CN108743446A - A kind of ultrafiltration membrane preparation method of cocoon sericin protein and its application - Google Patents
A kind of ultrafiltration membrane preparation method of cocoon sericin protein and its application Download PDFInfo
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- CN108743446A CN108743446A CN201810686464.0A CN201810686464A CN108743446A CN 108743446 A CN108743446 A CN 108743446A CN 201810686464 A CN201810686464 A CN 201810686464A CN 108743446 A CN108743446 A CN 108743446A
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
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- 238000003556 assay Methods 0.000 description 1
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- 238000002425 crystallisation Methods 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
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- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
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- 239000003797 essential amino acid Substances 0.000 description 1
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- 229930003935 flavonoid Natural products 0.000 description 1
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- 235000017173 flavonoids Nutrition 0.000 description 1
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- 235000013376 functional food Nutrition 0.000 description 1
- 229910000743 fusible alloy Inorganic materials 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
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- 229920002521 macromolecule Polymers 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
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- 239000002923 metal particle Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- QLWXZRVOHCYKKK-UHFFFAOYSA-N n-(cyclohexylideneamino)-2,4-dinitroaniline Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC=C1NN=C1CCCCC1 QLWXZRVOHCYKKK-UHFFFAOYSA-N 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
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- JCXJVPUVTGWSNB-UHFFFAOYSA-N nitrogen dioxide Inorganic materials O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
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- 239000002807 parathyroid gland hormone Substances 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 150000004968 peroxymonosulfuric acids Chemical class 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- CWHFDTWZHFRTAB-UHFFFAOYSA-N phenyl cyanate Chemical compound N#COC1=CC=CC=C1 CWHFDTWZHFRTAB-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
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- 229940067157 phenylhydrazine Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
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- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical class [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 235000011091 sodium acetates Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000013555 soy sauce Nutrition 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- 239000003643 water by type Substances 0.000 description 1
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- 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
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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Abstract
The present invention relates to a kind of ultrafiltration membrane preparation method of cocoon sericin protein and its applications, this method will strip silk cocoon surface filiform, remove remaining magazine in silk cocoon, it is spare to be cut into strip, petroleum ether degreasing is added, soaked in absolute ethyl alcohol is decolourized, it recycles aqueous sodium carbonate to boil, extracts, ultrafiltration, merging filtrate, centrifugation, supernatant with use molecular cut off for the ultrafiltration membrane of 30KDa, progress desalination, it is detained solution and continues to concentrate, and obtains refining high-quality sericine using spray drying or freeze-drying；It is shown by SDS-PAAG results：The sericine of extraction contains protein of the molecular weight distribution within 14.4KDa-66KDa.Have the characteristics that glue protein matter yield is high, while ultrafiltration membrane reaches a step desalination and concentration glue protein matter purpose, the cocoon sericin albumen that this method obtains can be used in the raw material of cosmetics, food and medicine；Removal fibroin makes silk gloss in silk, and the silk gum after removal, which is waste, becomes a kind of high value addition product.
Description
Technical field
The present invention relates to a kind of ultrafiltration membrane preparation method of cocoon sericin protein and its applications.
Background technology
China's silk oilfields are abundant, are the big countries of silk production and processing, fibroin is that mankind's early start uses
One of native protein enjoys always the good reputation of " fiber queen " from ancient times to the present.The silk yield in China is in rank first,
Account for about the half of Gross World Product.It will produce a large amount of leftover bits and pieces, the profit again of silk offcut in the process of manufacture of silk
With being important research direction.Fibroin albumen is the important component of silk, is a kind of natural protein polymer macromolecular,
Submissive with strand, good biocompatibility is easy film forming, and machinability is good, and the features such as be easy to molecular modification, in biology
There is important application prospect in the fields such as material, medicine, environmental treatment, nano material, functional material.The extraction of fibroin albumen and silk
The research of plain biomembrane material has important theory significance and potential application value.Fibroin from silk is as a kind of
Novel protein resource, not only with edible value but also with medical value.
Protein content in silk is up to 97.6%-98.5%, and structure is mainly by two fibroins (Fibroin) and turnover
Silk gum (Sericin) two parts of covering form, and silk peptide belongs to the intermediate product of Fibroin Hydrolysis.Fibroin (Fibroin, fibroin
Albumen), the also known as silk heart is the chief component of silk, accounts about 70% one the 80% of silk content, is with crystalline texture
Protein, be made of 18 kinds of amino acid, wherein 8 kinds of essential amino acids necessary to human body account for 7% or so.Except C, H, O, N tetra-
Outside kind essential element, fibroin albumen also contains other elements such as Ca, Si, Sr, P etc., these elements determine the one of fibroin
A little performances.The amino acid composed structure of fibroin is relatively simple, and packing is easy between the peptide molecule formed, leads to have certain crystallization
Property.Crystallinity is in 50%-60% or so.Polarity side group accounts for about 29.5% in fibroin, and nonpolar side group accounts for about 70.5%, the two ratio
It is 0.42.And ratio between two is far above fibroin in silk gum, and about 2.91, this is also that silk gum is soluble easily in water and fibroin is not soluble in water simultaneously
Basic reason with some strength.Side group in fibroin is simple glycine, serine, alanine, by certain sequence
Structural arrangement is predominantly located at the crystal region of fibroin albumen at the segment of rule.And phenylalanine, color ammonia with larger side group
Acid, tyrosine etc. are then primarily present in amorphous regions.The conformation of fibroin albumen mainly based on a random ball of string, in addition also contains
There are a small amount of β-corner, alpha-helix etc., molecular weight to generally believe between the ranges of 3.6-3.7 × 10.5.
