fat also occurs oxidation and produces serious tapinoma-odour, and consumer is difficult to connect
By these bad flavors, silkworm chrysalis is mainly used as feed at present, and it is affected into silkworm chrysalis directly as the less of raw-food material
The promotion of added value.
Invention content
In view of this, the purpose of the present invention is to provide a kind of preparation methods and pupa albumen of pupa albumen anti-oxidation peptide
The application of anti-oxidation peptide has stronger anti-oxidation function, and is easy to digest and assimilate, and can be used as food consumption.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of preparation methods of pupa albumen anti-oxidation peptide, include the following steps：
1) dried silkworm chrysalis meal is mixed, ultrasonic degreasing with petroleum ether, is separated by solid-liquid separation, obtained solid phase is dried, and obtains degreasing
Dried silkworm chrysalis meal；
2) degreased pupa powder, water and the pupa albumen hydrolase in the step 1) are mixed and is digested, obtain silkworm chrysalis
Powder enzymolysis liquid；
3) by the dried silkworm chrysalis meal enzymolysis liquid enzyme deactivation of the step 2), obtained supernatant is used 0.40~0.50 μm by centrifugation
Membrane filtration collects filtrate；
4) the ultrafiltration membrane ultrafiltration that the filtrate in the step 3) is used to 3000Da, collects permeate, obtains ultrafiltrate；
5) the ultrafiltration membrane ultrafiltration that the ultrafiltrate in the step 4) is used to 200Da, collects trapped substance；The trapped substance is
Pupa albumen anti-oxidation peptide.
Preferably, the mass ratio of degreased pupa powder, water and pupa albumen hydrolase is 5~8 in the step 2):95~
105:2.5~4；
The Rate activity of the pupa albumen hydrolase is 0.5 × 105~2.0 × 105U/g。
Preferably, the temperature digested in the step 2) is 50~60 DEG C, and the time of enzymolysis is 4~6h.
Preferably, the pH value in the step 2) when enzymolysis is 7.0~8.0.
Preferably, the quality of dried silkworm chrysalis meal and the volume ratio of petroleum ether are 1g in the step 1):6~10mL.
Preferably, the number of ultrasonic degreasing is 2~4 times in the step 1), and ultrasonic power is 100~150W, ultrasound
Time be 30~50min/ time, ultrasonic temperature be 35~45 DEG C.
Preferably, which is characterized in that the granularity of dried silkworm chrysalis meal is 140~160 mesh in the step 1).
Preferably, temperature dry in the step 1) is 50~60 DEG C, and the dry time is 45~60min.
Preferably, further include being freeze-dried the trapped substance after collection trapped substance in the step 5)；It is described cold
It is -40~-20 DEG C that dry temperature, which is lyophilized, and the time of the freeze-drying is 8~12h.
The present invention provides the pupa albumen anti-oxidation peptides that a kind of method described in said program is prepared to prepare food
Application in product or antioxidant drug.
The present invention provides a kind of preparation methods of pupa albumen anti-oxidation peptide, first take off dried silkworm chrysalis meal through petroleum ether
Fat prevents the fat in dried silkworm chrysalis meal from oxidation occurring in the preparation process of peptide and is emulsified with protein, obtains degreasing silkworm chrysalis
After powder recycle pupa albumen hydrolase digested, make aromatic amino acid related with antioxidant activity, branched-chain amino acid,
Acidic amino acid, basic amino acid ratio all increase, and make these amino acid residues expose, improve antioxidant activity.Into one
Step is filtered, ultrafiltration, so that essential amino acids content in the pupa albumen peptide being prepared is reached 43% or so, is met FAO/
Requirement of the essential amino acids content that WHO (FAO (Food and Agriculture Organization of the United Nation) FAO or World Health Organization WHO) is proposed 40% or so.
The pupa albumen peptide molecular weight being prepared simultaneously is easy to digest and assimilate in 200~3000Da.Embodiment the result shows that：It compares
The total antioxidant capacity of the pupa albumen peptide obtained when specific enzyme+flavor protease digests is hydrolyzed in using pupa albumen, to super
Oxygen anion (O2) Scavenging activity, to OH Scavenging activities and to the Scavenging activity of DPPH improve 73.8% successively respectively
~85.7%, 107.6%~112.6%, 74.1%~80.6% and 29.9%~32.53%；Compared to using pupa albumen
Hydrolyze specific enzyme+papain enzymolysis, preparation method of the invention in total antioxidant capacity, to superoxide anion (O2)
Improve 49.0%~59.2% in terms of Scavenging activity, Scavenging activity to OH Scavenging activities and to DPPH successively respectively,
50.6%~55.8%, 52.7%~58.4% and 34.2%~36.8%.
