CN106853249A - A kind of preparation method of the natural pigment member embedded object based on ferritin/shitosan - Google Patents
A kind of preparation method of the natural pigment member embedded object based on ferritin/shitosan Download PDFInfo
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- CN106853249A CN106853249A CN201510915980.2A CN201510915980A CN106853249A CN 106853249 A CN106853249 A CN 106853249A CN 201510915980 A CN201510915980 A CN 201510915980A CN 106853249 A CN106853249 A CN 106853249A
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Abstract
The invention discloses a kind of preparation method of new embedded object, comprise the following steps：Liquor ferri albuminati is placed in centrifuge tube, adjusting pH value with sodium hydroxide solution is denatured, stirring；Natural pigment class material is taken, ethanol solution is dissolved in；Polyphenols ethanol solution and above-mentioned liquor ferri albuminati are mixed, is stirred at room temperature, adjust pH value renaturation, stirred in chromatographing cabinet in 4 DEG C, the dialysis of above-mentioned solution is obtained final product into ferritin natural pigment embedded object three times.Then ferritin pigment embedded object is mixed by a certain percentage with shitosan, is stirred, quiet system, lucifuge dialysis obtains final product final products in chromatographing cabinet in 4 DEG C.Final products are a kind of new embedded objects that natural pigment member is loaded by ferritin/shitosan, both can serve as new food additive, can also be applied to improve medical curative effect, the constituent in cosmetics is also used as, the ferritin and natural pigment that embedded object is produced after decomposing also have certain biological action.This product takes full advantage of the function of ferritin and natural pigment, with practical application and promotional value very high.
Description
Technical field
A kind of preparation method of the natural pigment member embedded object based on ferritin/shitosan, belongs to field of food technology.
Background technology
Ferritin is the native iron storage protein that a class is widely present in animal, plant and microorganism.Due to its special hollow structure and the protein coat of uniqueness, it is widely used in the carrier of metal ion and organic molecule.Ferritin shell molecular structure is guarded, and is made up of 24 subunits, and antiparallel one group of the formation of each two subunit, an approximate regular octahedron is constituted, into the symmetrical hollow globular molecule of 4-3-2 solid axles, usual its internal diameter is about 8nm, external diameter is about 12nm, and thickness is about 2nm.In general, the ferritin internal cavities of 8nm can store about 4500 trivalent iron atoms, to form the iron core of compound state.As can be seen here, using ferritin under Denaturing (such as low pH or high pH) reversible self assembly characteristic, using its protein coat as nano material, there is sufficiently large space to load some nutrition small molecules, small-molecule substance can also be made from external interference, its water-soluble and stability is improved, with very big potentiality.
Natural pigment is the food coloring for having natural resources to obtain, and the pigment extracted mainly from plant, animal tissue and microorganism, wherein plant based colorant occupies the majority.Natural pigment not only has to the effect of food color, and considerable part natural pigment has physiologically active.Common natural colour have beta carotene, lutein, leaf green etc..Lutein is a kind of natural fat-soluble pigment being widely present in the plants such as vegetables, flowers, fruit.Contained natural carotenol is a kind of antioxidant of excellent performance in medical experiment proof plant, a certain amount of lutein is added in food can prevent cell ageing and biological organs exhaustion, while can also prevent the visual impairment that the elderly's eyeball retina macular degeneration causes with blindness.In addition, lutein is alternatively arranged as feed addictive for the coloring of poultry meat egg, while being also used as coloring and nutrition and health care agent in the food industry；Another common natural pigment rutin has anti-oxidant and anti-radical action, can be used for the treatment of cardiovascular and cerebrovascular disease, tumour, inflammation, therefore natural pigment has wider application prospect.But most of natural pigment poorly water-soluble, biological half-life is short, and the two properties limit most of natural pigment in food applications and the bioavailability of pharmaceuticals industry.Ferritin provides an opportunity for solving this problem well.Fat-soluble natural pigment molecule (such as lutein) is loaded into ferritin internal cavities and forms ferritin-pigment embedded object; natural pigment so can both have been protected from external interference; the water solubility and stability of natural pigment can be improved again, had great application prospect.
