CN102869271B - For the nano-particle of encapsulation compound and preparation thereof and application - Google Patents

Abstract

The present invention relates to the nano-particle for encapsulation compound, and prepare and application, this nano-particle includes casein substrate, basic amino acid and the metal selected from divalent metal, trivalent metal and combinations thereof.Described nano-particle can encapsulate water solublity or fat-soluble bioactive compound.Present invention could apply to food, pharmacy and cosmetic field and field of nanometer technology.

Description

For the nano-particle of encapsulation compound and preparation thereof and application

Technical field

The present invention relates to food, pharmacy and cosmetic field and field of nanometer technology, and include using casein to make Bioactive compound is encapsulated for coating agent.

Technical background

Food industry needs technical continuous progress thus meets new consumer demand.Nanotechnology exists makes food Industry changes huge potentiality, up hill and dale because this technology can encapsulate bioactive compound [BAC], such as spice, dimension Raw element, mineral, quintessence oil, antioxidant, probiotic bacteria etc., thus there is numerous advantage, for example, goods can be extended Pot-life；The consumption of BAC can be reduced；Its release can be controlled；Its bioavailability can be improved；Can cover and need not Taste, etc..

When design is applicable to the carrier encapsulating BAC, the material being correctly chosen for use as coating agent or substrate is very Important；To this end, inter alia, it is necessary in view of dosage form, toxicity, product (food, the cosmetic of preparation will be introduced Product, medicine, etc.) etc..In food nanotechnology field, do not recommend the polymer of synthesis, because they there may be Toxicity problem.Natural polymer does not have these shortcomings；But, their use necessarily makes the manufacture method of granule more multiple Miscellaneous；Additionally, in most cases, it is difficult to controlling the granularity (in many cases more than 100 μm) obtained, therefore consumer can Can be it will be noted that these nano-particle, and it may change the organoleptic attribute of objective food.

Traditionally, in these materials, protein is used as BAC coating agent.Use casein hydrophobic as carrier encapsulating Property BAC application in food, existing described (CA2649788 and EP2011472).

Folic acid (pteroic acid list glutamic acid or FA), the B race water soluble vitamins being included in folic acid group, for weight Biochemical process, the such as DNA synthesis wanted are highly important.Lack folic acid and megaloblastic anemia, Alzheimers Disease, mongolism, endocrine disturbance, certain type of cancer (colon cancer, cervical cancer, leukemia, cancer of pancreas), send out fetus Neural tube defect during educating, the complication in pregnancy and male infertility are relevant.But, folic acid can not lead to Cross organism to synthesize；Thus it must be supplied by different supplement or diet.

Although folic acid is naturally occurring in food (such as fruits and vegetables), but it is mainly with the shape of poly-glutamic acid salt Formula, their bioavailability is the most incomplete, and typically 50% or lower.Therefore, picked-up be added with the food of folic acid can be with shape Become the selection of a kind of complementarity, thus increase and take in taking the photograph of described vitamin in the case of less than recommended amounts at those folic acid Enter.But, in addition to other factor, because matrix effect, the bioavailability of the folic acid added to food is the most not exclusively (folic acid may be connected with food component, thus stops it to absorb), or any component being present in food to reduce it raw Thing availability.Additionally, when folic acid does not dissolves at enteral, it can not be well absorbed.By the side of capsule, tablet etc. Formula uses the supplement containing folic acid or hardening agent has following shortcoming, when it is degraded under one's belt due to gastric acid, and leaf Acid can precipitate, and is converted into its less soluble form, and the folic acid that the most only part is supplied can arrive intestinal.

Additionally, the strengthening containing folate or the food of folic acid is complicated process, because inter alia, folic acid Salt and their derivant and folic acid are the most sensitive for the change of temperature, light and pH value；Therefore their stability is subject to To the impact of food process condition, and the biologically active amount of the available vitamin of consumer may greatly reduce.By This, when utilizing described vitamin enrichment food, it is necessary to considers these aspects, because the loss of maximum is it may happen that eating In the storage of thing and preparation process.

