CN1654510A - Nano microgel and its preparing process and use - Google Patents
Nano microgel and its preparing process and use Download PDFInfo
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- CN1654510A CN1654510A CN 200510023646 CN200510023646A CN1654510A CN 1654510 A CN1654510 A CN 1654510A CN 200510023646 CN200510023646 CN 200510023646 CN 200510023646 A CN200510023646 A CN 200510023646A CN 1654510 A CN1654510 A CN 1654510A
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Abstract
The present invention provides one kind of nanometer micro gel prepared with protein and polysaccharide and its preparation process, and belongs to the fields of biochemistry, high molecular chemistry and medicine preparation. The nanometer micro gel is formed with two kinds of protein or one kind of protein and one kind of polysaccharide differently charged in the same pH conditioned, and through heating to denature protein. Differently charged protein and polysaccharide forms electrostatic composite, micelle or deposit, and through pH regulation and heating, nanometer micro gel in core-shell structure is obtained. The nanometer micro gel may have medicine or nutriment coated through coating/diffusion to obtain nanometer edible medicine or nutriment colloid. Or, it may have perfume, cosmetics or dye coated for releasing in special environment.
Description
Technical field
The invention belongs to biological chemistry, polymer chemistry and field of pharmaceutical preparations, a kind of protein/polysaccharide nano microgel is provided.The present invention also provides the preparation method and the application of this nano microgel.
Background technology
The pharmaceutical carrier of micron-scale and nano-scale has caused the extensive interest of scientific circles and industry member, and these carriers can be colloid, vesica or microballoon.They can load viable cell, enzyme, flavor oil, medicine, VITAMIN, agrochemicals, catalyzer etc., and have a lot of advantages: liquid can be embedded in the solid the inside, is used as solid to use; Toxicant can be handled safely; Unstable compounds can be protected; Smell and mouthfeel can be shielded effectively; Medicine can Be Controlled and discharge selectively etc. (InternationalJournal of Pharmaceutics 242 (2002) 163-166).In food, medicine and cosmetic industry, medicine/nutrition carrier that natural macromolecular forms is owing to having biological utilisation and biological degradability thereby being subjected to special favor.Up to the present, having by emulsification, spray-dired method, is (J.Agric.Food Chem.49 (2001) 1934-1938) with the soya-bean oil casein microcapsule of packing into; The method of condensing altogether in solution with two kinds of different protein or albumen-polysaccharide mixture prepares drug microcapsule embedding system (InternationalJournal of Pharmaceutics 242 (2002) l63-166; Journal Microencapsulation 19 (2002) 549-558); Casein carries out the reacted product of Maillard with the carbohydrate that contains reducing sugar and wraps up the oil (US Patent Application 20030185960) to oxygen sensitive with emulsification, exsiccant method; Calcium phosphate is as kernel, and medicine that will load or protein combine the back and with casein the surface wrapped up as oral pharmaceutical (United States Patent Application 20020054914) with calcium phosphate.Yet these medicine that is formed by biomacromolecule/nutrition carriers also have the following disadvantages: to the effectively embedding of charged hydrophilic small-molecule drug, enrichment and sustained release; The particle diameter of carrier is bigger than normal, all at micro-meter scale; The system instability that has need add stablizer; Prepare not easy.
Microgel has its special advantages as pharmaceutical carrier: 1, with respect to solid colloidal particle, the low density structures of microgel can carry the space for drug molecule provides bigger appearance; 2, with respect to the colloidal particle of hollow ball structure, the network structure of microgel can provide more medicine binding site, helps the high density enrichment of drug molecule; 3, the microgel that has weak acid/weak base group, the charge property of himself can change with the pH value of solution, therefore and the interaction that has between the small molecules of electric charge also change with pH value, so can be by the adjusting of pH enrichment and the charged drug molecule of release effectively.At present, utilize microgel as the research of about thing carrier more and more (Nature 394 (1998) 459-462).Body material as microgel mostly is synthetic macromolecule and partially modified natural polymer greatly, and the method for preparation has that letex polymerization, anionic copolymerization, adjacent polymer chains are crosslinked, conversed phase micro emulsion copolymerization etc. (Advances in Colloid and InterfaceScience 80 (1999) 1-25).Yet the enforcement of these methods all has certain degree of difficulty and is not suitable as oral pharmaceutical and the nutrition carrier.Therefore, be necessary to develop simpler, effectively and method in common prepare the biomacromolecule microgel of pure natural.