Silk is used as textile material in the past, gradually increases the research of silk with people in recent years, both at home and abroad to silkworm
The exploitation and utilization of silk and its product extend to the multiple fields such as food, material, drug, biomaterial and cosmetics.
1. the application of silk：
1.1 application in food：
Natural fibroin is a kind of azelon of macromolecular backbone, and quality is close, not soluble in water, is not only difficult to nozzle
It chews, human body stomach also can not directly absorb, only after fibroin molecule is degraded to lower molecular weight level by certain way,
Edible and the absorbable optimum efficiency utilized can be reached.Fibroin albumen through enzyme degrade or sour water solution after, can become oligopeptide with
The mixing of amino acid.Someone carried out with big white mouse animal experiments show that, big white mouse to enzyme degrade after silk fibroin powder digestion
Rate reaches 70%, reaches 91% to the digestibility of the silk fibroin powder after sour water solution；But if with silk fibroin water solution and not
The silk fibroin powder of degradation feeds big white mouse, and digestibility is respectively 47.4% and 27.6%.It can be seen that fibroin albumen
The fibroin albumen peptide and amino acid obtained after hydrolysis is easy to be digested absorption in vivo, this makes fibroin in functional food
Application aspect have larger potentiality to be exploited.Japan begins to set about the research of silk edibleization early in nineteen eighty-three, earliest
Product is that silk jelly has had developed a series of products using fibroin albumen by development for many years to current Japan.?
In the end of the year 1996, Japanese Jia Yue development companies come into effect the large-scale production of silk fibroin powder, and are sold to food producer.
There are many food of the kind containing fibroin powder such as gruel, beverage, fibroin albumen cake, noodles, candy, jelly pectin, soy sauce, ice rivers in Henan Province for oneself
The classes products such as leaching emerge, these products containing fibroin powder have become the health food of Japanese a new generation, and due to its unique product
Matter and it is favored by consumers.There is abundant waste silk resource, the research and development in relation to silk food also to have been reported in China.Lee
Moral is remote etc. will to pass through Na2CO3The fibroin protein obtained after solution degumming process is in CaCl2Further dissolution process in solution, warp
Silk protein solution is made in dialysis desalting.And beverage and jelly, good mouthfeel are prepared as raw material, transparent color and luster is eaten
It is safe and healthy.
1.2 application in the material：
In the traditional idea of people, silk is due to excellent wearability and environment amenable performance, making
The concern of people is constantly subjected to for a kind of natural fiber garment fabric.The hydrophilic-silk on silk surface is removed by degumming tech
After glue protein, the fibroin albumen of purifying can be obtained.Fibroin albumen has extremely excellent mechanical property：Tensile strength is reachable
To 0.5GPa, fracture elongation 15%.Excellent mechanical property possessed by fibroin albumen is inseparable with its own structure
's.Before nineteen ninety, Japan every year need to from national about 7.5 tons of the import green turtle crust such as Cuba and Indonesia, but due to
Green turtle is endangered wild animal type, thus since《Washington treaty》After forbidding green turtle to merchandise at the international level,
Japanese tortoise plastron association entrusts with Ministry of Commerce and Industry suffered by four countries of the counties Xiang Chuan industrial research, proceeds by and is made using fibroin
The experimental study of tortoise plastron substitute.On October 30th, 1992 in Asahi Shimbun, announces that research succeeds.The way of the method is
By transparent fibroin film well prepared in advance, suction press is carried out in water vapour, then obtains plate similar with tortoise plastron
Later when material adsorbing metal particle or after coloured with dyestuff the distinctive color of tortoise plastron can be presented, made of this method in material
The suture that craftwork enters behind market used in surgical operation welcomed by consumers generally is Silk Threads, although nearest Buddhist nun
The use of the synthetic fibers such as dragon, terylene is being continuously increased, but due to some superperformances such as fineness that silk suture has is small,
Easily knotting, tieing are not easy to scatter, and tensile strength is greatly, to human body without anaphylaxis etc. so that silk suture is in terms of some than closing
Advantage is had more at fiber, so the use of Silk Threads still occupies sizable ratio, cannot especially there is the ophthalmology of scar in wound
And plastic surgery, silk suture small using fineness, that tropism is good, intensity is big are more suitable.
1.3 application in medicine：
Fibroin albumen is also paid close attention to by more and more researchers in the application of biomedicine field, can be used as artificial skin
Skin, enzyme immobilizatio carrier, slow releasing carrier of medication and tissue engineering material etc..The hardness of cell culture medium be one in response to
The important physical factor of cell type, although the hydrogel prepared based on protein has been widely used as the culture of cell
Base, but their rigidity should further increase the Absorption Growth that can be just more conducive to cell.Lv et al. is with 1- (3- dimethylaminos
Propyl) for -3- ethyl-carbodiimide hydrochlorides by fibroin albumen and collagen induction crosslinking, preparation has the fibroin of appropriate hardness
Albumen/collagen mixing hydrogel.The storage modulus of the cross-linked hydrogel is higher than 3kPa, and even if above 10KPa, it still has very
High mechanical strength；In addition, cross-linked hydrogel remains to keep the conformation of its script not change at 80 DEG C, thus proving should
The heat-resistant stability of hydrogel is improved.Growth of the vascular smooth muscle cells in fibroin albumen/collagen hydrogels
Show that cross-linking reaction does not generate negative influence to the bioactivity of the hydrogel.The hydrogel will be in tissue engineering material
On have very wide application prospect.Its hydrolysate of fibroin albumen also has unique physiological activity and medical value, such as drops blood
Sugared effect, antioxidation, improves intestinal physiology function, the effect for preventing brain aging and cryoprotection work at norcholesterol effect
With etc..Therefore to fibroin albumen, this natural resources develops and utilizes, and not only improves warp of the fibroin as industry byproduct
Ji value, and there is larger Social benefit and economic benefit.