In addition, ferritin is used as amphiprotic substance, it is easily acted on from the polysaccharide with different electric charges, and compound agglutinating reaction occurs.Shitosan (Chitosan) is used as the unique band cation of nature (free-NH3 +) native amino polysaccharide, with good biocompatibility, security and low cost the features such as, have become medicine or food function component embedding ideal material.
Using soybean ferritin and shitosan as raw material, interacted using the reversible self-assembly property and protein-PS of ferritin and prepare dispersed, Nano grade ferritin-shitosan-natural pigment member embedded object through retrieving not disclosed report at home and abroad, the present invention at home and abroad has no report, not disclosed at home and abroad to use.
The content of the invention
An object of the present invention is to provide a kind of natural pigment member embedded object based on ferritin/shitosan and preparation method thereof, and with ferritin as carrier protein, shitosan is adjuvant, loads the natural pigment member material of stability difference.Final products are a kind of new embedded objects that natural pigment member is loaded by ferritin/shitosan, both can serve as new food additive, can also be applied to improve medical curative effect, the constituent in cosmetics is also used as, ferritin, shitosan and the natural pigment that embedded object is produced after decomposing also have certain biological action.This product takes full advantage of the function of ferritin, shitosan and natural pigment, with practical application and promotional value very high.
The invention provides a kind of preparation method of new embedded object, comprise the following steps：Liquor ferri albuminati is placed in centrifuge tube, adjusting pH value with sodium hydroxide solution is denatured, stirring；Natural pigment class material is taken, ethanol solution is dissolved in；Polyphenols ethanol solution and above-mentioned liquor ferri albuminati are mixed, is stirred at room temperature, adjust pH value renaturation, stirred in chromatographing cabinet in 4 DEG C, the dialysis of above-mentioned solution is obtained final product into ferritin pigment embedded object three times.Then ferritin pigment embedded object is mixed by a certain percentage with shitosan, is stirred, quiet system, lucifuge dialysis in chromatographing cabinet in 4 DEG C.
Further, the concentration of the liquor ferri albuminati is 2.0 μM, and its pH value is 8 or so.
Further, the liquor ferri albuminati pH value to 11.0 or so is adjusted with 1M NaOH.
Further, the liquor ferri albuminati 2h of denaturation is stirred at room temperature.
Further, molecule mol ratio when ferritin and natural pigment class solution mix is 1: 200.
Further, ferritin and Polyphenols solution mixed room temperature stirring 2h.
Further, solution ph to 7.5-8.0 renaturation is adjusted with 1M HCl.
Further, more than 2h is stirred in 4 DEG C of chromatography cabinets after renaturation.
Further, the bag filter aperture 1000kDa for being used during dialysis.
Further, dialyzate during dialysis：Tris-HCl buffer solutions (pH 7.5,50mM).
Further, dialysed three times in chromatographing cabinet at 4 DEG C, an elution buffer was changed every 6 hours.
Further, shitosan：The molar concentration ratio of ferritin pigment embedded object is 20: 1-40: 1
Further, stir 15 minutes, 20 DEG C of standing 2h,
Further, bag filter aperture is 10kDa, and dialyzate is Mops buffer solutions (pH 6.0,20mM), and lucifuge is dialysed four times in 4 DEG C of chromatography cabinets, and an elution buffer was changed every 8 hours.
The beneficial effect of this product is：Fat-soluble natural pigment can be embedded into the water solubility that internal cavities are remarkably improved fat-soluble natural pigment in conjunction with shitosan as a kind of new nano-carrier as a kind of ferritin of nutriment, and because the protein coat structure of stabilization can will be embedded into heat endurance and photostability that the food component isolation complicated with the external world of its internal unstable pigment molecular is opened and improve pigment.And this new embedded object can not only be used for functional food additives and nutritious supplementary pharmaceutical is used, and the bioavailability of natural pigment is substantially increased again, is widely used in food, medicine, cosmetics, with practical application and promotional value very high.