Protein is the important biomacromolecule of a class, has many physiology and biological function.Food protein is one of main nutrient of the mankind and plays an important role in Food science, as (Food Proteins and Their Applications such as thickening, gel, emulsification, foaming, bonding, combination water, grease and spices, S.Damodaran and A.Paraf, 1997, Marcel Dekker, Inc.:New York.1-24).Protein is the both sexes macromole, its molecule with electric charge relevant with the pH of solution: as pH during less than its iso-electric point, molecule is positively charged; PH is during greater than its iso-electric point, and molecule is electronegative; PH is when its iso-electric point, and the net charge of molecule is zero.After being heated, can destroy in protein its specific space structure; The hydrophobic grouping of protein molecule can be transferred to molecular surface and cause intermolecular hydrophobic association from intramolecule; The protein molecule that contains sulfydryl and intramolecular disulfide bond can form intermolecular disulfide linkage and cause molecular aggregates.For most of food protein aqueous solution, when heating under the pH value away from its iso-electric point, protein is the aggregate of sex change formation pearl shape at first, further assembles forming transparent macroscopical gelinite then; When the pH of solution value is near isoelectric point of protein, can form random aggregate behind the protein heat denaturation, further assemble forming muddy macroscopical gelinite then; When the pH of solution is between above-mentioned, protein can form above-mentioned two kinds of aggregates and translucent gelinite (Food Proteins and Their Applications when heat denatured, S.Damodaran and A.Paraf, 1997, Marcel Dekker, Inc.:New York.327-330).Polysaccharide is another kind of common biomacromolecule, also can form macroscopical gelinite after the aqueous solution heating of some polysaccharide.Natural food protein and polysaccharide are edible and cheap, are the desirable body materials of preparation microgel.Though food protein and polysaccharide are easy to form macroscopical gel when heating, up to the present, also do not find to utilize protein and polysaccharide to prepare the report of microgel.In addition, also do not find to utilize the simple heating method to prepare the report of nano microgel.
Summary of the invention
The purpose of this invention is to provide a kind of edible protein and polysaccharide microgel.
Another object of the present invention provides a kind of preparation method of simple, convenient, efficient, general protein/polysaccharide microgel.
A further object of the present invention provides the application of above-mentioned microgel.
The invention provides a kind of nano microgel, this nano microgel is by heating protein denaturation to be formed by the two kinds of protein that has different electric charges under identical pH condition or by a kind of protein and a kind of polysaccharide.
Microgel of the present invention has nuclear one shell structure, and nuclear contains a kind of protein, and shell contains another kind of protein or a kind of polysaccharide; Nuclear and shell with electric charge be subjected to the influence of pH value of solution: when microgel is made up of two kinds of different protein, pH is during less than two kinds of isoelectric point of protein, the nuclear of microgel and shell are all positively charged, as pH during greater than two kinds of isoelectric point of protein, the nuclear of microgel and shell are all electronegative, when pH is between two kinds of isoelectric point of protein, and the nuclear of microgel and shell oppositely charged, when pH equals to form the isoelectric point of protein of microgel shell, the shell neutral of microgel; When microgel is made up of protein and polysaccharide, pH is between isoelectric points of proteins and polysaccharide ionization constant pK the time, the nuclear of microgel and shell oppositely charged, for alkaline polysaccharide, as pH during greater than its pK+2, the shell neutral of microgel is for acidic polysaccharose, as pH during less than its pK-2, the shell neutral of microgel.
The preparation method of the above-mentioned microgel of work post also is provided in another aspect of this invention.
Utilize two kinds of protein and polysaccharide that have the protein of different iso-electric points or have different electric charges, under given conditions, the pH value of ie in solution is between these two kinds of isoelectric point of protein or when making protein/polysaccharide in the solution have opposite charges, these two kinds of molecules generate electrostatic complexes, micella or precipitation, the pH value that changes solution then is near a certain isoelectric point of protein, stirring or leave standstill for some time recombinates molecule: near the protein that is positioned at the iso-electric point tends to gathering because the electric charge of itself goes to zero, and away from the another kind of protein of iso-electric point or the polysaccharide that has an electric charge owing to self having a large amount of like charges and mutually exclusive tend to away from.Make protein denaturation with the solution heating this moment, be positioned near the protein of iso-electric point and can form charged hardly random aggregate, and another kind of protein or polysaccharide can form the aggregate of the linearity that contains a large amount of like charges around it, have therefore just formed the micro-gel of nanoscale.This micro-gel has certain nucleocapsid structure usually, is positioned at promptly that near the iso-electric point protein mainly is positioned at the inside of microgel and the outside that mainly is enriched in microgel away from the protein or the polysaccharide of iso-electric point.After microgel forms, because the crosslinked of intermolecular hydrophobic, hydrogen bond action and disulfide linkage taken place, thereby highly stable at the molecule of microgel inside; And the molecule of microgel outside is owing to have like charges and mutually exclusive, thereby makes the microgel particle can existence steady in a long-term in solution.