1.4 cosmetic field：
Fibroin albumen has good heat and moisture preserving, contributes to the moisture for adjusting skin, thus fibroin albumen can be used as
The good base material fibroin albumens of cosmetics are there are two types of the main application forms of cosmetic field：Fibroin powder and silk peptide.Naturally
Fibroin itself is not soluble in water, and fibroin powder remains the original structure composition of fibroin albumen, and therefore, fibroin powder inherits fibroin egg
While white distinctive sub-dued lustre, and the effect that ultraviolet light resists solar radiation that absorbs is had both.It is main in cosmetic field
Application be hydrolysis after relative molecular mass in 2KDa or less silk peptides.Wherein, the lower silk peptide of relative molecular mass
(300-800) can provide nutrient for the metabolism of skin and hair, and skin is contributed to resist the damage of chemistry and machinery with hair；Phase
It can then assign skin and hair natural gloss the silk peptide that molecular mass is 1000-2000Da, and then skin and hair is played
The effect of moisturizing.
2. hydrolysis and the separating-purifying of fibroin albumen：
The most crystal region of fibroin albumen macromolecular can not be utilized when not hydrolyzing, when it is hydrolyzed to molecular weight
When small molecule silk peptide less than 10KDa or so, the value of fibroin albumen could fully show.It, should be with fibroin in practical application
Based on the space structure of protein macromolecule main chain, using certain method for hydrolysis, fibroin albumen is hydrolyzed to average molecular matter
The smaller fibroin peptide product of amount, the silk peptide after hydrolysis is before biomedical, food and cosmetic field have wide application
Scape.The main method for hydrolysis of fibroin albumen has：Sour water solution, basic hydrolysis, enzyme hydrolysis etc..Each method has its advantage and disadvantage.Sour water
Solution can adequately be hydrolyzed fibroin albumen, but can lead to the destruction of amino acid；Basic hydrolysis is then easy to cause amino acid
Racemization, and then reduce fibroin albumen use value；Enzyme hydrolysis is due to having mild reaction condition and to Amino Acids Induced
Small advantage is expected to the best practice as hydrolytic degradation fibroin albumen.After fibroin albumen hydrolysis, should further it use corresponding
Separating-purifying means, the product after hydrolysis is purified.Currently, the method for protein hydrolysate separating-purifying is mainly wrapped
Include Flavonoids by Macroporous Adsorption Resin, gel filtration chromatography, high performance liquid chromatography and ultrafiltration etc..Macroporous absorbent resin itself has
Have a performance of absorption and screening, regenerating easily, have in purifies and separates bioactive substance simple equipment, mild condition and
Easy to operate advantage.Wu Jinhong, et al. using DA201-C macroporous absorbent resins to the sericin peptide taken of alcohol grading carry out desalination and
Separating-purifying, finally obtained polarity is different and has the sericin peptide taken of antioxidant activity, is the work(for being worth further investigation and exploitation
Energy product gel filtration chromatography has the advantages that simple, mild condition, and it is pure to carry out to be mainly based upon the molecular size range of sample
Change separation.Ni Li etc. isolates and purifies out silk peptide using gel filtration chromatography Sephadex-G15, finds alkali protease
When the degree of hydrolysis that Alcalase hydrolyzes fibroin is 20%, the ACE inhibitory activity of purifying gained silk peptide is most strong.Zhang Linghua is waited
Soya-bean polypeptides are isolated and purified using G-15 sephadexes.The application of high performance liquid chromatography (HPLC) not only can be with
It is completed in a short time separation purpose, it is often more important that HPLC can produce biologically active polypeptide on a preparative scale.It is super
Filter method is a kind of membrane separating method, is mainly used in genetic engineering field, is usually applied to desalination concentration, protein divides step by step
From and the processes such as buffer exchange.
Invention content
Present invention aims at provide ultrafiltration membrane preparation method and its application of a kind of cocoon sericin protein, this method
It will strip silk cocoon surface filiform, and remove remaining magazine in silk cocoon, it is spare to be cut into tiny strip, and oil is added at normal temperatures
Ether degreasing is decolourized after degreasing using soaked in absolute ethyl alcohol, and the sericine of the silk cocoon of degreasing, decoloration after dry is utilized carbonic acid
Sodium water solution boils, extraction, ultrafiltration, merging filtrate, centrifugation, supernatant with use molecular cut off for the ultrafiltration again of 30KDa
Film, rotating speed 3000rmp/min collect top, obtain glue protein extraction solution；It is shown through SDS-PAAG results：This method is extracted
Sericine contain protein of the molecular weight distribution within 14.4KDa-66KDa.The method of the invention has glue egg
The high feature of white matter yield, while ultrafiltration membrane reaches a step desalination and concentration glue protein matter purpose；Be a green, high yield,
Efficiently and it is easy to amplify a kind of high efficiency preparation method for the cocoon sericin protein implemented；The glue protein that this method obtains can be with
Used in cosmetics, the raw material of food and medicine；Removal fibroin makes silk gloss in silk, and the silk gum after removal, which is waste, becomes a kind of
High value addition product.
