CN103923965A - Fibroin micro-molecule polypeptide preparing method - Google Patents
Fibroin micro-molecule polypeptide preparing method Download PDFInfo
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- CN103923965A CN103923965A CN201410194925.4A CN201410194925A CN103923965A CN 103923965 A CN103923965 A CN 103923965A CN 201410194925 A CN201410194925 A CN 201410194925A CN 103923965 A CN103923965 A CN 103923965A
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Abstract
The invention relates to a fibroin micro-molecule polypeptide preparing method. Fibroin is used for producing micro-molecule polypeptide. The method comprises the following steps that first, fat of silkworms is separated out; fibroin is processed into powder of 200-300 meshes after fat separation; fibroin and 20%-50% of papain are subjected to enzymolysis, and during enzymolysis, 5%-10% of selenium enrichment enzymes are added for fermentation treatment for 24 hours; then fibroin micro-molecule polypeptide after fermentation is subjected to freeze drying treatment, and the water content of the fibroin micro-molecule polypeptide does not exceed 13%-15%; and finally product packaging is carried out, and the packaging requirement is that nitrogen-filled packaging needs to be used. According to the fibroin micro-molecule polypeptide preparing method, the bio-orientation enzymolysis technology is used for obtaining fibroin micro-molecule polypeptide from silkworm protein, the fibroin micro-molecule polypeptide has the advantages that anti-aging performance is achieved, digestive absorption is easy, the biological regulation effect on a human body is achieved, and antigenicity is low.
Description
Technical field
Invention relates to silkworm protein peptide, is specifically related to a kind of preparation method of fibroin micromolecule polypeptide.
Background technology
Silkworm egg is a kind of function macro-molecular protein in human plasma in vain, is to maintain plasma osmolarity, increase Q volume of blood, the important component of amino acid is provided, and be the indispensable nutritive substance of human body.The composition of amino acid that silkworm egg is white is closely similar, has 20 whole seed amino acids, and its biological value, up to 94, is outstanding protein.The small-molecular peptides that silkworm egg obtains by enzymolysis in vain, it is more conducive to absorption of human body, and body is had to multiple physiological regulation function, as resisting oxidation and delaying senility, enhancing body immunizing power, resistance Zinc metallopeptidase Zace1 produce the effect of reducing blood pressure etc.
It is white that silkworm protein peptide derives from silkworm egg, utilizes the bioactive small-molecular peptides that has that modern biotechnology extracts and extract through a series of processing technologies through prozyme directional enzymatic, and those skilled in the art are devoted to a problem of research and development just.
Summary of the invention
Task of the present invention is to provide a kind of fibroin micromolecule polypeptide preparation method, from silkworm egg is white, produce fibroin micromolecule polypeptide, utilize bio-orientation zymolysis technique to produce fibroin micromolecule polypeptide, that fibroin micromolecule polypeptide has is anti-ageing, be easy to digest and assimilate, biological regulation effect to human body and the feature of low antigenicity.
Technical solution of the present invention is as follows:
A kind of fibroin micromolecule polypeptide preparation method, utilizes silk protein to produce micromolecule polypeptide, comprises the following steps:
First self grease of silkworm is separated;
Silk protein separates post-treatment through grease and becomes 200 order～300 object pulvis;
Carry out enzymolysis by 20%～50% of fibroin and papoid, add 5%～10% rich selenium ferment enzyme to carry out fermentative processing when enzymolysis, the fermentative processing time is 24 hours;
Then the fibroin micromolecule polypeptide fermenting is carried out to lyophilize processing, fibroin micromolecule polypeptide water content is no more than 13%～15%;
Finally carry out the packing of product, packing instructions are to adopt nitrogen-filled packaging.
Described fibroin micromolecule polypeptide pulvis is creamy white or micro-yellow, odorless tasteless, can be dissolved in water completely and be water white transparency state.
The content > 80% of described fibroin micromolecule polypeptide.
Described fibroin micromolecule polypeptide contains protein 30%～45%, and class silkworm egg is white 12%, albumen and MUC1 4%, the white egg 8% of ball, the lytic enzyme 3.4% of solubilized bacterial cell wall.