Based on above-mentioned principle, the invention provides a kind of general method for preparing protein microgel or protein and polysaccharide microgel.This method may further comprise the steps:
(1) will under identical pH condition, have the two kinds of protein or the protein of different electric charges and polysaccharide is mixed with the aqueous solution and it is mixed forms electrostatic complexes, micella or precipitation respectively;
(2) secondly, adjust the potential of hydrogen of mixing solutions, make that any isoelectric point of protein difference is 0～3 in its pH value and the mixing solutions, stir or leave standstill so that resolution of precipitate or that electrostatic complexes is spread is even, but electrostatic complexes is dissociated fully;
(3) last, the heating mixing solutions makes protein denaturation wherein, thereby obtains microgel.
When using two kinds of protein (1), these two kinds of protein have different iso-electric points; When using multiple proteins, wherein have at least two kinds of protein to have different iso-electric points; When using protein and polysaccharide, the two has opposite electric charge in certain pH range.
Can adopt different protein combinations or protein/polysaccharide combination among the present invention.Protein can be any water miscible protein, and water-soluble food protein is preferable, polysaccharide must be the polysaccharide that has electric charge, formed protein combination or protein and polysaccharide combination must have opposite charges when mixing, perhaps under a certain pH condition, have opposite charges, as oralbumin and hen egg white lysozyme, bovine serum albumin and hen egg white lysozyme, α s-casein and ovum Siderophilin, oralbumin and ovum Siderophilin, beta-casein and hen egg white lysozyme, α
S-casein and hen egg white lysozyme, κ-casein and hen egg white lysozyme, casein and hen egg white lysozyme, chitosan and oralbumin, chitosan and α
S-casein, alginates and hen egg white lysozyme combination or the like.
In the present invention, when being mixed with the aqueous solution (1), earlier protein or polysaccharide are mixed with mass percentage concentration greater than 0 and less than 10% the aqueous solution, will have the protein of different electric charges or protein and polysaccharide again and be in molar ratio 0.05～20 mixed.The concentration of aqueous solution of preparation is preferable below 8%.
Polysaccharide and protein the time may need to add the pH value that extra acid (as acetic acid) or alkali (as sodium hydroxide) are helped its dissolving or adjusted its solution in dissolving.
In the present invention, when two kinds of solution mixed, the pH value of mixing solutions should make two kinds of protein or protein and polysaccharide produce the intensive electrostatic attraction in (1); Preferably, make mixing solutions pH value be the intermediate value of any two isoelectric points of proteins wherein, or the intermediate value of any one isoelectric points of proteins and polysaccharide ionization constant pK.
In the present invention, adjust the pH value of mixing solutions with diluted acid or diluted alkaline.Used acid is any in hydrochloric acid, acetic acid, phosphoric acid or the citric acid when regulating mixing solutions pH value; Used alkali is any in sodium hydroxide, yellow soda ash, sodium bicarbonate or the sodium-acetate.Concentration can adopt 0.01～1.0mol/L.
And then the pH value of mixing solutions adjusted near any one isoelectric point of protein, determining of concrete pH value is relevant with selected protein and polysaccharide with churning time, the standard that the pH value is determined is between two protein or the electrostatic interaction power between protein and polysaccharide is very weak or it is zero to be, making the pH value of mixing solutions and one of them isoelectric point of protein difference usually is 0～3; Preferably, both differences are 0～2; More preferably, both differences are 0～1.
In the present invention, after regulating the potential of hydrogen of mixing solutions, can stir or leave standstill 0-24 hour, stir or standard that time of repose is determined be macroscopical resolution of precipitate between two kinds of molecules or electrostatic complexes diffusion evenly but electrostatic complexes is dissociated fully.Overlong time can not obtain good target product.
The microgel of the present invention's preparation obtains protein denaturation by heating, and the choice criteria of Heating temperature and heat-up time is the fully sex change of protein that makes in the solution.In the present invention, can adopt 50～100 ℃, and keep 1 minute～3 hours; More preferably, adopt 70～90 ℃ of heating 30～120 minutes.
The microgel of the present invention preparation has nucleocapsid structure, and nuclear mainly is made up of a kind of protein, and shell mainly is made up of another kind of protein or polysaccharide, the nuclear of microgel and shell with electric charge different along with the change of pH value of solution.For example, use two kinds of microgels with protein preparation of different iso-electric points, when the pH of solution was lower than these two kinds of isoelectric point of protein, the nuclear of microgel and shell all had positive charge; When the pH of solution was higher than these two kinds of isoelectric point of protein, the nuclear of microgel and shell all had negative charge; When the pH of solution is between these two kinds of isoelectric point of protein, the electric charge that the nuclear of microgel is opposite with the shell band; When the pH of solution value had been positioned near the isoelectric point of protein of microgel shell, the shell neutral of microgel thereby microgel can further be assembled, but this gathering meeting spreads out again along with the variation of pH value of solution.Fig. 2 utilizes the prepared microgel (embodiment 1) of oralbumin (iso-electric point 4.7) and hen egg white lysozyme (iso-electric point 10.7) charged synoptic diagram when different pH.