Specific implementation mode
The present invention provides a kind of preparation methods of pupa albumen anti-oxidation peptide, include the following steps：
1) dried silkworm chrysalis meal is mixed, ultrasonic degreasing with petroleum ether, is separated by solid-liquid separation, obtained solid phase is dried, and obtains degreasing
Dried silkworm chrysalis meal；
2) degreased pupa powder, water and the pupa albumen hydrolase in the step 1) are mixed and is digested, obtain silkworm chrysalis
Powder enzymolysis liquid；
3) by the dried silkworm chrysalis meal enzymolysis liquid enzyme deactivation of the step 2), obtained supernatant is used 0.40~0.50 μm by centrifugation
Membrane filtration collects filtrate；
4) the ultrafiltration membrane ultrafiltration that the filtrate in the step 3) is used to 3000Da, collects permeate, obtains ultrafiltrate；
5) the ultrafiltration membrane ultrafiltration that the ultrafiltrate in the step 4) is used to 200Da, collects trapped substance；The trapped substance is
Pupa albumen anti-oxidation peptide.
The present invention mixes dried silkworm chrysalis meal with petroleum ether, ultrasonic degreasing, is separated by solid-liquid separation, and obtained solid phase is dried, obtains
Degreased pupa powder.In the present invention, the quality of the dried silkworm chrysalis meal and the volume ratio of petroleum ether are preferably 1g:6~10mL, more preferably
1g:8mL.In the present invention, the granularity of the dried silkworm chrysalis meal is preferably 140~160 mesh, more preferably 150 mesh.It is described in the present invention
Filature silkworm chrysalis is preferably crushed at 40~50 DEG C after dry 120~180min, is sieved by the preparation method of dried silkworm chrysalis meal.The reel silk from cocoons
The drying temperature of silk silkworm chrysalis is preferably 45 DEG C.The drying time of the filature silkworm chrysalis is preferably 150min.The present invention is to the reel silk from cocoons
The source of silk silkworm chrysalis is not particularly limited, using this field conventional commercial product.In the present invention, the boiling range of the petroleum ether
Preferably 60~90 DEG C.The present invention is not particularly limited the source of the petroleum ether, is using this field conventional commercial product
It can.The petroleum ether used in the embodiment of the present invention is purchased from Chengdu Ke Long chemical reagents factory.
In the present invention, the number of the ultrasonic degreasing is preferably 2~4 times, more preferably 3 times.The power of the ultrasound is excellent
It is selected as 100~150W, more preferably 120W.The time of the ultrasound is preferably 30~50min/ times, more preferably 40min/ times.
The temperature of the ultrasound is preferably 35~45 DEG C, more preferably 40 DEG C.In the present invention, temperature when obtained solid phase is dried
Preferably 50~60 DEG C, more preferably 55 DEG C.The time that obtained solid phase is dried is preferably 45~60min, more preferably
50min.The present invention mixes dried silkworm chrysalis meal with petroleum ether, ultrasound, is conducive to remove the fat in dried silkworm chrysalis meal, prevent in subsequent water
Fat, which influences the dissolving of protein in aqueous solution, in solution preocess influences the acquisition of peptide, while being also prevented from the follow-up process
Oxidation occurs for fat and fat is emulsified with protein, influences the separation of polypeptide products quality and protein solution.
After the solid phase is dried, the present invention preferably crushes the solid phase after obtained drying.The crushing
Granularity be preferably 190~210 mesh, more preferably 200 mesh.The present invention, which crushes the solid phase after drying, can make solid phase more
Uniformly, it is conducive to subsequent enzymolysis.The present invention is not particularly limited the ultrasound and the dry equipment used, normal using this field
Advise commercial product.
After obtaining degreased pupa powder, the degreased pupa powder, water and pupa albumen hydrolase are mixed and carry out enzyme by the present invention
Solution, obtains dried silkworm chrysalis meal enzymolysis liquid.In the present invention, the mass ratio of the dried silkworm chrysalis meal, water and pupa albumen hydrolase is preferably 5~8:
95~105:2.5~4, more preferably 6:100:3.The Rate activity of the pupa albumen hydrolase is preferably 0.5 × 105~2.0
×105U/g, more preferably 1.0 × 105U/g.The temperature of the enzymolysis is preferably 50~60 DEG C, more preferably 52 DEG C.The enzyme
The time of solution is preferably 4~6h, more preferably 5h.The present invention is not particularly limited the source of the pupa albumen hydrolase,
Using this field conventional commercial product.The pupa albumen hydrolase used in the embodiment of the present invention is purchased from the Nanning roads Dong Henghua
Bio tech ltd.
The pH value of mixed liquor is preferably 7.0~8.0 when enzymolysis, and more preferably 7.2.It is excellent in enzymolysis process in the present invention
Every 25~35min is selected to detect a pH value.In the present invention, the solution for adjusting pH value is preferably that hydrochloric acid solution or sodium hydroxide are molten
Liquid.The concentration of the hydrochloric acid solution is preferably 1mol/L.The concentration of the sodium hydroxide solution is preferably 1mol/L.
In the present invention, using pupa albumen hydrolase carry out enzymolysis make aromatic amino acid related with antioxidant activity,
Branched-chain amino acid, acidic amino acid, basic amino acid ratio all increase, and make these amino acid residues expose, improve antioxygen
Change activity.