Described fibroin micromolecule polypeptide is made oral powder or liquor.
Adopt fibroin micromolecule polypeptide preparation method of the present invention, utilize bio-orientation zymolysis technique to produce fibroin micromolecule polypeptide from silkworm egg is white, fibroin micromolecule polypeptide pulvis is milky white or micro-yellow, odorless tasteless, is dissolved in water completely and is water white transparency state.That fibroin micromolecule polypeptide has is anti-ageing, be easy to digest and assimilate, biological regulation effect to human body and the feature of low antigenicity.
Brief description of the drawings
Fig. 1 is the pupa worm peptide inspection after construction report of producing by fibroin micromolecule polypeptide preparation method of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention is elaborated.
By a kind of fibroin micromolecule polypeptide preparation method of the present invention, utilize silk protein to produce micromolecule polypeptide, comprise the following steps:
First self grease of silkworm is separated, because the oleaginousness of silkworm itself can affect the explained hereafter of producing polypeptide.The grease that silkworm is separated also has very high value, can be made full use of.
Silk protein separates post-treatment through grease and becomes 200 order～300 object pulvis.Fibroin micromolecule polypeptide pulvis is creamy white or micro-yellow, odorless tasteless, can be dissolved in water completely and be water white transparency state.
20%～50% of silkworm egg is white and papoid carries out enzymolysis again, adds 5%～10% rich selenium ferment enzyme to carry out fermentative processing when enzymolysis, and the fermentative processing time is 24 hours.
Then the fibroin micromolecule polypeptide fermenting is carried out to lyophilize processing, fibroin micromolecule polypeptide water content is no more than 13%～15%, the content > 80% of fibroin micromolecule polypeptide.
Fibroin micromolecule polypeptide contains protein 30%～45%, and class silkworm egg is white 12%, albumen and MUC1 4%, the white egg 8% of ball, the lytic enzyme 3.4% of solubilized bacterial cell wall.
Finally carry out the packing of product, packing instructions are to adopt nitrogen-filled packaging, otherwise product can go bad.Quality guaranteed period can reach 2～3 years to the fibroin micromolecule polypeptide product of nitrogen-filled packaging in the ordinary course of things.
The fibroin micromolecule polypeptide of preparing by the inventive method can be made into oral powder, particle, capsule or liquor, and oral functional peptides has biological regulation effect to human body.
Fig. 1 has shown the pupa worm peptide inspection after construction report of producing by fibroin micromolecule polypeptide preparation method of the present invention.Pupa worm peptide is silkworm protein peptide, and its outward appearance is brown ceramic powder, without the visible impurity of naked eyes, free from extraneous odour.Wherein moisture content 2.98g, ash oontent 6.8g, protein (butt) 73.1g, polypeptide (butt) 72.0g, total reducing sugar 6.2g, the massfraction of the shared total peptide section of≤relative molecular mass peptide section is 92.05%.Check conclusion is qualified.
The fibroin micromolecule polypeptide of preparing by the inventive method has following concrete physicochemical property:
1, the content of fibroin micromolecule polypeptide: > 80%.
2, silkworm protein peptide relative molecular mass scope: HPLC method, chromatographic condition is Agilent 1100, TSKge l2000SWxl, 300 × 7.8mm(TOSH company); Moving phase: acetonitrile: water: trifluoro water acid=20:80:0.1,220nm, 0.5ml/min.Application GPC software analysis is known, and silkworm protein peptide relative molecular mass mark scope is: be less than 1.0 × 10
6account for 84%, 1.0 × 10
6～2.0 × 10
6account for 13%, be greater than 2.0 × 10
6account for 3% left and right, be mainly distributed in 1.0 × 10
6below.
3, the white peptide ammino acid composition of silkworm egg: in percentage composition (%), wherein Asp 7.48, Ser 6.82, Gly 2.92, Ala 4.72, Tyr 3.26, Val 5.34, Phe 6.72, Leu 8.64, Pro 2.60, Glu 10.94, His 1.80, Thr 3.20, Arg 4.50, Cys 1.16, Met 3.46, Ile 4.27, Lys 5.36.