The microgel that utilizes the present invention to obtain, has good monodispersity, utilize dynamic light scattering, transmission electron microscope and atomic force microscope to characterize microgel and be approximately spherical, particle diameter changes with the composition and the ratio of two kinds of protein or protein and polysaccharide, greatly between 40～700nm.
Utilize the inventive method can obtain 1% or the higher microgel aqueous solution of concentration.Prepared microgel can be in the medium-term and long-term preservation of the aqueous solution, and after Fig. 1 demonstrated and utilizes oralbumin and the prepared microgel (embodiment 1) of hen egg white lysozyme to deposit in the aqueous solution 120 days, its particle diameter and size distribution did not almost change.Prepared microgel can be preserved the dried powder that obtains through after freezing the draining, and this powder can be distributed in the aqueous solution once more and keep its original particle diameter and size distribution.
Preparation method of the present invention does not need specific installation, and product does not need to separate, and can directly be used for embedding medicinal.
On the other hand, the present invention also provides above-mentioned microgel preparation method's application, and the microgel that promptly obtains is wrapped in small molecules in the microgel by diffusion way.The condition of diffusion process is determined on a case-by-case basis, and for example, small molecules to be wrapped is dissolved in the microgel solution, perhaps microgel is dissolved in the small molecules solution to be wrapped.
The microgel of the present invention's preparation can be used as medicine and nutraceutical carrier.
Because the microgel of the present invention's preparation belongs to nano material (specifically seeing Fig. 3), and whole process all adopts natural macromolecular and edible bronsted lowry acids and bases bronsted lowry, do not add other chemical substances, therefore not only its embedding is effective for prepared microgel, edible assimilation effect is also good, can be used for the nano-carrier of oral pharmaceutical.
In like manner, the microgel of the present invention's preparation can be used as nutraceutical nano-carrier.
The microgel of the present invention preparation can discharge medicine or nutrition by people's the digestive tube or the hydrolysis method of proteolytic enzyme after being wrapped in medicine or nutrition wherein from microgel.
The microgel of the present invention's preparation can be used to prepare dyestuff, spices and makeup nano-carrier.
After the microgel of the present invention's preparation is rolled in medicine, nutrition, dyestuff, spices, toiletry bag wherein, can it be discharged from microgel by the mode that changes solution acid alkalinity.
The microgel that utilizes the present invention to prepare has good embedding and releasing effect to charged small molecules.Microgel for example shown in Figure 2 can be when pH 4.7 have the small-molecule drug of negative charge by the diffusion enrichment; Solution is remained on acidity or neutral pH, can not discharged from microgel by the small molecules of embedding; Have only when the pH of solution greater than 10.7 the time, electronegative small-molecule drug just can discharge; On the other hand, utilize the microgel of food protein and polysaccharide preparation can be degraded, therefore can discharge the small-molecule drug of institute's embedding at people's digestive tube.
The present invention can have particular core by selecting pairing of different protein-protein or protein-polysaccharide pairing to prepare, the microgel of shell charge property, thus can the multiple medicine of embedding, nutrition, dyestuff, spices and makeup.
Utilize the present invention to prepare microgel, whole process all adopts natural macromolecular and edible bronsted lowry acids and bases bronsted lowry, does not add other chemical substances, and therefore prepared microgel can be used as oral pharmaceutical/nutrition carrier, not only its embedding is effective, and edible assimilation effect is also good.
The microgel that utilizes the present invention to obtain has parcel charged small-molecule drug/nutraceutical good result, because therefore its edibility has its wide application prospect as medicine/nutraceutical carrier.Especially the very suitable utilization embedding of the microgel that utilizes the present invention to obtain is to the material of environment sensitive.
Description of drawings
Fig. 1 is particle diameter and the size distribution figure when depositing 120 days with the microgel of oralbumin and hen egg white lysozyme preparation in the aqueous solution.
Fig. 2 is with microgel in the different pH aqueous solution the charged synoptic diagram of oralbumin with the hen egg white lysozyme preparation.
Fig. 3 is the transmission electron microscope picture with the microgel of oralbumin and hen egg white lysozyme preparation.
Fig. 4 is particle diameter and the size distribution with the microgel aqueous solution of oralbumin and hen egg white lysozyme preparation.
Embodiment
Example 1. oralbumins (Ovalbumin) and hen egg white lysozyme (Lysozyme) form microgel in alkaline aqueous solution.
Oralbumin and hen egg white lysozyme are dissolved in respectively in the deionized water, are made into the protein soln of 2mg/mL and 0.624mg/mL.Under magnetic agitation, the albuminous aqueous solution of 1.2mL is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.1mol/L NaOH regulator solution be 10.3 and agitation as appropriate made it to mix in 60 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 1.5 hours, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: median size is 147nm, and polydispersity coefficient is 0.127 (Fig. 4).Transmission electron microscope is measured this microgel its median size after super-dry and is had only 80nm (Fig. 3).This microgel solution is extremely stable, has deposited 120 days no obvious variation (shown in Figure 1) in 4 ℃ refrigerator.