There is document to describe how to make food rich in folic acid, be mainly in milk product and cereal product.Rich in leaf Acid or the dietary supplement (EP2002839) of folate or food, such as, cook the meat (ES2302571) of sausage, milk product (EP1941804), children's (baby) youngster's food (US4753926), or even Carnis Gallus domesticus, Carnis Sus domestica or canned beef food (RU2223672 and RU2213493) is also existing described.But, in the case of described, all do not account for vitamin and food The possible interaction of thing substrate and its bioavailability.

Prior art also discloses a kind of for obtaining alginate and the method for pectin microcapsule, and this microcapsule contains folic acid Thus avoid the infringement of the environmental factors (such as stomach environment) that folic acid protected it to exempt to cause it to degrade, it is achieved folic acid its at intestinal Interior release.But, the microcapsule obtained is too big, it has impact on the organoleptic attribute of target food.Prior art also designs A kind of for folic acid being encapsulated in poly lactic coglycolic acid (PLGA) Nano microsphere and realizing what it discharged constantly Method；Although result is positive, but the infringement that the application that this design is in food is used by polymer, because it is doctor Learn and pharmaceutical field is prohibited from use.

Therefore, it is necessary to exploitation BAC encapsulation system, the most water miscible, more preferably acid water solublity BAC, such as leaf Acid, it completely or partially overcomes drawbacks described above.

Summary of the invention

Currently, it has unexpectedly been found that utilize casein to be formed and farther include basic amino acid (such as arginine or bad Propylhomoserin) and it is applicable to the nano-particle of metal (such as calcium) of food, form a kind of new bag of bioactive compound (BAC) Envelope and stabilisation systems, this bioactive compound can be water solublity and fat-soluble, the most water miscible, more preferably acid The water solublity BAC of property, it can be applied in food, cosmetics and medicine.

Thus, on the one hand, the present invention relates to nano-particle, it includes casein substrate, basic amino acid and selected from bivalence The metal of the food stage of metal, trivalent metal and combinations thereof.Described nano-particle can be used as processing additive；They are further Having encapsulating BAC, preferred water solublity BAC, the ability of more preferably acid water solublity BAC, such as vitamin B group or C race are tieed up Raw element, such as folic acid, pantothenic acid and ascorbic acid, or other hydrophilic compounds, although they can also encapsulate fat-soluble BAC。

Described nano-particle is stable and BAC can be protected to avoid in Product processing (such as food, medicine or change Cosmetic) and storing process in degraded by external factor, such as light, pH value change, oxidation etc., and additionally, when they are applied Time in food, they protection BAC, from the impact of stomach sour environment, prevent it from discharging along gastropore, thus avoid BAC to sink Form sediment, and thus avoid bioavailability to reduce.It is situated between additionally it was found that described nano-particle can be dissolved in (simulation) intestines In matter, promote that BAC, in the release completely of enteral, makes it correctly absorb, and further avoid any kind of toxicity and ask Topic.Advantageously, described nano-particle is inert in the food introduced, therefore avoid BAC and substrate different component it Between reaction, and avoid reducing its bioavailability.

It addition, one of several most important feature of this nano-particle that the present invention provides be to use casein as with Damage in protection BAC from environmental condition and stomach condition, promote that it discharges in intestines and thus improves its bioavailability Natural carrier because casein itself has been proved to have nutritive peculiarity, so that it supplements the useful of BAC self Effect.

On the other hand, the present invention relates to a kind of method manufacturing described nano-particle.Described method is simple and energy Enough industrialization large-scale application.Advantageously, described method does not includes being not approved for the synthesis into food additive or reaction Property polymer, minimize the surfactant or emulsifying agent comprised, and the method allow to obtain and has controlled granularity Nano level nano-particle.

In the specific embodiment of one, described method farther includes to be dried the suspension comprising described nano-particle Thus obtain the preparation of powder type, maintain stablizing of BAC as time goes by；Especially, such powder formulation Use be applicable to food.Advantageously, described dried performs in the presence of nano-particle is protectant.Thus obtain The nano-particle comprising BAC obtained can be readily suspended in aqueous medium, and protection BAC avoids degrading in the solution.Obtain End article is stable and protects BAC in whole long-term storing process, and it can also be applied to dissimilar Food, either liquid food (such as beverage etc.) or food.