4, be easy to digest and assimilate: in recent years, peptide matters is at the existing newer research report of the absorption process of human body, confirmed that intestinal mucosa exists the delivery system that absorbs low peptide, intestinal absorption is not only with amino acid whose form, and is mostly to be absorbed with the form of oligopeptides.In diet, the peptide section of different lengths (2～3 amino acid are 10～51 amino acid even) all can be to some extent through intestinal absorption, and can be used as biologically active peptides and organizing the biological effect that causes body in level.
5, the biological regulation effect of oral polypeptide to human body: the peptide of verified low relative molecular masses can directly enter blood after by intestinal absorption, produces biological effect.In addition the polypeptide that, some relative molecular masses are larger also can also be produced effect through cross-cell membrane approach, paracellular pathway, M cellular pathways by intestinal absorption in vivo with complete form.According to research reports, give rat 125ug throtropin releasing hormone (TRH, tripeptides) give TRH50ug comparison with vein through intestines, the thyrotropic hormone (TSH) that can produce same intensity in blood is replied.Give metakentrin through intestines and discharge (LHRH, 10 peptides) and vein and give LHRH comparison, in blood, measure reply hormone FSH be vein give 50%.Above-mentioned experiment shows, oral have bioactive functional peptides and can produce regulating effect to human body.
6, low antigenicity: verified, except supplementing the nutrients, because fibroin micromolecule polypeptide is small-molecule substance, while producing biological effect in vivo, can't cause the anaphylaxis of health, this has more significant using value for infant, the elderly, after being ill decubation and gastrointestinal function impaired patients.
It is anti-ageing or delay senility that the fibroin micromolecule polypeptide product of preparing by the inventive method can be applicable to polypeptide, by oral micromolecule polypeptide albumen, and can be by the transhipment passage of small intestine directly by cell, histoorgan be utilized.Fibroin micromolecule polypeptide also has antioxygenation, enhancing immunologic function and the endocrine regulation effect to human body.
Fibroin micromolecule polypeptide has Antioxidation in vitro: in the white hydrolyzate of protease of silkworm egg, have the bioactive peptide with oxidation-resistance, a large amount of accumulation that the active Toplink of this class resists machine interior free yl, play certain effect to improving free radical scavenging mechanism, thereby can eliminate to a certain extent different physiological roles obstacle in body, delay the aging of body, reduce the generation of various geriatric diseases.Experimental results show that: the resistance of oxidation of fibroin micromolecule polypeptide strengthens with the increase of its concentration, in experimental concentration (1～100mg/ml) scope, free radical is all had to scavenging(action), and along with concentration increases, its effect of removing free radical strengthens.In the time that concentration exceedes 50mg/ml, clearance rate increases slowly.The fibroin micromolecule polypeptide that different process obtains, its resistance of oxidation is not identical yet.The fibroin micromolecule polypeptide anti-oxidant vigor being obtained by alkaline enzymic hydrolysis is stronger than the fibroin micromolecule polypeptide anti-oxidant vigor being obtained by protease hydrolysis.
The antioxygenation of silkworm protein peptide to aging model mouse: adopt the method for the subcutaneous injection D-of nape portion semi-lactosi to prepare aging model, then fill with the silkworm protein peptide 45d that feeds various dose, measure Hemoglobin Value, MDA value and the SOD activity in blood, the SOD activity of hepatic tissue.Result shows: three dosage groups of silkworm protein peptide all can improve the content of the oxyphorase of mouse; 20 times and 30 times groups of silkworm protein peptide have the effect that reduces MDA content in blood; In 20 times of group blood of silkworm protein peptide, the activity of SOD is significantly higher than the blank group of model (p < 0.05); 20 times and 30 times group silkworm protein peptide all can improve the SOD activity of murine liver tissue.Point out thus silkworm protein peptide to mice aging model have anti-oxidant, prolong old and feeble effect.
Fibroin micromolecule polypeptide has immuno-potentiation to mouse body: fibroin micromolecule polypeptide contains 30%～45% protein, 12% class silkworm egg white (Transferrins,iron complexes), 14% albumen and mucoprotein, 8% the white egg of ball, the lytic enzyme of 3.4% solubilized bacterial cell wall, and some other protein.