After obtaining dried silkworm chrysalis meal enzymolysis liquid, obtained supernatant is adopted the dried silkworm chrysalis meal enzymolysis liquid enzyme deactivation, centrifugation by the present invention
With 0.40~0.50 μm of membrane filtration, filtrate is collected.In the present invention, the mode of the enzyme deactivation is preferably high temperature enzyme deactivation.It is described to go out
The temperature of enzyme is preferably 95~110 DEG C, more preferably 100 DEG C.The time of the enzyme deactivation is preferably 4~8min, more preferably
6min.After enzyme deactivation, the present invention preferably cools down enzymolysis solution after enzyme deactivation.The mode of the cooling is preferably circulating water punching
It washes.Since high temperature, partial moisture evaporation improve to make the volume before and after measurement remain unchanged and measure knot in enzymolysis process
Coolant liquid is preferably added water to be supplemented to the volume before digesting by the accuracy of fruit, after the cooling period, the present invention.In the present invention, it is described from
The rotating speed of the heart is preferably 3500~4500r/min, more preferably 4000r/min.The time of the centrifugation is preferably 10~
20min, more preferably 15min.In the present invention, the mode of the membrane filtration preferably filters.The vacuum degree when suction filtration
Preferably 0.08~0.10Mpa, more preferably 0.09Mpa.The aperture of the filter membrane is preferably 0.45 μm.The present invention will be after enzyme deactivation
Dried silkworm chrysalis meal enzymolysis liquid centrifuged, filter the large granular impurity that can be removed in enzymolysis liquid, prevent in subsequent ultra-filtration process
In block fenestra, influence membrane flux.
The present invention is not particularly limited the source of the filter membrane, using this field conventional commercial product.The present invention
The filter membrane used in embodiment is purchased from Tianjin Jin Teng experimental facilities Co., Ltd.
After obtaining filtrate, the filtrate is used the ultrafiltration membrane ultrafiltration of 3000Da by the present invention, is collected permeate, is obtained ultrafiltration
Liquid.The temperature when ultrafiltration is preferably room temperature.The pressure when ultrafiltration is preferably 0.25Mpa.
In the present invention, 3000Da polypeptides below can be collected using the ultrafiltration membrane ultrafiltration of 3000Da.
The present invention is not particularly limited the source of the ultrafiltration membrane of the 3000Da, is using this field conventional commercial product
It can.In the embodiment of the present invention using Shanghai rub fast science equipment Co., Ltd MSM-2008 experiment ultrafiltration micro-filtration UF membranes dress
Set carry out ultrafiltration.
After obtaining ultrafiltrate, the ultrafiltrate is used the ultrafiltration membrane ultrafiltration of 200Da by the present invention, collects trapped substance.It is described
Temperature when ultrafiltration is preferably room temperature.The pressure when ultrafiltration is preferably 0.4Mpa.In the present invention, using the ultrafiltration of 200Da
Film ultrafiltration can remove the polypeptide that molecular weight is less than 200Da, so that the molecular weight of polypeptide is controlled in 200~3000Da, improve antioxygen
The property changed.
The present invention is not particularly limited the source of the ultrafiltration membrane of the 200Da, is using this field conventional commercial product
It can.In the embodiment of the present invention using Shanghai rub fast science equipment Co., Ltd MSM-2008 experiment ultrafiltration micro-filtration UF membranes dress
Set carry out ultrafiltration.
Due to being continuously added NaOH in pupa albumen enzymolysis process, part salt is formed in enzymolysis process.In the present invention, collect
After trapped substance, it is preferred to use molecule interception is the NF membrane nanofiltration of 100~200Da, desalting processing is carried out, after obtaining desalination
Trapped substance.
In the present invention, after collecting trapped substance, the trapped substance is preferably carried out vacuum freeze drying by the present invention, obtains silkworm chrysalis
Protein antioxidant peptide.The temperature of the vacuum freeze drying is preferably -40~-20 DEG C, more preferably -25 DEG C.The vacuum is cold
It is preferably 8~12h that the dry time, which is lyophilized, more preferably 10h.
The present invention is not particularly limited the equipment used when the vacuum freeze drying, is produced using this field conventional commercial
Product.The ALPAAI-4LSC vacuum freeze driers of SIGMA are used in the embodiment of the present invention.
Amino acid content is 51.86%~56.09% in the pupa albumen peptide that the present invention is prepared, it is necessary to which amino acid contains
Amount is higher than requirement of the essential amino acids content of FAO/WHO propositions 40% or so 41% or more.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
The dried silkworm chrysalis meal that 100g granularities are 140 mesh is weighed, is wrapped, is put into glass container with filter paper, is added in glass container
Enter the petroleum ether that 600mL boiling ranges are 60~90 DEG C, ultrasonic degreasing 4 times under the conditions of 100W, 45 DEG C, each 30min will after ultrasonic
It is taken out using the dried silkworm chrysalis meal of filter paper package, dry 60min, obtains degreased pupa powder, the degreased pupa powder that will be obtained at 50 DEG C
After being crushed to 190 mesh again, degreased pupa powder, water and pupa albumen hydrolase are pressed 5:105:2.5 mass ratio mixing, will
The mixed liquor arrived digests 6h at 50 DEG C, and (control ph (detects the pH of a solution per 25min, works as hair for 7.0 in enzymolysis process
When changing, be adjusted using the hydrochloric acid or sodium hydroxide of 1mol/L) after the enzyme deactivation 8min at 95 DEG C again.Using stream after enzyme deactivation
Water makes enzymolysis solution after enzyme deactivation cool down, and adds in enzymolysis solution after enzyme deactivation and to add water to enzymolysis solution after enzyme deactivation volume and restore to enzyme
Volume before solution.20min is centrifuged with the rotating speed of 3500r/min again, by obtained supernatant using 0.40 μm of filter membrane in vacuum degree
To be filtered under conditions of 0.08Mpa, filtrate is collected.Obtained filtrate is used to the ultrafiltration membrane ultrafiltration of 3000Da, collects permeate,
Obtain ultrafiltrate.Obtained ultrafiltrate is used to the ultrafiltration membrane ultrafiltration of 200Da, collects trapped substance.By obtained trapped substance -20
Vacuum freeze drying 12h at DEG C.