A kind of ultrafiltration membrane preparation method of cocoon sericin protein of the present invention, follows these steps to carry out：
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape；
B, the silk cocoon 100g that weighs that treated, with being dried after petroleum ether degreasing 48h, then again with soaked in absolute ethyl alcohol for 24 hours,
Small molecule ingredient and pigment are removed, is dried；
C, by silk cocoon obtained by step b in 100 DEG C of temperature, the sodium carbonate liquor refluxing extraction 2-4 of a concentration of 0.05-0.1%
It is secondary, it 20 minutes every time, must slightly be guided and supported；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 3-5 times using Rotary Evaporators, centrifuge successively, supernatant
Spray drying or freeze-drying, sieving, ultraviolet sterilization, packaging is to get to 30KDa above section cocoon sericin protein.
Purposes of the cocoon sericin protein that the method obtains in the drug for preparing antibacterium bioactivity.
Purposes of the cocoon sericin protein that the method obtains in the food for preparing antimycotic bioactivity.
Purposes of the cocoon sericin protein that the method obtains in cosmetics.
Purposes of the cocoon sericin protein that the method obtains in preparing food additives.
A kind of ultrafiltration membrane preparation method of cocoon sericin protein of the present invention, this method by ultrafiltration membrane desalination and
Glue protein matter is concentrated, is a green, high yield, height that is efficient and being easy to a kind of protein of silkworm cocoon (glue protein) that amplification is implemented
Imitate preparation method；What this method obtained silk gum has multiple biological activities and pharmacological action, as antibacterial and its digestive function, suppression
Ultraviolet radiation processed, hypoglycemic effect inhibit tyrosinase activity the natural material of isoreactivity ingredient, and protein content reaches
2204.1 μ g/mL, 88.1%.Pass through the SDS-PAAG electrophoresis showeds of sericine：Sericine contains molecular weight distribution
Within 14.4KDa-66KDa, mainly in 20KDa, 27KDa, 35KDa, 45KDa or so, cosmetics, food and doctor can be used in
The raw material of medicine, removal fibroin makes silk gloss in silk, and the silk gum after removal, which is waste, becomes a kind of high value addition product.
Description of the drawings
Fig. 1 be sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the present invention-silver staining chromatic graph, 13% separation gel, 4%
Glue is concentrated, wherein A is protein molecular weight standard, 66KDa, bovine serum albumin(BSA) .45KDa, ovalbumin .35KDa, pig stomach egg
White enzyme .27KDa, phosphotriose isomerase .20KDa, trypsin inhibitor .14.4KDa, lysozyme .9.5KDa, parathyroid gland
Hormone .6.5KD, Aprotinin, water body glue protein (upper ocean buffer solution), B is sample protein band (silver staining after example 1 is lyophilized
Color)；
Fig. 2 is sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the present invention-coomassie brilliant blue staining figure, and wherein A is
Protein molecular weight standard, 66KDa, bovine serum albumin(BSA) 45KDa, ovalbumin 35KDa, porcine pepsin 27KDa, phosphoric acid third
Sugared isomerase 20KDa, trypsin inhibitor 14.4KDa, lysozyme 9.5KDa, parathyroid hormone 6.5KD, Aprotinin, water
Body glue protein (upper ocean buffer solution), B are 1 sample of example, and C is that 3KD concentrates position, and D is that 3KD penetrates position.
Specific implementation mode
Embodiment 1
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape；
B, the silk cocoon 100g that weighs that treated, with being dried after petroleum ether degreasing 48h, then again with soaked in absolute ethyl alcohol for 24 hours,
Small molecule ingredient and pigment are removed, is dried；
C, by silk cocoon obtained by step b in 100 DEG C of temperature, a concentration of 0.05% sodium carbonate liquor refluxing extraction 4 times, every time
20 minutes, obtain crude extract；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 3 times using Rotary Evaporators, centrifuge successively, supernatant spray
Mist is dried, sieving, ultraviolet sterilization, packaging, you can obtain 3KD above section cocoon sericin protein powders 19.6g.
Embodiment 2
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape.
B, it is dried after the precision silk cocoon 100g that weighs that treated, petroleum ether degreasing 48h, then uses soaked in absolute ethyl alcohol again
Small molecule ingredient and pigment are removed for 24 hours, are dried；
C, by silk cocoon obtained by step b in 100 DEG C of temperature, a concentration of 0.1% sodium carbonate liquor refluxing extraction 3 times, every time
20 minutes, obtain crude extract；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 4 times using Rotary Evaporators, centrifuge successively, supernatant liquid cooling
It is lyophilized dry, sieving, ultraviolet sterilization, packaging is to get to 30KDa above section cocoon sericin albumen powders 20.1g.
Embodiment 3
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape；
B, precision weighs that treated then silk cocoon 100g uses soaked in absolute ethyl alcohol again with being dried after petroleum ether degreasing 48h
For 24 hours, small molecule ingredient and pigment are removed, is dried；
C, by silk cocoon obtained by step b in 100 DEG C of temperature, a concentration of 0.08% sodium carbonate liquor refluxing extraction 2 times, every time
20 minutes, obtain crude extract；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 5 times using Rotary Evaporators, centrifuge successively, supernatant spray
Mist is dried, and sieving, ultraviolet sterilization, packaging is to get to 30KDa above section cocoon sericin albumen powders 17.9g.
Embodiment 4
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape；
B, precision weighs that treated then silk cocoon 100g uses soaked in absolute ethyl alcohol again with being dried after petroleum ether degreasing 48h
For 24 hours, small molecule ingredient and pigment are removed, is dried；
C, by silk cocoon obtained by step b in 100 DEG C of temperature, a concentration of 0.07% sodium carbonate liquor refluxing extraction 4 times, every time
20 minutes, obtain crude extract；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 3 times using Rotary Evaporators, centrifuge successively, supernatant spray
Mist is dry or freeze-drying, sieving, ultraviolet sterilization are packed, you can obtain 30KDa above section glue protein powder 18.1g.