The product (silkworm protein peptide or protein peptide) of silkworm egg white matter specific protein enzymic hydrolysis has multiple physicochemical property and function, and as gelation, emulsifying property, whipability, thermo-coagulating etc., these characteristics make it in much food, become desirable additive.Silkworm protein peptide has the effect of enhancing nucleic acid at intestinal absorption and promotion body spleen lymphocyte proliferation.By methods such as series filtration, chromatography, purifying, the immunoglobulin (Ig) during silkworm egg is white can be reinforced and separate, and has the ability that improves body resist the disease.
By the result of study of cell and humoral immune function is shown, three dosage of silkworm protein peptide and control group comparison, have no the weight of animals and body weight gain had to obvious impact.Thymus gland/body weight ratio of three dosage groups and control group comparison, difference all has statistical significance.Results of immunologic detection confirms, the swollen degree of delayed allergy test ear and control group comparison, 10 times, 20 times, 30 multiple dose group difference highly significants; The dark blood pH-value determination pH of mouse half, 10 times, 20 times, HC50 value and the control group comparing difference of 30 multiple dose groups have statistical significance; Mouse is huge bite rate and silkworm bite index all the utmost point be significantly higher than control group, the phagocytic rate of 10 multiple dose groups is significantly higher than control group; NK cytoactive detection shows, with control group comparison, 30 multiple dose groups significantly raise, and result is positive.Above results suggest silkworm protein peptide has the effect that significantly improves mouse cell and humoral immune function.
The silkworm protein peptide being produced by cocconase solution is except having the effect of supplementary proteinaceous nutrient and indispensable amino acid, human body is also possessed to multiple regulatory function, as regulate immunity, promote various nutrition speciality in food the absorption of enteron aisle, anti-oxidant, resist the multiple physiological regulation functions such as Zinc metallopeptidase Zace1 activity decreased blood pressure.Study and show recently, silkworm protein peptide can make the serum immunoglobulin level of antigen immune mouse significantly raise.External Schanbacher report, silkworm protein peptide can strengthen the phagolysis of mouse peritoneal generation phagocyte to sheep red blood cell in testing in vitro, and intravenous injection can make mouse strengthen the ability that anti-klebsiella pneumoniae infects.Researchist injects 93 routine leukaemics by silkworm protein peptide, finds that the white Toplink of silkworm egg significantly strengthens leukaemic's anti-infection ability, reduces its case fatality rate.The polypeptide form that discovery silkworm protein peptide can be complete is at present by intestinal absorption, these micromolecule polypeptides can stimulate the propagation of body Autologous lymphocyte, the release that promotes cytokine, the huge ability of biting of enhancing body scavenger cell, and the activating macrophage silkworm under very low dose bites the effect of function, its mechanism of action is by bringing out a large amount of hyperplasia of rat lymphocyte, strengthening lymphocyte moves to inflammation part, and induction of lymphocyte secretion lymphokine, thereby promote body's immunity to strengthen.
Through experimental observation, silkworm protein peptide has the nucleic acid of promotion in the absorption of small intestine and in the distribution of the immune organs such as thymus gland, and the silkworm protein peptide various aminoacid components that contain equiblibrium mass distribution, point out thus silkworm protein peptide to the enhancement of immunologic function may with immune cell propagation is provided, to break up required nutritive substance relevant.In addition, the reports such as external Lillard, silkworm protein peptide has the lymphocytic proliferative response of CD4T and the release of cytokine and the expression of costimulating factor of remarkable enhancing CD3 induction.Prompting silkworm protein peptide also may, by promoting the generation of the biologically active substance relevant with immunity, reach the effect that improves immunizing power.Experimental result show silkworm protein peptide for building up health, preventing disease and promote that rehabilitation is after being ill favourable.