Using the full-automatic amino-acid analyzer of L-8900 types, in the pupa albumen hydrolysate that Hitachi detections are prepared
Amino acid forms and content, and the results are shown in Table 1.
Amino acid composition and content in 1 pupa albumen hydrolysate of table
As can be seen from Table 1, the amino acid content for the pupa albumen hydrolysate that the present invention is prepared is 54.20%.
According to the testing result of amino acid in table 1, the distribution of amino acid classes is counted, the results are shown in Table 2.
The Species distributing of amino acid in 2 pupa albumen hydrolysate of table
| Hydrophobic amino acid % | 21.42 |
| Essential amino acid % | 43.69 |
| Aromatic amino acid % | 20.15 |
| Branched-chain amino acid % | 24.31 |
| Acidic amino acid % | 13.60 |
| Basic amino acid % | 14.20 |
As can be seen from Table 2, in the pupa albumen hydrolysate that the present invention is prepared essential amino acids content 43% with
On, it is higher than requirement of the essential amino acids content of FAO/WHO propositions 40% or so.
Embodiment 2
The dried silkworm chrysalis meal that 100g granularities are 160 mesh is weighed, is wrapped, is put into glass container using filter paper, in glass container
The petroleum ether that 1000mL boiling ranges are 60~90 DEG C, ultrasonic degreasing 2 times under the conditions of 150W, 35 DEG C, each 50min, ultrasound is added
It will be taken out afterwards using the dried silkworm chrysalis meal of filter paper package, dry 45min, obtains degreased pupa powder, the degreasing silkworm that will be obtained at 60 DEG C
After pupa powder is crushed to 210 mesh again, degreased pupa powder, water and pupa albumen hydrolase are pressed 8:95:4 mass ratio mixing, will
Obtained mixed liquor digested at 60 DEG C 4h (in enzymolysis process control ph be 8.0 (per 35min detect a solution pH, when
When changing, be adjusted using the hydrochloric acid or sodium hydroxide of 1mol/L) after the enzyme deactivation 4min at 105 DEG C again.It is adopted after enzyme deactivation
So that enzymolysis solution after enzyme deactivation is cooled down with flowing water, and adds in enzymolysis solution after enzyme deactivation and add water to the recovery of enzymolysis solution after enzyme deactivation volume
Volume before to enzymolysis.10min is centrifuged with the rotating speed of 4500r/min again, by obtained supernatant using 0.50 μm of filter membrane true
Reciprocal of duty cycle filters under conditions of being 0.1Mpa, collects filtrate.Obtained filtrate is used to the ultrafiltration membrane ultrafiltration of 3000Da, collects and penetrates
Liquid obtains ultrafiltrate.Obtained ultrafiltrate is used to the ultrafiltration membrane ultrafiltration of 200Da, collects trapped substance.The trapped substance that will be obtained
The vacuum freeze drying 8h at -40 DEG C.
Using the full-automatic amino-acid analyzer of L-8900 types, in the pupa albumen hydrolysate that Hitachi detections are prepared
Amino acid forms and content, and the results are shown in Table 3.
Amino acid composition and content in 3 pupa albumen peptide of table
As can be seen from Table 3, the amino acid content for the pupa albumen hydrolysate that the present invention is prepared is 51.86%.
According to the testing result of amino acid in table 3, the distribution of amino acid classes is counted, the results are shown in Table 4.
Amino acid composition and content in 4 pupa albumen peptide of table
As can be seen from Table 4, essential amino acids content about 42% in the pupa albumen hydrolysate that the present invention is prepared is high
In requirement of the essential amino acids content that FAO/WHO is proposed 40% or so.