Embodiment 5
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape.
B, then the precision silk cocoon 100g that weighs that treated uses soaked in absolute ethyl alcohol again with being dried after petroleum ether degreasing 48h
Small molecule ingredient and pigment are removed for 24 hours, are dried；
C, by step b gained silk cocoons at 100 DEG C, a concentration of 0.1% sodium carbonate liquor refluxing extraction 3 times, 20 points every time
Clock obtains crude extract；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 5 times using Rotary Evaporators, centrifuge successively, supernatant spray
Mist is dry or freeze-drying, sieving, ultraviolet sterilization are packed, you can obtain 30KDa above section glue protein powder 21.5g.
Embodiment 6
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape；
B, precision weighs that treated then silk cocoon 100g uses soaked in absolute ethyl alcohol again with being dried after petroleum ether degreasing 48h
Small molecule ingredient and pigment are removed for 24 hours, are dried；
C, by step b gained silk cocoons at 100 DEG C, a concentration of 0.1% sodium carbonate liquor refluxing extraction 2 times, 20 points every time
Clock obtains crude extract；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 3 times using Rotary Evaporators, centrifuge successively, supernatant spray
Mist is dry or freeze-drying, sieving, ultraviolet sterilization are packed, you can obtain 30KDa above section glue protein powder 21.9g.
Embodiment 7
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape.
B, then the precision silk cocoon 100g that weighs that treated uses soaked in absolute ethyl alcohol again with being dried after petroleum ether degreasing 48h
Small molecule ingredient and pigment are removed for 24 hours, are dried；
C, by step b gained silk cocoons at 100 DEG C, a concentration of 0.07% sodium carbonate liquor refluxing extraction 3 times, 20 points every time
Clock obtains crude extract；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 3 times using Rotary Evaporators, centrifuge successively, supernatant spray
Mist is dry or freeze-drying, sieving, ultraviolet sterilization are packed, you can obtain 30KDa above section glue protein powder 20g.
Embodiment 8
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape；
B, then the precision silk cocoon 100g that weighs that treated uses soaked in absolute ethyl alcohol again with being dried after petroleum ether degreasing 48h
Small molecule ingredient and pigment are removed for 24 hours, are dried；
C, by silk cocoon obtained by step b in 100 DEG C of temperature, a concentration of 0.1% sodium carbonate liquor refluxing extraction 4 times, every time
20 minutes, obtain crude extract；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant,
Obtain glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 4 times using Rotary Evaporators, centrifuge successively, supernatant spray
Mist is dry or freeze-drying, sieving, ultraviolet sterilization are packed, you can obtain 30KDa above section glue protein powder 20.5g.
Embodiment 9
The measurement of protein concentration：
It is measured using bicinchoninic acid (bicinchoninic acid, BCA) method and implements 3KD above section glue obtained by 1-8
Protein concentration in albumen powder；
The drafting of standard curve operates according to BCA protein measurement kit specifications and prepares working solution, every 50 times of volumes
BCA reagent A solution adds the BCA reagent B solutions of 1 times of volume, mix well it is spare, according to table 1 draw kit in protein standard
Product bovine serum albumin, is diluted with water, and shakes up, and each pipe takes 25 μ L in 96 orifice plates respectively, each that 175 μ L BCA working solutions are added, gently
Micro oscillation, mixing incubate 30min in 37 DEG C of insulating boxs of temperature, take out 96 orifice plates, measure absorbance with microplate reader, measure wave
A length of 562nm.It is each do three groups it is parallel, using absorbance as ordinate, protein concentration is that abscissa carries out regressing calculation；
Table 1 is serially diluted bovine serum albumin(BSA) (BSA) standard items
The measurement of sample：
It is accurate to weigh 2.5mg3KD above section glue protein powder samples, 1mL is added and distills water dissolution, 10000 revs/min of centrifugations
2min takes 25 μ L samples solution in 96 orifice plates according to standard curve operating method, each that 175 μ L BCA working solutions are added, slightly
Oscillation, mixing incubate 30min in 37 DEG C of insulating boxs of temperature, take out 96 orifice plates, measure absorbance with microplate reader, measure wavelength