Result shows, the oral silkworm protein peptide of mouse and nucleic acid group with take nucleic acid group merely and compare, silkworm protein peptide has the sorption of remarkable promotion Nucleotide, shows that, in same detection time, Nucleotide increases in liver and thymus gland.Liver is the metabolic most important organ of body, and nucleic acid raw material, in this enrichment, shows to be conducive to body cell reparation and delaying senility function.Thymus gland is immune maincenter organ, and nucleic acid, in the enrichment of thymus gland, shows to be conducive to immune cell in processes such as this maturation, differentiation, ensures renewal herein of immunocyte and required various base substances thereof and the supply of raw material.
In sum, adopt fibroin micromolecule polypeptide preparation method of the present invention, utilize bio-orientation zymolysis technique to produce fibroin micromolecule polypeptide from silkworm egg is white, that fibroin micromolecule polypeptide has is anti-ageing, be easy to digest and assimilate, biological regulation effect to human body and the feature of low antigenicity.
Certainly, those skilled in the art in the art will be appreciated that, above-described embodiment is only for the present invention is described, and not as limitation of the invention, as long as within the scope of connotation of the present invention, variation, the modification etc. of above-described embodiment all will be dropped in the scope of the claims in the present invention.
Claims (5)
1. a fibroin micromolecule polypeptide preparation method, is characterized in that, utilizes silk protein to produce micromolecule polypeptide, comprises the following steps:
First self grease of silkworm is separated;
Silk protein separates post-treatment through grease and becomes 200 order～300 object pulvis;
Carry out enzymolysis by 20%～50% of fibroin and papoid, add 5%～10% rich selenium ferment enzyme to carry out fermentative processing when enzymolysis, the fermentative processing time is 24 hours;
Then the fibroin micromolecule polypeptide fermenting is carried out to lyophilize processing, fibroin micromolecule polypeptide water content is no more than 13%～15%;
Finally carry out the packing of product, packing instructions are to adopt nitrogen-filled packaging.
2. fibroin micromolecule polypeptide preparation method according to claim 1, is characterized in that: described fibroin micromolecule polypeptide pulvis is creamy white or micro-yellow, odorless tasteless, can be dissolved in water completely and be water white transparency state.
3. fibroin micromolecule polypeptide preparation method according to claim 1, is characterized in that: the content > 80% of described fibroin micromolecule polypeptide.
4. fibroin micromolecule polypeptide preparation method according to claim 1, it is characterized in that: described fibroin micromolecule polypeptide contains protein 30%～45%, class silkworm egg is white 12%, albumen and MUC1 4%, the white egg 8% of ball, the lytic enzyme 3.4% of solubilized bacterial cell wall.
5. fibroin micromolecule polypeptide preparation method according to claim 1, is characterized in that: described fibroin micromolecule polypeptide is made oral powder or liquor.
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| CN104961817A (en) * | 2015-07-28 | 2015-10-07 | 苏州普罗达生物科技有限公司 | Female silkworm fibroin polypeptide 2, and preparation and application thereof |
| CN104961805A (en) * | 2015-07-28 | 2015-10-07 | 苏州普罗达生物科技有限公司 | Fibroin polypeptide, and preparation and application thereof |
| CN104987375A (en) * | 2015-07-28 | 2015-10-21 | 苏州普罗达生物科技有限公司 | Regenerated silk protein polypeptide, preparation method therefor and application thereof |
| CN104987364A (en) * | 2015-07-28 | 2015-10-21 | 苏州普罗达生物科技有限公司 | Regenerated natural-silk protein polypeptide and preparation method therefor and application thereof |
| CN104987373A (en) * | 2015-07-28 | 2015-10-21 | 苏州普罗达生物科技有限公司 | Regenerated silk fibroin polypeptide, and preparation method and application thereof |