In above-mentioned system, the mol ratio of albumin and N,O-Diacetylmuramidase is controlled in 0.05～5 the scope, in the scope of the pH regulator to 9.0 of solution～11.0, all can obtain narrow dispersive, particle diameter is the stable microgel in 80～300nm scope.
With freezing the draining of above-mentioned microgel solution, with the pressed powder deionized water dissolving of gained, the HCl of usefulness 0.1mol/L or the pH value of NaOH regulator solution still can obtain the microgel solution of aforementioned stable to acid or alkaline.
Example 2. oralbumins (Ovalbumin) and hen egg white lysozyme (Lysozyme) form microgel in acidic aqueous solution.
Oralbumin and N,O-Diacetylmuramidase are dissolved in respectively in the deionized water, are made into the protein soln of 2mg/mL and 0.624mg/mL.Under magnetic agitation, the albuminous aqueous solution of 600 μ L is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.06mol/L NaOH regulator solution be 10.0 and agitation as appropriate made it to mix in 3 minutes, pH with 0.01mol/L HCl quick adjustment solution is 5.0 again, rapidly the solution that mixes is placed 80 ℃ water-bath to heat then 10 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: median size is 71.4nm, and polydispersity coefficient is 0.224.
In above-mentioned system, the mol ratio of albumin and N,O-Diacetylmuramidase is controlled in 0.05～5 the scope, in the scope of the pH regulator to 4.0 of solution～6.5, all can obtain particle diameter and be the stable microgel in 50～300nm scope.
Example 3. bovine serum albumins (Bovine Serum Albumin) and N,O-Diacetylmuramidase (Lysozyme) form microgel in alkalescence or acidic aqueous solution.
Bovine serum albumin and N,O-Diacetylmuramidase are dissolved in respectively in the deionized water, are made into the protein soln of 2.838mg/mL and 0.624mg/mL.The aqueous solution with the 1.5mL bovine serum albumin under magnetic agitation is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.1mol/L NaOH regulator solution be 8.90 and agitation as appropriate made it to mix in 15 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 1 hour, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 151.8nm, and polydispersity coefficient is 0.0880.
In above-mentioned system, the mol ratio of bovine serum albumin and N,O-Diacetylmuramidase is controlled in 0.05～5 the scope, in the pH regulator to 4.0 of solution～6.5 or 8.0～11.0 the scope, all can obtain particle diameter and be the stable microgel in 50～300nm scope.
Example 4. α s-caseins (α s-Casein) and ovum Siderophilin (Ovotransferrin) form microgel in neutral aqueous solution.
α s-casein and ovum Siderophilin are dissolved in respectively in the deionized water, are made into the protein soln of 4mg/mL and 0.5mg/mL.Under magnetic agitation, the caseic aqueous solution of 38.4 μ L is added drop-wise in the aqueous solution of 3mL ovum Siderophilin, with the pH of 0.04mol/L NaOH regulator solution be 6.97 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 1 hour, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 138nm, and polydispersity coefficient is 0.214.
In above-mentioned system, the mol ratio of α s-casein and ovum Siderophilin is controlled in 0.05～5 the scope, in the scope of the pH regulator to 5.3 of solution～8.5, all can obtain particle diameter and be the stable microgel in 50～500nm scope.
Example 5. oralbumins (Ovalbumin) and ovum Siderophilin (Ovotransferrin) form microgel in neutral aqueous solution.
Oralbumin and ovum Siderophilin are dissolved in respectively in the deionized water, are made into the protein soln of 11.2mg/mL and 0.5mg/mL.Under magnetic agitation, the albuminous aqueous solution of 40 μ L is added drop-wise in the aqueous solution of 3mL ovum Siderophilin, with the pH of 0.04mol/L NaOH regulator solution be 7.0 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 1 hour, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 142.4nm, and polydispersity coefficient is 0.161.
In above-mentioned system, in the mol ratio of oralbumin and ovum Siderophilin is controlled at 0.05～10 scope, in the scope of the pH regulator to 5.3 of solution～8.5, all can obtain particle diameter and be the stable microgel in 50～500nm scope.
Example 6. chitosans (Chitosan) and oralbumin (Ovalbumin) form microgel in acidic aqueous solution.
Chitosan is dissolved in 2% the acetic acid, is made into concentration and is 2% heavy-gravity chitosan aqueous acetic acid; Oralbumin is dissolved in the deionized water, is made into the protein soln of 0.5mg/mL.Under magnetic agitation, be added drop-wise to the aqueous acetic acid of 161 μ L chitosans in the albuminous aqueous solution of 3mL and agitation as appropriate made it to mix in 10 minutes, be 5.30 and stirred 5 minutes with the pH of 0.2mol/LNaOH regulator solution.Then the solution that mixes is placed 80 ℃ water-bath to heat 20 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 508.9nm, and polydispersity coefficient is 0.386.