For 562nm, each sample do three groups it is parallel, the content of protein is calculated according to standard curve；
Experimental result：
By Fig. 2 and table 2 it is found that being extracted regardless of solution, using liquid nitrogen grinding to the extract protein of feedstock processing
Content is extract obtained higher than stirring means processing raw material；Using the extract of same material processing, phosphate is utilized
The protein content of buffer solution extraction is higher than to be extracted using sodium chloride；Protein content is reduced after removing salting-in-protein, therefore, profit
Raw material is crushed with liquid nitrogen grinding method, extract obtained protein content highest is extracted with phosphate buffer, up to 2204.1 μ
G/mL accounts for the 88.1% of extract, is shown in Table 2
2 protein extracting ratio of table is the glue protein percentage amounts extracted per 100g silk cocoons
| Experiment condition | Protein extracting ratio | Protein content | |
| Example 1 | 0.05% sodium carbonate, 3KD, 48h | 19.6% | 0.88mg/ml |
| Example 2 | 0.05% sodium carbonate, 3KD, 72h | 20.1% | 0.78mg/ml |
| Example 3 | 0.05% sodium carbonate, 8KD, 48h | 17.9% | 0.81mg/ml |
| Example 4 | 0.05% sodium carbonate, 8KD, 72h | 18.1% | 0.59mg/ml |
| Example 5 | 0.1% sodium carbonate, 3KD, 48h | 21.5% | 0.63mg/ml |
| Example 6 | 0.1% sodium carbonate, 3KD, 72h | 21.9% | 0.61mg/ml |
| Example 7 | 0.1% sodium carbonate, 8KD, 48h | 20.0% | 0.59mg/ml |
| Example 8 | 0.1% sodium carbonate, 8KD, 72h | 20.5% | 0.68mg/ml |
Embodiment 10
SDS-PAAG collection of illustrative plates freezes 1 protein of embodiment using sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Dry-eye disease (Fig. 1), ultrafiltration membrane ultrafiltration upper bit and non-ultrafiltration solution (Fig. 2) carry out protein classes determination：
Preparation of reagents and sample treatment：
Sample buffer：0.5mL trishydroxymethylaminomethanes and mixed in hydrochloric acid buffer solution (0.5moL/L pH=6.8),
10% lauryl sodium sulfate, 1mL glycerine, the beta -mercaptoethanol of 0.5mL, the water of 1mL, the micro bromophenol blue of 2mL；
The processing of sample：Precision weighs 3 milligrams of sample and is dissolved in 1mL distilled water, shakes up, and is mixed in equal volume with sample buffer
It closes, water proof boils 10 minutes, 10000 revs/min, centrifuges 5 minutes；
Electrode buffer：By 15.1 grams of trishydroxymethylaminomethanes, 10% dodecyl sulphate of 94 grams of glycine, 50mL
Sodium dissolves and is settled to 4L；
Sodium dodecyl sulfate-polypropylene acrylamide gel storing liquid：30% (W/V) extra-pure grade acrylamide, 0.8% (W/
V) N, N- methylene bisacrylamide；
The separation gel of 12% sodium dodecyl sulfate-polypropylene acrylamide gel is prepared：30% (the V/ of H2O, 4mL of 3.3mL
V) sodium dodecyl sulfate-polypropylene acrylamide gel storing liquid, 2.5mL 1.5moL/L trishydroxymethylaminomethane and hydrochloric acid
Cocktail buffer (pH=8.8), 10% (W/V) lauryl sodium sulfate of 0.1mL, 0.1mL 10% (W/V) ammonium persulfate,
The tetramethylethylenediamine of 0.006mL；
The spacer gel of 5% sodium dodecyl sulfate-polypropylene acrylamide gel is prepared：The 30% of H2O, 0.17mL of 0.68mL
(V/V) sodium dodecyl sulfate-polypropylene acrylamide gel storing liquid, 0.13mL trishydroxymethylaminomethanes and mixed in hydrochloric acid buffer
10% (W/V) persulfuric acid of liquid (0.5moL/L pH=6.8), 10% (W/V) lauryl sodium sulfate of 0.01mL, 0.01mL
The tetramethylethylenediamine of ammonium, 0.001mL；
Deposition condition：75 volts of constant pressure, 40 minutes, then 150 volts of constant pressure, 90 minutes；Fixer：25% isopropanol, 10%
Glacial acetic acid, 65% water；Silver staining (uses this method) to preferably observe protein band after freeze-drying；It impregnates：75ml ethyl alcohol,
17g sodium acetates, 1.25ml25% glutaraldehydes, 0.5g sodium thiosulfate 5H2O dH2250ml 30min are added to after O dissolvings；Drift
It washes：Use dH2O is rinsed 3 times 5min/ times；Silver staining：0.25g silver nitrates, 50ul formaldehyde add to 250ml 20min with dH2O；Colour developing：
6.25g sodium carbonate, 25ul formaldehyde, uses dH2O adds to 250ml 2-10min opsin bands；Show dark-brown；It terminates：
3.65gEDTA-Na2·2H2O uses dH2O adds to 250ml 10min；Rinsing：Use dH2O is rinsed 3 times 5min/ times；It preserves：25ml
Glycerine dH2O adds to 250ml 30min taking-ups and dries；
Experimental result：There are many containing in 1 Silver stain of embodiment in sodium dodecyl sulfate-polyacrylamide gel electrophoresis figure
Activated protein, wherein sericine contain molecular weight distribution within 14.4KDa-66KDa.