| CN104987374A (en) * | 2015-07-28 | 2015-10-21 | 苏州普罗达生物科技有限公司 | Female silkworm silk fibroin polypeptide, and preparation method and application thereof |
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| CN105017400A (en) * | 2015-07-28 | 2015-11-04 | 苏州普罗达生物科技有限公司 | Female silkworm protein polypeptide 1 as well as preparation method and application of female silkworm protein polypeptide 1 |
| CN105061563A (en) * | 2015-07-28 | 2015-11-18 | 苏州普罗达生物科技有限公司 | Fibroin polypeptide and preparation and application thereof |
| CN105111292A (en) * | 2015-09-08 | 2015-12-02 | 苏州普罗达生物科技有限公司 | Taro mucoprotein peptide and application thereof |
| CN106706524A (en) * | 2017-02-22 | 2017-05-24 | 大连大学 | Method of quickly detecting oxygen content in gas by using fibroin membrane |
| CN107699603A (en) * | 2017-11-28 | 2018-02-16 | 张夏洋 | A kind of extracting method of fibroin |
| CN110184152A (en) * | 2019-07-04 | 2019-08-30 | 金寨新丝路纺织有限公司 | A kind of brewing method with wound repair silk cocoon yellow rice wine |
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| CN104961817A (en) * | 2015-07-28 | 2015-10-07 | 苏州普罗达生物科技有限公司 | Female silkworm fibroin polypeptide 2, and preparation and application thereof |
| CN104961805A (en) * | 2015-07-28 | 2015-10-07 | 苏州普罗达生物科技有限公司 | Fibroin polypeptide, and preparation and application thereof |
| CN104987375A (en) * | 2015-07-28 | 2015-10-21 | 苏州普罗达生物科技有限公司 | Regenerated silk protein polypeptide, preparation method therefor and application thereof |
| CN104987364A (en) * | 2015-07-28 | 2015-10-21 | 苏州普罗达生物科技有限公司 | Regenerated natural-silk protein polypeptide and preparation method therefor and application thereof |
| CN104987373A (en) * | 2015-07-28 | 2015-10-21 | 苏州普罗达生物科技有限公司 | Regenerated silk fibroin polypeptide, and preparation method and application thereof |
| CN104987374A (en) * | 2015-07-28 | 2015-10-21 | 苏州普罗达生物科技有限公司 | Female silkworm silk fibroin polypeptide, and preparation method and application thereof |
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| CN105017400A (en) * | 2015-07-28 | 2015-11-04 | 苏州普罗达生物科技有限公司 | Female silkworm protein polypeptide 1 as well as preparation method and application of female silkworm protein polypeptide 1 |
| CN105061563A (en) * | 2015-07-28 | 2015-11-18 | 苏州普罗达生物科技有限公司 | Fibroin polypeptide and preparation and application thereof |
| CN105111292A (en) * | 2015-09-08 | 2015-12-02 | 苏州普罗达生物科技有限公司 | Taro mucoprotein peptide and application thereof |
| CN106706524A (en) * | 2017-02-22 | 2017-05-24 | 大连大学 | Method of quickly detecting oxygen content in gas by using fibroin membrane |
| CN106706524B (en) * | 2017-02-22 | 2019-04-19 | 大连大学 | A method of using oxygen content in the quick detection gas of fibroin film |
| CN107699603A (en) * | 2017-11-28 | 2018-02-16 | 张夏洋 | A kind of extracting method of fibroin |
| CN110184152A (en) * | 2019-07-04 | 2019-08-30 | 金寨新丝路纺织有限公司 | A kind of brewing method with wound repair silk cocoon yellow rice wine |
| CN110937595A (en) * | 2020-02-10 | 2020-03-31 | 江苏科技大学 | Method for preparing polypeptide-graphene composite material by utilizing silk fibroin oligopeptide water-phase stripping graphite and application thereof |
| CN110937595B (en) * | 2020-02-10 | 2023-04-18 | 江苏科技大学 | Method for preparing polypeptide-graphene composite material by utilizing silk fibroin oligopeptide water phase to strip graphite and application of polypeptide-graphene composite material |
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| CN112409469A (en) * | 2020-11-04 | 2021-02-26 | 付俊 | Extraction method of silk peptide |
| CN112409469B (en) * | 2020-11-04 | 2023-09-26 | 付俊 | Extraction method of silk peptide |
| CN115590807A (en) * | 2022-09-14 | 2023-01-13 | 杭州千岛湖天鑫有限公司（Cn） | Hydrolyzed silk fermentation product and preparation method and application thereof |
| CN115590807B (en) * | 2022-09-14 | 2023-10-20 | 杭州千岛湖天鑫有限公司 | Hydrolyzed silk fermentation product and preparation method and application thereof |
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