In above-mentioned system, in the scope of weight ratio 0.1～10 of chitosan/oralbumin, in the scope of the pH regulator to 4.0 of solution～8.0, all can obtain particle diameter is the interior stable microgel of 100～1000nm scope.
(β-Casein) and N,O-Diacetylmuramidase (Lysozyme) form microgel to example 7. beta-caseins in acidic aqueous solution.
Beta-casein and N,O-Diacetylmuramidase are dissolved in respectively in the deionized water, are made into the protein water soln of 10mg/mL and 0.624mg/mL.The aqueous solution with 120/ μ L beta-casein under magnetic agitation is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.01mol/L HCl regulator solution be 4.46 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 222.6nm, and polydispersity coefficient is 0.0692.
In above-mentioned system, in the scope of mol ratio 0.05～10 of beta-casein/N,O-Diacetylmuramidase, the pH value all can obtain stable microgel particle at 4.0～7.5 o'clock.
(β-Casein) and N,O-Diacetylmuramidase (Lysozyme) form microgel to example 8. beta-caseins in alkaline aqueous solution.
Beta-casein and N,O-Diacetylmuramidase are dissolved in respectively in the deionized water, are made into the protein soln of 10mg/mL and 0.624mg/mL.The aqueous solution with 120 μ L beta-caseins under magnetic agitation is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.06mol/L NaOH regulator solution be 10.28 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 195.9nm, and polydispersity coefficient is 0.177.
In above-mentioned system, in the scope of mol ratio 0.05～10 of beta-casein/N,O-Diacetylmuramidase, the pH value all can obtain stable microgel particle at 8.5～11.0 o'clock.
Example 9. α
S-casein (α
S-Casein) and N,O-Diacetylmuramidase (Lysozyme) in acidic aqueous solution, form microgel.
With α
S-casein and N,O-Diacetylmuramidase are dissolved in respectively in the deionized water, are made into the protein solution of 4mg/mL and 0.624mg/mL.Under magnetic agitation with 300 μ L α
S-caseic the aqueous solution is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.01mol/L HCl regulator solution be 5.30 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 191nm, and polydispersity coefficient is 0.125.
In above-mentioned system, work as α
SThe mol ratio of-casein/N,O-Diacetylmuramidase is in 0.05～10 scope, and the pH value all can obtain stable microgel particle at 4.0～7.0 o'clock.
Example 10. α
S-casein (α
S-Casein) and N,O-Diacetylmuramidase (Lysozyme) in alkaline aqueous solution, form microgel.
With α
S-casein and N,O-Diacetylmuramidase are dissolved in respectively in the deionized water, are made into the protein solution of 4mg/mL and 0.624mg/mL.Under magnetic agitation with 300 μ L α
S-caseic the aqueous solution is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.04mol/L NaOH regulator solution be 10.2 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 172.9nm, and polydispersity coefficient is 0.207.
In above-mentioned system, in the scope of mol ratio 0.05～10 of beta-casein/N,O-Diacetylmuramidase, the pH value all can obtain stable microgel particle at 8.5～11.0 o'clock.
Example 11. κ-casein (α
S-Casein) and N,O-Diacetylmuramidase (Lysozyme) in acid and neutral aqueous solution, form microgel.
κ-casein and N,O-Diacetylmuramidase are dissolved in respectively in the deionized water, are made into the protein solution of 8.2mg/mL and 0.624mg/mL.Under magnetic agitation with 120 μ L α
S-caseic the aqueous solution is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.01mol/L HCl regulator solution be 4.74 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 288.8nm, and polydispersity coefficient is 0.0828.
In above-mentioned system, in the scope of mol ratio 0.05～10 of κ-casein/N,O-Diacetylmuramidase, the pH value all can obtain stable microgel particle at 4.0～8.0 o'clock.
Example 12. caseins (Casein) and N,O-Diacetylmuramidase (Lysozyme) form microgel in acidic aqueous solution.
Casein is added in the deionized water, and add 0.1mol/L NaOH and stir to spend the night and make it dissolving, the pH value that is made into 10.0mg/mL is 7.2 caseic aqueous solution.N,O-Diacetylmuramidase is dissolved in the deionized water, is made into the protein solution of 0.624mg/mL.Under magnetic agitation, the caseic aqueous solution of 120 μ L is added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, with the pH of 0.01mol/L HCl regulator solution be 4.50 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 230.6nm, and polydispersity coefficient is 0.121.
In above-mentioned system, in the scope of mol ratio 0.05～10 of casein/N,O-Diacetylmuramidase, the pH value all can obtain stable microgel particle at 4.0～7.0 o'clock.