Brief description of the drawings
Fig. 1 is the Technology Roadmap of ferritin-shitosan-natural pigment member embedded object.
Fig. 2 is the Technology Roadmap of ferritin-shitosan-natural pigment member embedded object (by taking lutein as an example).
Fig. 3 is the Technology Roadmap of ferritin-shitosan-natural pigment member embedded object (by taking rutin as an example).
Fig. 4 is the electron microscope of ferritin-shitosan-lutein embedded object.
Fig. 5 is the electron microscope of ferritin-shitosan-rutin embedded object.
Specific embodiment
The specific embodiment of the invention will be described in detail with reference to specific accompanying drawing herein.It should be noted that the combination of the technical characteristic or technical characteristic described in following embodiments should not be considered as isolated, they can be mutually combined so as to reach superior technique effect.In the accompanying drawing of following embodiments, the same-sign that each accompanying drawing occurs represents identical feature or part, in can be applied to different embodiments.
Embodiment 1 is by taking lutein as an example
The accurate lutein standard items for weighing 20mg are dissolved in 100ml absolute ethyl alcohols and are prepared into the mother liquor that concentration is 351.6 μM, 4 DEG C keep in dark place it is stand-by.2.0 μM of liquor ferri albuminatis of 5mL (pH 7.8, Tris buffer solution) are placed in the centrifuge tube of 10mL.Solution ph is adjusted to 11.0 or so with 1M NaOH, and after 2h is stirred at room temperature, being slowly added to 5.68mL lutein mother liquor (4 DEG C keep in dark place) makes ferritin: the molecule mol ratio of lutein is 1: 200, and 2h is stirred at room temperature, and is sufficiently mixed it.Solution ph to 7.5-8.0 then is adjusted with 1M HCl, more than 2h is stirred in chromatographing cabinet in 4 DEG C.Solution after renaturation is placed in the bag filter that aperture is 1000kDa, lucifuge is dialysed three times in chromatographing cabinet at 4 DEG C using Tris-HCl buffer solutions (pH 7.5,50mM), and an elution buffer was changed every 6 hours.Passage aperture (about 0.3nm) due to lutein molecular weight more than ferritin, so that part lutein is embedded in ferritin internal cavities, free lutein is then removed in dialysis procedure.The solution dialysed is taken out, is clarified with the hydrophilic cellulose membrane filtration of 0.45um.To finally be embedded with the conditions of the iron protein solution of lutein is placed on 4 DEG C and keep in dark place.Then, different amounts of shitosan is added into ferritin-(1.0 μM of lutein embedded object in proportion, 2.0mL, pH6.0) in solution, make shitosan: the molar concentration ratio of ferritin-lutein embedded object is 20: 1-40: 1, stirring makes it fully act in 15 minutes, 20 DEG C of standing 2h, then, the solution of reactant is placed in the bag filter that aperture is 10kDa, use Mops buffer solutions (pH 6.0, lucifuge is dialysed four times in 20mM) chromatographing cabinet at 4 DEG C, an elution buffer was changed every 8 hours, to remove uncombined shitosan, obtain ferritin-shitosan-lutein embedded object, kept in dark place at being placed in 4 DEG C.