Embodiment 3
The dried silkworm chrysalis meal that 100g granularities are 150 mesh is weighed, is wrapped, is put into glass container using filter paper, in glass container
The petroleum ether that 800mL boiling ranges are 60~90 DEG C, ultrasonic degreasing 3 times under the conditions of 120W, 40 DEG C, each 40min, after ultrasonic is added
It will be taken out using the dried silkworm chrysalis meal of filter paper package, dry 50min, obtains degreased pupa powder, the degreasing silkworm chrysalis that will be obtained at 55 DEG C
After powder is crushed to 100 mesh again, degreased pupa powder, water and pupa albumen hydrolase are pressed 6:100:3 mass ratio mixing, will
The mixed liquor arrived digests 6h at 55 DEG C, and (control ph (detects the pH of a solution per 30min, works as hair for 7.7 in enzymolysis process
When changing, be adjusted using the hydrochloric acid or sodium hydroxide of 1mol/L) after the enzyme deactivation 6min at 100 DEG C again.It is used after enzyme deactivation
Flowing water makes enzymolysis solution after enzyme deactivation cool down, and add in enzymolysis solution after enzyme deactivation add water to enzymolysis solution after enzyme deactivation volume restore to
Volume before enzymolysis.15min is centrifuged with the rotating speed of 4000r/min again, by obtained supernatant using 0.45 μm of filter membrane in vacuum
Degree filters under conditions of being 0.09Mpa, collects filtrate.Obtained filtrate is used to the ultrafiltration membrane ultrafiltration of 3000Da, collects and penetrates
Liquid obtains ultrafiltrate.Obtained ultrafiltrate is used to the ultrafiltration membrane ultrafiltration of 200Da, collects trapped substance.The trapped substance that will be obtained
The vacuum freeze drying 10h at -25 DEG C.
Using the full-automatic amino-acid analyzer of L-8900 types, in the pupa albumen hydrolysate that Hitachi detections are prepared
Amino acid forms and content, and the results are shown in Table 5.
Amino acid composition and content in 5 pupa albumen hydrolysate of table
As can be seen from Table 5, the amino acid content for the pupa albumen hydrolysate that the present invention is prepared is 56.09%.
According to the testing result of amino acid in table 5, the distribution of amino acid classes is counted, the results are shown in Table 6.
Amino acid composition and content in 6 pupa albumen hydrolysate of table
As can be seen from Table 6, essential amino acids content exists in the pupa albumen hydrolysate that the present invention is prepared
40.90%, meet requirement of the essential amino acids content of FAO/WHO propositions 40% or so.
Comparative example 1
The dried silkworm chrysalis meal that 100g granularities are 150 mesh is weighed, is wrapped, is put into glass container using filter paper, in glass container
The petroleum ether that 800mL boiling ranges are 60~90 DEG C, ultrasonic degreasing 3 times under the conditions of 120W, 40 DEG C, each 40min, after ultrasonic is added
It will be taken out using the dried silkworm chrysalis meal of filter paper package, dry 50min, obtains degreased pupa powder, the degreasing silkworm chrysalis that will be obtained at 55 DEG C
After powder is crushed to 100 mesh again, by degreased pupa powder, water and complex enzyme, (complex enzyme is that pupa albumen hydrolyzes specific enzyme and flavor
The mass ratio of enzyme, the two is 1:1) 6 are pressed:100:Obtained mixed liquor, is digested 6h (enzymolysis by 3 mass ratio mixing at 55 DEG C
Control ph is the 8.0 (pH of solution of every 30min detections, when a change, using the hydrochloric acid or hydrogen of 1mol/L in the process
Sodium oxide molybdena is adjusted) after the enzyme deactivation 6min at 100 DEG C again.Enzymolysis solution after enzyme deactivation is set to cool down using flowing water after enzyme deactivation, and
Add in enzymolysis solution after enzyme deactivation and adds water to enzymolysis solution after enzyme deactivation volume and restore the volume to enzymolysis.Again with 4000r/min's
Rotating speed centrifuges 15min, uses 0.45 μm of filter membrane to be detached under conditions of vacuum degree is 0.09MPa obtained supernatant, collects
Filtrate.Obtained filtrate is used to the ultrafiltration membrane ultrafiltration of 3000Da, permeate is collected, obtains ultrafiltrate.The ultrafiltrate that will be obtained
Using the ultrafiltration membrane ultrafiltration of 200Da, trapped substance is collected.By obtained trapped substance at -25 DEG C vacuum freeze drying 10h.
Comparative example 2
The dried silkworm chrysalis meal that 100g granularities are 150 mesh is weighed, is wrapped, is put into glass container using filter paper, in glass container
The petroleum ether that 800mL boiling ranges are 60~90 DEG C, ultrasonic degreasing 3 times under the conditions of 120W, 40 DEG C, each 40min, after ultrasonic is added
It will be taken out using the dried silkworm chrysalis meal of filter paper package, dry 50min, obtains degreased pupa powder, the degreasing silkworm chrysalis that will be obtained at 55 DEG C
After powder is crushed to 100 mesh again, by degreased pupa powder, water and complex enzyme, (complex enzyme is that pupa albumen hydrolyzes specific enzyme and pawpaw
The mass ratio of protease, the two is 1:1) 6 are pressed:100:3 mass ratio mixing, 6h is digested by obtained mixed liquor at 50 DEG C
(control ph is the 7.5 (pH of solution of every 30min detections, when a change, using the salt of 1mol/L in enzymolysis process
Acid or sodium hydroxide are adjusted) after the enzyme deactivation 6min at 100 DEG C again.Keep enzymolysis solution after enzyme deactivation cold using flowing water after enzyme deactivation
But, and in enzymolysis solution after enzyme deactivation add and add water to enzymolysis solution after enzyme deactivation volume and restore the volume to enzymolysis.Again with
The rotating speed of 4000r/min centrifuges 15min, uses 0.45 μm of filter membrane in vacuum degree for the condition of 0.09MPa obtained supernatant
Under detach, collect filtrate.Obtained filtrate is used to the ultrafiltration membrane ultrafiltration of 3000Da, permeate is collected, obtains ultrafiltrate.Will
The ultrafiltrate arrived uses the ultrafiltration membrane ultrafiltration of 200Da, collects trapped substance.By obtained trapped substance, vacuum refrigeration is done at -25 DEG C
Dry 10h.