Example 13. caseins (Casein) and N,O-Diacetylmuramidase (Lysozyme) form microgel in alkaline aqueous solution.
Casein is added in the deionized water, and add 0.1mol/L NaOH and stir to spend the night and make it dissolving, the pH value that is made into 10.0mg/mL is 7.2 caseic aqueous solution.N,O-Diacetylmuramidase is dissolved in the deionized water, is made into the protein solution of 0.624mg/mL.Under magnetic agitation the caseic aqueous solution of 120 μ L being added drop-wise in the aqueous solution of 3mL N,O-Diacetylmuramidase, is that 10.3l and agitation as appropriate made it to mix in 5 minutes with the pH of 0.04mol/L NaOH regulator solution.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 224.1nm, and polydispersity coefficient is 0.208.
In above-mentioned system, in the scope of mol ratio 0.05～10 of beta-casein/N,O-Diacetylmuramidase, the pH value 9.0～11.0 the time, all can obtain stable microgel particle.
Example 14. chitosans (Chitosan) and α
S-casein (α
S-Casein) in acidic aqueous solution, form microgel.
Chitosan is dissolved in 2% the acetic acid, is made into concentration and is 2% heavy-gravity chitosan aqueous acetic acid; With α
S-casein is dissolved in the deionized water, is made into the protein soln of 1.03mg/mL.Aqueous acetic acid with 161 μ L chitosans under magnetic agitation is added drop-wise to 3mL α
SIn-caseic the aqueous solution and agitation as appropriate made it to mix in 10 minutes, be 4.20 and stirred 5 minutes with the pH of 0.2mol/L NaOH regulator solution.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 262.8nm, and polydispersity coefficient is 0.316.
In above-mentioned system, as chitosan/α
S-caseic mass ratio 0.1～10 scope in, in the scope of the pH regulator to 3.5 of solution～7.5, all can obtain stable microgel particle.
Example 15. egg albumen powders (Chicken Albumin) form microgel in the aqueous solution.
Egg albumen powder is dissolved in the deionized water, is made into the mixed protein solution of 2mg/mL, with the pH of 0.02mol/L NaOH regulator solution be 6.0 and agitation as appropriate made it to mix in 5 minutes.Then the solution that mixes is placed 80 ℃ water-bath to heat 30 minutes, can obtain stable microgel solution.With the resulting microgel solution of dynamic laser light scattering measurement: particle diameter is 161.4nm, and polydispersity coefficient is 0.350.
In above-mentioned system, when the pH of solution value 4.0～10.5 the time, all can obtain stable microgel particle.
Claims (19)
1. nano microgel is characterized in that making protein denaturation formed by the two kinds of protein that has different electric charges under identical pH condition or by a kind of protein and a kind of polysaccharide by heating.
2. microgel according to claim 1 is characterized in that this microgel has nucleocapsid structure, and nuclear contains a kind of protein, and shell contains another kind of protein or a kind of polysaccharide; Nuclear and shell with electric charge be subjected to the influence of pH value of solution: when microgel is made up of two kinds of different protein, pH is during less than two kinds of isoelectric point of protein, the nuclear of microgel and shell are all positively charged, as pH during greater than two kinds of isoelectric point of protein, the nuclear of microgel and shell are all electronegative, when pH is between two kinds of isoelectric point of protein, and the nuclear of microgel and shell oppositely charged, when pH equals to form the isoelectric point of protein of microgel shell, the shell neutral of microgel; When microgel is made up of protein and polysaccharide, pH is between isoelectric points of proteins and polysaccharide ionization constant pK the time, the nuclear of microgel and shell oppositely charged, for alkaline polysaccharide, as pH during greater than its pK+2, the shell neutral of microgel is for acidic polysaccharose, as pH during less than its pK-2, the shell neutral of microgel.
3. preparation method of nano microgel according to claim 1 is characterized in that this method may further comprise the steps:
(1) will have the protein of different electric charges or protein and polysaccharide under identical pH condition is mixed with the aqueous solution and makes it form electrostatic complexes or micella or precipitation;
(2) secondly, adjust the potential of hydrogen of mixing solutions, make that any isoelectric point of protein is 0～3 in its pH value and the mixing solutions, stir or leave standstill so that micella or resolution of precipitate or that electrostatic complexes is spread is even, but not exclusively dissociate;
(3) last, the heating mixing solutions makes protein denaturation wherein, thereby obtains microgel.
4. the preparation method of microgel according to claim 3 is characterized in that two kinds of protein that use in (1) have different iso-electric points; When using multiple proteins, wherein have at least two kinds of protein to have different iso-electric points; When using protein and polysaccharide, the two has opposite electric charge in certain pH range.
5. the preparation method of microgel according to claim 3, when it is characterized in that the solution of mixing protein or polysaccharide, make mixing solutions pH value be the intermediate value of any two isoelectric points of proteins wherein, or the intermediate value of any one isoelectric points of proteins and polysaccharide ionization constant pK.