Embodiment 2 is by taking rutin as an example
The accurate rutin standard items for weighing 20mg are dissolved in 80% absolute ethyl alcohol and are prepared into the mother liquor that concentration is 323.0 μM, 4 DEG C keep in dark place it is stand-by.2.0 μM of liquor ferri albuminatis of 5mL (pH 7.8, Tris buffer solution) are placed in the centrifuge tube of 10mL.Solution ph is adjusted to 11.0 or so with 1M NaOH, and after 2h is stirred at room temperature, being slowly added to 3.7mL rutins titer (4 DEG C keep in dark place) makes ferritin: the molecule mol ratio of rutin is 1: 120, and 1h is stirred at room temperature, and is sufficiently mixed it.Solution ph to 7.5-8.0 then is adjusted with 1M HCl, more than 2h is stirred in chromatographing cabinet in 4 DEG C.Solution after renaturation is placed in the bag filter that aperture is 1000kDa, lucifuge is dialysed three times in chromatographing cabinet at 4 DEG C using Tris-HCl buffer solutions (pH 7.5,50mM), and an elution buffer was changed every 6 hours.Passage aperture (about 0.3nm) due to rutin molecular weight more than ferritin, so that part rutin is embedded in ferritin internal cavities, free rutin is then removed in dialysis procedure.The solution dialysed is taken out, centrifuging and taking supernatant is the liquor ferri albuminati that can obtain being embedded with rutin.Then, different amounts of shitosan is added into ferritin-(1.0 μM of natural pigment embedded object in proportion, 2.0mL, pH 6.0) in solution, make shitosan: the molar concentration ratio of ferritin-rutin embedded object is 20: 1-40: 1, stirring makes it fully act in 15 minutes, 20 DEG C of standing 2h, then, the solution of reactant is placed in the bag filter that aperture is 10kDa, use Mops buffer solutions (pH 6.0, lucifuge is dialysed four times in 20mM) chromatographing cabinet at 4 DEG C, an elution buffer was changed every 8 hours, to remove uncombined shitosan, obtain ferritin-shitosan-rutin embedded object, kept in dark place at being placed in 4 DEG C.
The invention provides a kind of preparation method of new embedded object, fat-soluble natural pigment can be embedded into after internal cavities the water solubility that fat-soluble natural pigment is remarkably improved in conjunction with shitosan as a kind of ferritin of nutriment as a kind of new nano-carrier, and because the protein coat structure of stabilization can will be embedded into heat endurance and photostability that the food component isolation complicated with the external world of its internal unstable pigment molecular is opened and improve pigment.Given this advantage, this technology is respectively provided with wide market prospects in medicine, foods and cosmetics.
Although some embodiments of the present invention are had been presented for herein, it will be appreciated by those of skill in the art that without departing from the spirit of the invention, can be changed to embodiments of the invention.Above-described embodiment is exemplary, should not be using the embodiments herein as the restriction of interest field of the present invention.
Claims (10)
1. a kind of preparation method of the natural pigment member embedded object based on ferritin shitosan, it is characterised in that comprise the following steps：Liquor ferri albuminati is placed in centrifuge tube, adjusting pH value with sodium hydroxide solution is denatured, stirring；Big so pigment molecular class material is taken, ethanol solution is dissolved in；Natural pigment member class ethanol solution and above-mentioned liquor ferri albuminati are mixed, is stirred at room temperature, adjust pH value renaturation, stirred in chromatographing cabinet in 4 DEG C, the dialysis of above-mentioned solution is obtained final product for three times.Then ferritin pigment embedded object is mixed by a certain percentage with shitosan, is stirred, quiet system, lucifuge dialysis in chromatographing cabinet in 4 DEG C.
2. the preparation method of ferritin as claimed in claim 2/shitosan natural pigment member embedded object, it is characterised in that the concentration of the liquor ferri albuminati is 2.0 μM, its pH value is 8 or so.
3. the preparation method of the big so pigment molecular embedded object of ferritin/shitosan as claimed in claim 3, it is characterised in that adjust the liquor ferri albuminati pH value to 11.0 or so with 1M NaOH.
4. the preparation method of ferritin as claimed in claim 4/shitosan natural pigment member embedded object, it is characterised in that the liquor ferri albuminati 2h of denaturation is stirred at room temperature.
5. the preparation method of ferritin as claimed in claim 5/shitosan natural pigment member embedded object, it is characterised in that molecule mol ratio when ferritin and natural pigment member class solution mix is 1: 200, and 2h is stirred at room temperature.
6. the preparation method of ferritin as claimed in claim 6/shitosan natural pigment member embedded object, it is characterised in that adjust solution ph to 7.5-8.0 renaturation with 1M HCl, more than 2h is stirred after renaturation in 4 DEG C of chromatography cabinets.