Embodiment 4
Using Examples 1 to 3 as experimental group, comparative example 1 and 2 measures degree of hydrolysis, total respectively as control group 1 and 2 respectively
Reducing power, to superoxide anion (O2) Scavenging activity, to OH Scavenging activities and to the Scavenging activity of DPPH, specifically
The results are shown in Table 7.Specific assay method is as follows for These parameters：
The measurement of degree of hydrolysis
Amino acid nitrogen content is measured using formol titration, calculates degree of hydrolysis, specific formula is：
The measurement of reducing power
The silkworm chrysalis polypeptide liquid of gained is diluted into 100 times of progress reducing power measurement.The phosphate for being 6.6 in 2.5mLpH values is slow
Add the sample liquid 2.5mL of various concentration in fliud flushing, the potassium ferricyanide solution 2.5mL of mass fraction 1%, mixture is in 50 DEG C of constant temperature
After 20min, the solution of trichloroacetic acid of 2.5mL volume fractions 10% is added, 10min is then centrifuged with 3000r/min, is taken
Supernatant liquor 5mL adds distilled water 5mL and mass fraction 0.1%FeC13Solution 0.5mL measures light absorption value at 700nm, to inhale
Light value indicates reducing power.Absorbance is higher, and reducing power is stronger, while making reagent blank and positive control.Superoxide anion
(O2) Scavenging activity measurement
10 times of dilutions of silkworm chrysalis polypeptide hydrolyzate of gained are subjected to O2The evaluation of clearance rate.
0.1mol/LTris-HCL buffer solutions (the pH8.2, wherein containing 1mmol/ of 2.5mL is added with 1mL distilled water
LEDTA it) returns to zero, the pyrogallol (25 DEG C of water-bath preheatings) of 10 μ L50mmol/L, rapid mixing, every 30s at 320nm is added
It is primary to read absorbance, terminates after 5min, the regression equation changed over time as light absorption value, slope is mouse thymus cells
Rate V1。
The 0.1mol/L Tris-HCL buffer solutions (pH8.2) of 2.5mL are added in pipette samples 1mL, mixing are shaken, 25
The pyrogallol (25 DEG C of water-baths preheatings) of 10ul 50mmol/L is added after DEG C water-bath heat preservation 10min, rapid mixing simultaneously starts to count
When, light absorption value A is measured at 320nm, is terminated after 30s reads light absorption value A320,5min.It is changed over time as light absorption value
Regression equation, slope are sample mouse thymus cells rate V2.The clearance rate of superoxide anion (O2) calculates as follows：
In formula：
V1Control group mouse thymus cells rate
V2Sample mouse thymus cells rate
The measurement of hydroxy radical (OH) Scavenging activity
The silkworm chrysalis polypeptide hydrolyzate of gained is diluted to the evaluation of 10 times of progress OH clearance rates.
In 10mL test tubes, it is separately added into 2mL6mmol/L salicylic acids-ethanol solution, 2mL6mmol/LFeSO4 (contains
6mmol/LEDTA), 2mL sample solutions are eventually adding 2mL6mmol/LH2O2 startups and react, after 37 DEG C of water-bath 30min,
Light absorption value is measured at 510nm.Measure the light absorption value of blank control group respectively under same reaction system, color sample group is interfered
The clearance rate calculating of light absorption value, OH is as follows：
In formula：
A1The light absorption value of blank control group
A2Sample sets absorbance
A3The light absorption value of color sample group interference.
The measurement of DPPH.free radical (DPPH) Scavenging activity
The silkworm chrysalis polypeptide hydrolyzate of gained is diluted to the evaluation of 100 times of progress DPPH clearance rates.
20 μ g/LDPPH solution are prepared with ethyl alcohol, are kept in dark place spare.Take the prepare liquid of 3mL hydrolyzates in test tube respectively
In, 3mL20 μ g/LDPPH solution is added, shakes up, its light absorption value is measured at 517nm after placing 30min.In same reaction body
The light absorption value of blank control group, the light absorption value of color sample group interference are measured under system respectively, the clearance rate calculating of 0H is as follows：
In formula：
A1The light absorption value of blank control group
A2Sample sets absorbance
A3The light absorption value of color sample group interference.