6. the preparation method of microgel according to claim 3, it is characterized in that in (1) earlier protein or polysaccharide are mixed with mass percentage concentration greater than 0 and less than 10% the aqueous solution, will have the protein-protein of different electric charges or protein and polysaccharide again and be in molar ratio 0.05～20 mixed.
7. the preparation method of microgel according to claim 6 is characterized in that earlier protein or polysaccharide are mixed with mass percentage concentration greater than 0 and less than 8% the aqueous solution, remix.
8. the preparation method of microgel according to claim 3 is characterized in that the pH value of mixing solutions is regulated with weak acid or weak base.
9. the preparation method of microgel according to claim 8, used acid is any in hydrochloric acid, acetic acid, phosphoric acid or the citric acid when it is characterized in that regulating mixing solutions pH value; Used alkali is any in sodium hydroxide, yellow soda ash, sodium bicarbonate or the sodium-acetate.
10. the preparation method of microgel according to claim 8, the weak acid or the weak base concentration that it is characterized in that regulating mixing solutions pH value are 0.01～1.0mol/L.
11. the preparation method of microgel according to claim 3 is characterized in that some isoelectric point of protein differences are 0～2 in the pH value of mixing solutions and the mixing solutions.
12. the preparation method of microgel according to claim 3 is characterized in that some isoelectric point of protein differences are 0～1 in the pH value of mixing solutions and the mixing solutions.
13. the preparation method of microgel according to claim 3 after it is characterized in that regulating the potential of hydrogen of mixing solutions, stirred or leaves standstill 0-24 hour.
14. the preparation method of microgel according to claim 3 is characterized in that heating mixing solutions to 50～100 ℃, and kept 1 minute～3 hours.
15. the preparation method of microgel according to claim 14 is characterized in that heating mixing solutions to 70～90 ℃, heats 30～120 minutes.
16. the application of microgel according to claim 1, it is characterized in that microgel forms after, by diffusion way small molecules is wrapped in the microgel.
17. the application of microgel according to claim 1 is characterized in that this microgel as medicine or nutraceutical carrier.
18. the application of microgel according to claim 1 is characterized in that this microgel is used for oral pharmaceutical or nutraceutical nano-carrier.
19. the application of microgel according to claim 1 is characterized in that this microgel is used to prepare the nano-carrier of dyestuff, spices or makeup.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103269605A (en) * | 2010-12-23 | 2013-08-28 | 帝斯曼知识产权资产管理有限公司 | Compositions of fat-oluble active ingredients containing plant protein soy polysaccharide complexes |
| CN104394715A (en) * | 2012-06-27 | 2015-03-04 | 帝斯曼知识产权资产管理有限公司 | Nanogel comprising water-soluble active ingredients |
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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 |
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| CN103269605A (en) * | 2010-12-23 | 2013-08-28 | 帝斯曼知识产权资产管理有限公司 | Compositions of fat-oluble active ingredients containing plant protein soy polysaccharide complexes |
| CN104394715A (en) * | 2012-06-27 | 2015-03-04 | 帝斯曼知识产权资产管理有限公司 | Nanogel comprising water-soluble active ingredients |
| EP2866588B1 (en) * | 2012-06-27 | 2016-07-20 | DSM IP Assets B.V. | Nanogel comprising water-soluble active ingredients |
| CN107603241A (en) * | 2017-09-25 | 2018-01-19 | 中原工学院 | A kind of preparation method of fusiformis nano-micelle |
| CN107603241B (en) * | 2017-09-25 | 2019-09-13 | 中原工学院 | A kind of preparation method of shuttle shape nano-micelle |
| 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 |
| CN108771076A (en) * | 2018-05-17 | 2018-11-09 | 南京农业大学 | A kind of compound curcumin fribrillin solid beverage prepares and redissolves method |
| CN108783149A (en) * | 2018-05-17 | 2018-11-13 | 南京农业大学 | A kind of production method of curcumin-ovalbumin compound anti-oxidation protein beverage |
| CN108771076B (en) * | 2018-05-17 | 2021-09-03 | 南京农业大学 | Preparation and redissolution method of composite curcumin myofibrillar protein solid beverage |
| CN109222069A (en) * | 2018-08-10 | 2019-01-18 | 江苏省农业科学院 | A kind of anthocyanidin nano-complex and preparation method thereof of " core-shell structure copolymer " structure |
| CN111420478A (en) * | 2020-04-08 | 2020-07-17 | 东莞市艾尔佳过滤器制造有限公司 | Long-acting slow-release fragrance air conditioner filter element and processing method thereof |
| CN114568674A (en) * | 2022-02-17 | 2022-06-03 | 江南大学 | Preparation method of high-embedding-rate microcapsules for powdering spice essential oil |
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