Implementation column 11
Silk-fibroin amino acid analysis
This method is enlightening equine skill application experiment room respectively using phenyl isothiocyanate, 2,4- dinitrofluorobenzene as derivating agent
Protein hydrolyzate and free amine group acid injection are derived, then detached using Diamonsil AAA columns, it can
Meet the analysis of 19 kinds of natural amino acids, each component separating degree is higher, quantitative result is accurate and stablizes；
Reagent：0.1% phenol+6mol/l HCl/water solution：It weighs 0.1g phenol and is placed in 100ml volumetric flasks, 50ml is added
Concentrated hydrochloric acid (36%-38%, molar concentration are about 12mol/l), then plus water is settled to 100ml, 0.1mol/l HCl/water solution：
8.3ml concentrated hydrochloric acids are measured, are then settled to 1000ml amino acid stocks with pure water：A certain amount of amino acid standard is weighed, is used
0.1mol/l HCl/water solution dissolves, cystine 0.01mol/l, tyrosine 0.02mol/l, other amino acid are
0.05mol/l；
Amino acid uses liquid：Storing solution 0.1mol/l HCl/water solution is diluted, obtains a concentration of 0.002mol/l's
Amino acid list mark and mixed mark (cystine concentration 0.001mol/l) are used for PITC derivatization methods；By storing solution 0.1mol/l HCl
Aqueous solution dilutes, and obtains the amino acid list mark of a concentration of 0.0005mol/l and mixed mark (cystine concentration 0.00025mol/l)
For DNFB derivatization methods；
Nor-leucine internal standard solution：Using nor-leucine as internal standard compound.A certain amount of nor-leucine is weighed, 0.1mol/L is dissolved in
HCl/water solution obtains the nor-leucine internal standard solution of 0.02mol/L；
Phenyl isothiocyanate solution：250 μ l phenyl isothiocyanates are settled to 10ml with acetonitrile, obtain the different sulphur of 0.2mol/L
Phenyl-cyanate solution；
Triethylamine solution：1.4ml triethylamines are settled to 10ml with acetonitrile, obtain 1.0mol/L triethylamine solutions 1%
DNFB acetonitrile solutions：0.5ml 2,4-dinitrofluorobenzene (DNFB) is dissolved in 50ml acetonitriles, and volume fraction 1% is approximately equal to
0.08mol/l；0.1mol/L Na2B4O7 aqueous solutions：1.91g Na2B4O710H2O are weighed, are dissolved with 50ml pure water, solution
PH is about 9.50；
Sample treatment：Protein hydrolyzes (acid-hydrolysis method)：Protein example (feed, milk powder, animal tissue etc.)：For dynamic
Object sample, crushed after being dried is at fine particle；For feed, it is ground into fine particle；Milk powder is not required to pre-process, and sample is placed in
20m spiral covers (liner PTFE dottle pins) narrow-mouthed bottle is added 0.1% phenol+6mol/l HCl/water solution of 10ml, screws lid, vibrates
Mixing is reacted for 24 hours at 110 DEG C of temperature, and reaction finishes, and reaction solution is transferred completely into 100ml cucurbits, subtracts at 75 DEG C of temperature
Pressure distillation is done to close, to remove HCl remaining in reaction solution, is then dissolved in three times with 12ml 0.1mol/L HCl/waters solution
Residue, and 20ml glass tool plug scale test tubes are transferred to, then it is settled to 20ml with pure water, it waits deriving；
Injection processing：When using PITC derivatization methods, sample is diluted to 4g/l with 0.1mol/l HCl/water solution；When
When deriving using DNFB, sample is diluted to 2g/L with 0.1mol/L HCl/water solution；
The analysis condition of amino acid-PITC derivatives：
Chromatographic column：Diamonsil AAA amino acid analysis columns, 250 × 4.6mm, 5 μm of (CAT#:99751)；
Guard column：EasyGuard C18,10 × 4.0mm, 5 μm (CAT#:6201)；
Mobile phase A：0.05mol/l aqueous sodium acetate solutions (it is 6.50 ± 0.05 that glacial acetic acid, which adjusts pH value)；
Mobile phase B：Methanol:Acetonitrile:Water=20:60:20 (volume ratios)；
Flow velocity：1.0ml/min, column temperature：35℃；Sampling volume：10 μ l, Gradient program；Time/min 0,39,40,45,
46,60；Mobile phase A/%95,52,0,0,95,95 Mobile phase Bs/%5,48,100,100,5,5；
3 Gradient program of table
| Time/min | 0 | 39 | 40 | 45 | 46 | 60 |
| Mobile phase A/% | 95 | 52 | 0 | 0 | 95 | 95 |
| Mobile phase B/% | 5 | 48 | 100 | 100 | 5 | 5 |
Experimental result is shown in Table 4：
4 glue protein aminoacid ingredient of table and assay result
Embodiment 13
Determination of elemental analysis：
The method of carbon, hydrogen, nitrogen and sulphur or oxygen (C, H, N, S/O) constituent content in organic matter is measured with elemental analyser, is fitted
| || micro type elemental analyser for adsorbing separation and chromatographic isolation formula；
Method And Principle
Carbon, hydrogen, nitrogen and sulphur in organic matter or oxygen (C, H, N, S/O) element, after catalysis oxidation (or cracking)-reduction
It is transformed into carbon dioxide, vapor, nitrogen and sulfur dioxide or carbon monoxide (CO2, H2O, N2, SO2/CO) respectively.Then exist
Under the promotion of carrier gas, each component of method sequentially determining is examined with adsorbing separation-thermal conductivity difference；Or mixed gas is detached with chromatography
Afterwards, the response signal value of component is measured respectively with thermal conductivity detector (TCD) or infrared absorption detector.According to the signal value of component (or color
Spectrum peak) and corresponding element sensitivity (or correction) factor K value, calculate separately the content of various elements in sample；
Reagent and material：
Standard substance：Benzoic acid, antifebrin, cyclohexanone -2,4- dinitrophenylhydrazone, chlorination S- benzyl thiocarbamides, p-aminophenyl
Sulfonic acid；
ω × 102 of carrier gas (helium or argon gas) purity >=99.99, moisture≤10-6g/L；
ω × 102 of oxygen purity >=99.99, moisture≤10-6g/L；
Instrument forms：Instrument is mainly by filling the oxidation tube of oxidation catalyst and filling with reduction tube and the heating of reducing agent
At stove, separation and the separation of detection oxidation (or cracking)-reduzate CO2, H2O, N2 and SO2 or CO and detecting system, data
Reason machine or microcomputer workstation, micro autoelectrinic balance (see appendix A) and groups of printers at；