The measurement result of pupa albumen peptide oxidation resistance prepared by 7 distinct methods of table
| Degree of hydrolysis (%) | Total reducing power | O2(%) | OH (%) | DPPH (%) | |
| Embodiment 1 | 20.71±0.21 | 0.75±0.03 | 34.54±1.85 | 43.77±1.32 | 79.38±0.54 |
| Embodiment 2 | 20.53±0.54 | 0.73±0.01 | 34.21±1.01 | 42.34±0.94 | 78.76±0.36 |
| Embodiment 3 | 21.02±0.36 | 0.78±0.02 | 35.04±0.46 | 43.91±1.02 | 80.34±0.24 |
| Comparative example 1 | 28.44±0.79 | 0.42±0.01 | 16.48±0.67 | 24.32±1.44 | 60.62±0.37 |
| Comparative example 2 | 26.81±0.49 | 0.49±0.01 | 16.07±1.00 | 27.72±0.85 | 58.71±0.60 |
As can be seen from Table 7, using double enzymes (pupa albumen hydrolyze specific enzyme+food flavor enzyme or pupa albumen hydrolysis specific enzyme+
Papain) enzymolysis pupa albumen degree of hydrolysis be higher than individually with pupa albumen hydrolyze specific enzyme, when using the present invention
Pupa albumen hydrolysis specific enzyme is digested, the total antioxidant capacity of obtained protein peptides, to superoxide anion (O2) it is clear
Removing solid capacity is above use double enzymes enzymolysis using comparative example 1 and 2 to OH Scavenging activities and to the Scavenging activity of DPPH
Effect.73.8%~85.7%, 107.6%~112.6%, 74.1%~80.6% is improved successively respectively compared to comparative example 1
With 29.9%~32.53%；Compared to comparative example 2 respectively successively improve 49.0%~59.2%, 50.6%~55.8%,
52.7%~58.4% and 34.2%~36.8%.The result shows that：The pupa albumen peptide that the present invention is prepared has stronger
Oxidation resistance.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of pupa albumen anti-oxidation peptide, includes the following steps：
1) dried silkworm chrysalis meal is mixed, ultrasonic degreasing with petroleum ether, is separated by solid-liquid separation, obtained solid phase is dried, and obtains degreasing silkworm chrysalis
Powder；
2) degreased pupa powder, water and the pupa albumen hydrolase in the step 1) are mixed and is digested, obtain dried silkworm chrysalis meal enzyme
Solve liquid；
3) by the dried silkworm chrysalis meal enzymolysis liquid enzyme deactivation of the step 2), obtained supernatant is used 0.40~0.50 μm of filter membrane by centrifugation
Filtrate is collected in filtering；
4) the ultrafiltration membrane ultrafiltration that the filtrate in the step 3) is used to 3000Da, collects permeate, obtains ultrafiltrate；
5) the ultrafiltration membrane ultrafiltration that the ultrafiltrate in the step 4) is used to 200Da, collects trapped substance, and the trapped substance is silkworm chrysalis
Protein antioxidant peptide.
2. preparation method according to claim 1, which is characterized in that degreased pupa powder, water and silkworm chrysalis in the step 2)
The mass ratio of proteolytic enzyme is 5~8:95~105:2.5~4；
The Rate activity of the pupa albumen hydrolase is 0.5 × 105~2.0 × 105U/g。
3. preparation method according to claim 1, which is characterized in that the temperature digested in the step 2) is 50~60
DEG C, the time of enzymolysis is 4~6h.
4. according to the preparation method described in claims 1 to 3 any one, which is characterized in that in the step 2) when enzymolysis
PH value is 7.0~8.0.
5. preparation method according to claim 1, which is characterized in that the quality and petroleum ether of dried silkworm chrysalis meal in the step 1)
Volume ratio be 1g:6~10mL.
6. preparation method according to claim 1, which is characterized in that the number of ultrasonic degreasing is 2~4 in the step 1)
Secondary, ultrasonic power is 100~150W, and the ultrasonic time is 30~50min/ times, and ultrasonic temperature is 35~45 DEG C.
7. according to the preparation method described in claim 1,5~6 any one, which is characterized in that dried silkworm chrysalis meal in the step 1)
Granularity be 140~160 mesh.
8. preparation method according to claim 1, which is characterized in that dry temperature is 50~60 in the step 1)
DEG C, the dry time is 45~60min.
9. the preparation method according to claim 1 or 8, which is characterized in that also wrapped after collecting trapped substance in the step 5)
It includes and is freeze-dried the trapped substance；The temperature of the freeze-drying is -40~-20 DEG C, the time of the freeze-drying
For 8~12h.