On the other hand, the present invention relates to a kind of include that the compositions of described nano-particle is at food, medicine or cosmetics In application.It practice, described nano-particle can be bonded in Emulsion, gel and hydrogel, thus obtain and be applicable to this The stable cosmetic formulations in one field.Described nano-particle can also utilize and be applicable to use described nanometer by topic route The excipient of granule is prepared.

On the other hand, the present invention relates to a kind of food, including institute based on casein nano-particle provided by the present invention State compositions.In the specific embodiment of one, described food is the form of liquid, semisolid or solid.

Accompanying drawing explanation

Fig. 1 shows the schematic diagram of a kind of specific embodiment describing the inventive method, and it comprises leaf for acquisition The casein nano-particle of acid.

Fig. 2 shows transmission electron microscope (TEM) image of the casein nano-particle of sky.It is positioned at the bottom of this image The secret note of left side edge part corresponds to 100nm.

Fig. 3 shows transmission electron microscope (TEM) image of the casein nano-particle comprising folic acid.It is positioned at this image The secret note of lower left side marginal portion corresponding to 100nm.

Folic acid amount that Fig. 4 shows encapsulating and the ratio added between the casein amount of the folic acid of every milligram in said preparation Example.In all of preparation, the weight ratio of lysine and this protein was 1:12 before adding folic acid solution.

Fig. 5 shows the casein nano-particle comprising folic acid and the scanning electron microscopy in the formulation with lysine Mirror (SEM) microgram, is respectively and does not carries out HIGH PRESSURE TREATMENT (A and B), process 5 minutes (C) under 100MPa, place under 400MPa Manage 5 minutes (D) and under 800MPa, process 5 minutes (E).

Fig. 6 shows in the casein nano-particle comprising folic acid and preparation have arginic scanning electron microscope (SEM) microgram, it processes 5 minutes under 400MPa.

Fig. 7 shows in the simulated gastric fluid (SGF) at 37 ± 1 DEG C (in first 2 hours: 0-2 hour) and at mould After intending intestinal juice (SIF) (2-24 hour) is hatched, without the release from casein nano-particle of folic acid during HIGH PRESSURE TREATMENT.This number According to showing meansigma methods ± standard deviation (n=6).

Fig. 8 shows at 37 ± 1 DEG C in simulated gastric fluid (SGF) (in first 2 hours: 0-2 hour) and in simulation After intestinal juice (SIF) (2-8 hour) is hatched, under (A) 150MPa HIGH PRESSURE TREATMENT 5 minutes and under (B) 400MPa HIGH PRESSURE TREATMENT 5 Minute time, folic acid release from casein nano-particle.The data show meansigma methods ± standard deviation (n=4).

Fig. 9 shows after using different vitamin preparations in experimental animal, according to the Serum Folic Acid Concentration of time (ng/mL) change.The results show meansigma methods ± standard deviation (n=5).

A) intravenous injection, dosage 1mg/kg.

B) oral route, dosage 1mg/kg: the non-encapsulated folic acid (●) being dissolved in water；Be dispersed in water is encapsulated in Folic acid (■) in casein nano-particle；Be dispersed in water with the leaf being encapsulated in casein nano-particle of HIGH PRESSURE TREATMENT Acid (▲).

Detailed Description Of The Invention

The present invention provides casein nano-particle and the method for encapsulating bioactive compound (BAC), thus prevents it Degrade due to external factor, such as light, pH value change, oxidation etc..

Definition

Understanding the purpose of the present invention for helping, some term used in present invention and the implication of statement are such as Lower described.

As it is used in the present context, " basic amino acid " includes lysine, arginine and histidine.

As it is used in the present context, " casein " relates to associated proteins, it forms the total milk protein of about 80%.At ball egg As defined in the range of Bai, casein is the protein of phosphoprotein type, solvable, has high water holding capacity and in 20 DEG C about Precipitate under the pH value of 4.6.Casein is by four essential parts (α s1-casein, α s2-casein, beta-casein and κ-cheese egg Being formed in vain), it is each other by their aminoacid ingredient, their CHARGE DISTRIBUTION and they formation in the presence of calcium The trend of agglomerate and be distinguish between.In emulsion, casein forms a diameter of 50 to 600nm(the most about 150nm) claimed Big colloidal solid for " micellar casein ".These granules are by hydrophobic interaction and by by being present in casein knot Calcium phosphate that phosphoserine free radical in structure is compound and formed.Highly stable colloid is formed at micelle described in emulsion System, this is to form its color, heat stability and the one of the main reasons condensed due to Chymosin.