Sample：Sample should be free from the homogeneous solid particle or liquid of adsorption moisture, volatile samples low-melting alloy
Container sealing weighs, and corrosive liquids is weighed with low-melting glass capillary seal, and when oxidation should have explosion precaution；
K values measure：Standard sample 1mg-3mg is weighed, 0.001mg is accurate to, by sample evaluation procedures METHOD FOR CONTINUOUS DETERMINATION two
Above standard sample, until result reaches the specified value of instrumentation regulation；
K values calculate：The K values of i elements press (1-a) formula (adsorption separation method) or (1-b) formula (chromatographic isolation in standard sample
Method) it calculates：
The signal value of μ V-standard sample i element unit quality in formula；M-standard sample quality；W-standard sample i members
Plain true value percentage；
M-standard sample quality in formula；W-standard sample i element true value percentages；A-standard sample i elements peak face
Product；The K values of Ki=i (C, H, N and S or O) element in formula (1) and formula (2)；
Sample measures：Sample 1mg-3mg is weighed, 0.001mg is accurate to, by sample evaluation procedures METHOD FOR CONTINUOUS DETERMINATION 2 or more
Sample, until result reaches the requirement of analytical error；
Experimental result is shown in Table 5：
5 glue protein constituent content of table
| Weight | C% | H% | S% | N% |
| 1mg | 41.01 | 5.922 | 0.280 | 14.73 |
Embodiment 14
Antioxidant activity is tested：
Experiment reagent：(2,2 '-hexichol are for bitter taste by DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate)
Acyl group phenylhydrazine), it is a kind of lipid free radical centered on nitrogen of stabilization, contains 3 phenyl ring, methanol or ethyl alcohol in structure
Solution is in purple, has strong absorption near 517nm, in the presence of having the antioxidant of hydrogen supply capacity, color can be by dark purple discoloration
It is light yellow[4], the absorbance at 517nm becomes smaller.The degree that its absorbance becomes smaller is proportionate with the ability for removing free radical
Property, thus can be used for detecting antioxidant activity；
Experimental method：
Prepare 6 × 10-5M DPPH (Sigma) methanol solution：1.2mg DPPH are weighed, 500 μ L methanol solutions is dissolved in, is made into
100 × liquid is preserved, it is placed in 4 DEG C of refrigerators of temperature and preserves, answered at working solution, DPPH working solutions with after 100 times of methanol dilution using preceding
It is kept in dark place as possible, it is now with the current；
Be dissolved in ultra-pure water polypeptide sample be added DPPH working solutions, system be 100 μ L, room temperature avoid light place 30min, in
Light absorption is detected at 517nm, free radical is reacted in the reduction relative to blank control (with the ultra-pure water of sample same volume) light absorption
Removing situation, each concentration samples do 3 it is parallel, ultraviolet specrophotometer return to zero when use methanol；
DPPH free radical scavenging activities are calculated by the following formula：
Clearance rate=[(A0-A1)/A0] × 100
Wherein, A0 is blank control absorbance value, and A1 is that sample sets absorbance value is added
Experimental result is shown in Table 6
6 glue protein of table removes the capability result table of free radical
As can be seen from the table：The cocoon sericin protein that the present invention obtains has the activity for removing free radical.
Claims (5)
1. a kind of ultrafiltration membrane preparation method of cocoon sericin protein, it is characterised in that follow these steps to carry out：
A, the pre-treatment of silk cocoon：The filiform on silk cocoon surface is peelled off, remaining impurity in silk cocoon is removed, is cut into tiny item
Shape；
B, the silk cocoon 100g that weighs that treated, with being dried after petroleum ether degreasing 48h, then again with soaked in absolute ethyl alcohol for 24 hours, remove
Small molecule ingredient and pigment, dry；
C, by silk cocoon obtained by step b in 100 DEG C of temperature, the sodium carbonate liquor refluxing extraction of a concentration of 0.05-0.1% 2-4 times, often
It secondary 20 minutes, must slightly be guided and supported；
D, after the crude extract concentration obtained step c, under 12000 revs/min, 4 DEG C of centrifugation 10min of temperature take supernatant, obtain
Glue protein extracting solution；
E, the glue protein crude extract for obtaining step d uses molecular cut off for the ultrafiltration membrane again of 30KDa, rotating speed
3000rmp/min collects top, obtains glue protein extraction solution；
F, step e is obtained into glue protein and extracts solution, be concentrated into 3-5 times using Rotary Evaporators, centrifuge successively, supernatant spraying
Dry or freeze-drying, sieving, ultraviolet sterilization, packaging is to get to 30KDa above section cocoon sericin protein.
2. use of the cocoon sericin protein that method as described in claim 1 obtains in the drug for preparing antibacterium bioactivity
On the way.
3. use of the cocoon sericin protein that method as described in claim 1 obtains in the food for preparing antimycotic bioactivity
On the way.
4. purposes of the cocoon sericin protein that method as described in claim 1 obtains in cosmetics.
5. purposes of the cocoon sericin protein that method as described in claim 1 obtains in preparing food additives.
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| CN114404568A (en) * | 2022-01-16 | 2022-04-29 | 重庆理工大学 | Sericin polypeptide injection and application thereof |
| CN114989285A (en) * | 2022-06-16 | 2022-09-02 | 华南农业大学 | Multifunctional mulberry silk collagen and application thereof in biomedical materials |
| CN115531284A (en) * | 2022-09-15 | 2022-12-30 | 江苏华佳丝绸股份有限公司 | Preparation method of silk mask containing sericin |
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| CN115531284A (en) * | 2022-09-15 | 2022-12-30 | 江苏华佳丝绸股份有限公司 | Preparation method of silk mask containing sericin |
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