7. the preparation method of ferritin as claimed in claim 7/shitosan natural pigment member embedded object, it is characterised in that the bag filter aperture 1000kDa used during dialysis, dialyzate during dialysis：Tris-HCl buffer solutions (pH 7.5,50mM), are dialysed three times in chromatographing cabinet at 4 DEG C, and an elution buffer was changed every 6 hours.
8. the preparation method of the big so pigment molecular embedded object of ferritin/shitosan as claimed in claim 8, it is characterised in that shitosan：The molar concentration ratio of ferritin pigment embedded object is 20: 1-40: 1.
9. the preparation method of ferritin as claimed in claim 9/shitosan natural pigment member embedded object, it is characterized in that, stirring 15 minutes, 20 DEG C of standing 2h, bag filter aperture is 10kDa during dialysis, and dialyzate is Mops buffer solutions (pH 6.0,20mM), lucifuge is dialysed four times in 4 DEG C of chromatography cabinets, and an elution buffer was changed every 8 hours.
10. the big so pigment molecular embedded object of ferritin/shitosan as claimed in claim 10, it is characterised in that the ferritin/shitosan natural pigment member embedded object is prepared by the method for claim any one of 1-9.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108741097A (en) * | 2018-05-17 | 2018-11-06 | 华南理工大学 | A kind of albumen self assembly embedding difficult resolving active material nanometer products and preparation method thereof |
| CN109517866A (en) * | 2018-11-01 | 2019-03-26 | 天津科技大学 | A method of ferritin-chitosan complexes are prepared using transglutaminase and chitosan |
| CN111150063A (en) * | 2020-01-07 | 2020-05-15 | 天津科技大学 | Method for simultaneously improving hydrophobicity and stability of water-soluble active ingredients by using cage-shaped plant ferritin and application |
| CN113367335A (en) * | 2021-06-24 | 2021-09-10 | 中国农业大学 | Improve astaxanthin water-solubilitySex and protect it from Fe2+Method for oxidative degradation |
| CN114456251A (en) * | 2021-11-25 | 2022-05-10 | 中国农业大学 | Micromolecule-ferritin embedding substance and method for embedding micromolecules through ferritin one-step deferrization |
| CN115226895A (en) * | 2022-06-28 | 2022-10-25 | 海南大学 | Preparation method of areca seed nanocellulose-quercetin compound |
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2015
- 2015-12-09 CN CN201510915980.2A patent/CN106853249A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108741097A (en) * | 2018-05-17 | 2018-11-06 | 华南理工大学 | A kind of albumen self assembly embedding difficult resolving active material nanometer products and preparation method thereof |
| CN109517866A (en) * | 2018-11-01 | 2019-03-26 | 天津科技大学 | A method of ferritin-chitosan complexes are prepared using transglutaminase and chitosan |
| CN109517866B (en) * | 2018-11-01 | 2022-04-08 | 天津科技大学 | Method for preparing ferritin-chitosan compound by using transglutaminase and chitosan |
| CN111150063A (en) * | 2020-01-07 | 2020-05-15 | 天津科技大学 | Method for simultaneously improving hydrophobicity and stability of water-soluble active ingredients by using cage-shaped plant ferritin and application |
| CN113367335A (en) * | 2021-06-24 | 2021-09-10 | 中国农业大学 | Improve astaxanthin water-solubilitySex and protect it from Fe2+Method for oxidative degradation |
| CN113367335B (en) * | 2021-06-24 | 2022-05-06 | 中国农业大学 | Improve astaxanthin water-solubilitySex and protect it from Fe2+Method for oxidative degradation |
| CN114456251A (en) * | 2021-11-25 | 2022-05-10 | 中国农业大学 | Micromolecule-ferritin embedding substance and method for embedding micromolecules through ferritin one-step deferrization |
| CN114456251B (en) * | 2021-11-25 | 2022-10-14 | 中国农业大学 | Micromolecule-ferritin embedding substance and method for embedding micromolecules by ferritin one-step deferrization |
| CN115226895A (en) * | 2022-06-28 | 2022-10-25 | 海南大学 | Preparation method of areca seed nanocellulose-quercetin compound |
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