10. the pupa albumen anti-oxidation peptide that method as described in any one of claim 1 to 9 is prepared is preparing food or is resisting
Aoxidize the application in drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810464907.1A CN108456707A (en) | 2018-05-16 | 2018-05-16 | A kind of preparation method of pupa albumen anti-oxidation peptide and the application of pupa albumen anti-oxidation peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810464907.1A CN108456707A (en) | 2018-05-16 | 2018-05-16 | A kind of preparation method of pupa albumen anti-oxidation peptide and the application of pupa albumen anti-oxidation peptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108456707A true CN108456707A (en) | 2018-08-28 |
Family
ID=63214450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810464907.1A Pending CN108456707A (en) | 2018-05-16 | 2018-05-16 | A kind of preparation method of pupa albumen anti-oxidation peptide and the application of pupa albumen anti-oxidation peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108456707A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110100984A (en) * | 2019-05-24 | 2019-08-09 | 邓光 | Health care blueberry concentrate and its production technology |
| CN110241159A (en) * | 2019-05-08 | 2019-09-17 | 中山大学 | A kind of technique of enzymatic hydrolysis fly pupa preparation antioxidant activity polypeptide |
| CN114395605A (en) * | 2022-02-21 | 2022-04-26 | 吉林省蚕业科学研究院 | Preparation method of tussah pupa protein peptide, tussah pupa protein peptide and application thereof |
| CN114656522A (en) * | 2022-03-08 | 2022-06-24 | 江苏科技大学 | Antioxidant peptide and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104277975A (en) * | 2014-09-28 | 2015-01-14 | 江苏科技大学 | Pretreatment method for culturing oleaginous microorganisms from degreased silkworm chrysalis residues, product obtained by using pretreatment method and applications of product |
-
2018
- 2018-05-16 CN CN201810464907.1A patent/CN108456707A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104277975A (en) * | 2014-09-28 | 2015-01-14 | 江苏科技大学 | Pretreatment method for culturing oleaginous microorganisms from degreased silkworm chrysalis residues, product obtained by using pretreatment method and applications of product |
Non-Patent Citations (2)
| Title |
|---|
| 丁顺杰 等: ""酶解缫丝蚕蛹蛋白抗氧化肽的分离与稳定性研究"", 《食品科学》 * |
| 秦樱瑞 等: ""缫丝蚕蛹蛋白酶解产物体外抗氧化能力研究"", 《食品工业科技》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241159A (en) * | 2019-05-08 | 2019-09-17 | 中山大学 | A kind of technique of enzymatic hydrolysis fly pupa preparation antioxidant activity polypeptide |
| CN110100984A (en) * | 2019-05-24 | 2019-08-09 | 邓光 | Health care blueberry concentrate and its production technology |
| CN114395605A (en) * | 2022-02-21 | 2022-04-26 | 吉林省蚕业科学研究院 | Preparation method of tussah pupa protein peptide, tussah pupa protein peptide and application thereof |
| CN114656522A (en) * | 2022-03-08 | 2022-06-24 | 江苏科技大学 | Antioxidant peptide and preparation method and application thereof |
| CN114656522B (en) * | 2022-03-08 | 2024-03-19 | 江苏科技大学 | Antioxidant peptide and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108456707A (en) | A kind of preparation method of pupa albumen anti-oxidation peptide and the application of pupa albumen anti-oxidation peptide | |
| CN104745663B (en) | A kind of method of PINPROL comprehensive utilization | |
| Osborne | THE PROTEIDS OF BARLEY. | |
| CN101928742B (en) | Whey protein active peptide with antioxidant activity and preparation method thereof | |
| CN102174627B (en) | Method for preparing rapeseed bioactive peptide | |
| CN104513843B (en) | A kind of combined preparation process of polysaccharide and protein peptides | |
| US20180258147A1 (en) | Pearl protein preparation method and a water-soluble pearl protein and acid-soluble pearl protein obtained by adopting this method | |
| CN109385457B (en) | Preparation method of giant salamander Maillard peptide with antioxidant activity | |
| CN110240627B (en) | Method for extracting small molecule active peptide from pearl powder by utilizing complex enzyme | |
| CN107446978A (en) | A kind of stem of noble dendrobium small-molecular peptides and preparation method thereof | |
| CN113249421B (en) | Golden-silk-royal chrysanthemum protein polypeptide and preparation and application thereof | |
| CN109371092A (en) | A kind of preparation method of collagen peptide | |
| CN109439717A (en) | A kind of extracting method of small molecule rhizoma polygonati polypeptide | |
| CN104152519A (en) | Preparation method of pepsin-soluble high-purity superhelical-structured type-I collagen | |
| CN110746515A (en) | Lycium barbarum polysaccharide, lycium barbarum red element and lycium barbarum polypeptide prepared by synchronous separation and preparation method thereof | |
| CN107619411B (en) | Heme extraction method | |
| CN104531813B (en) | A kind of preparation method of cornu caprae hircus active peptides | |
| CN107529554A (en) | A kind of production technology of pig blood ball peptide | |
| CN114517218A (en) | Sea-buckthorn oligopeptide powder and preparation method and application thereof | |
| CN108300752A (en) | A method of it is sticked together using donkey-hide gelatin and prepares small molecule donkey-hide gelatin peptide | |
| CN206494880U (en) | A kind of Household integral collagen preparation facilities | |
| CN116035218A (en) | Pea oligopeptide health care product and preparation method thereof | |
| CN113481259B (en) | Method for extracting starch syrup and rice protein isolate by using broken rice | |
| CN110172490A (en) | A method of except grease in donkey-hide gelatin peptide | |
| CN109457008A (en) | A kind of preparation method of ginseng chelating peptide |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180828 |