As it is used in the present context, " bioactive compound " or " BAC " relates to the most fat-soluble and water solublity Tool is nutritious, treatment and/or the compound of cosmetic activity.The nonrestrictive explanation of the BAC according to the present invention The example of property include aminoacid, antimicrobial, aromatizer, preservative, sweeting agent, steroid, medicine, hormone, lipid, peptide, Polynucleotide, polysaccharide, protein, Dan Baiduotang proteoglycan PG, aromatic, vitamin etc..

As it is used in the present context, " water-soluble bioactive compound " or " water-soluble BAC " relate to having nutritious, Treatment and/or cosmetic activity and water soluble solution (fairly soluble, diffluent, solvable, indissoluble or Sl. sol.) compound, it defines according to the standard of imperial family of Spain pharmacopeia:

The nonrestrictive illustrative example of water solublity BAC includes vitamin, such as vitamin B group or C vitamin and Its derivant, salt or ester；Hyaluronic acid, chondroitin sulfate, thioctic acid, its salt or ester, etc..At the specific embodiment of one In, described water solublity BAC select free folic acid, PABA, nicotinic acid, pantothenic acid, monophosphate thiamine, diphosphothiamine, Thiamine triphosphate, ascorbic acid, pteroyl polyglutamic acid (folic acid derivatives: folic acid poly-glutamic acid salt；Poly-glutamic acid folic acid Salt), folic acid, nicotinic acid, hyaluronic acid, thioctic acid (alpha lipoic acid), p-coumaric acid, caffeic acid, its pharmacy or cosmetics can accept Or the derivant of food stage, ester or salt, and mixture constitute group in.

As it is used in the present context, " lipophilic bioactive compound " or " fat-soluble BAC " relates to having nutrition , treatment and/or cosmetic activity and dissolve in the (fairly soluble, diffluent, solvable, indissoluble of fat and oil Or sl. sol.) compound, it defines according to the standard of imperial family of Spain pharmacopeia.The nonrestrictive explanation of fat-soluble BAC Property example includes vitamin, such as A race, D race, E race, K vitamin and derivant thereof, phospholipid, carotenoid (carotene, Lycopene, phylloxanthin, capsanthin, cryptoxanthin etc.), omega-3 fatty acid (docosahexenoic acid (DHA), 20 carbon five Olefin(e) acid (EPA) etc.), plant sterol and phytosterol (sitosterol, campesterol, stigmasterol etc.), polyphenol (Quercitroside, rutin, Resveratrol, nimbecetin, myricetin, isorhamnetin etc.) and derivant.

As fruit product for humans and animals food in time be safe, then according to country or tissue food standard These goods are known as " food stage ", such as United Nations Food and Agricultural Organization (FAO) or the World Health Organization (WHO)； Therefore, the goods of " food stage " are " using be applicable to food " nontoxic goods, and use the most in the present note Two kinds of statements are synonyms, and do not distinguish.

As it is used in the present context, " divalent metal " includes any metallic element that quantivalence is 2, such as alkaline earth gold Belong to, such as calcium, magnesium, zinc etc., if or its there is multiple quantivalence, when one of them is 2, such as ferrum etc., same to limit It is pharmaceutically or cosmetics are acceptable, or be applicable to use food.

As it is used in the present context, " trivalent metal " includes any metallic element that quantivalence is 3, if or its tool There is multiple quantivalence, when one of them is 3, such as ferrum etc., with limit, it is pharmaceutically or cosmetics are acceptable, or Person is be applicable to use food.

As it is used in the present context, " nano-particle " relates to the spherical or colloid system of analogous shape, its granularity is little In 1 micron (μm), preferably from about 10 to 900 nanometers (nm).

As it is used in the present context, " particle mean size " relates to the nano-particle colony moved together in an aqueous medium Average diameter.The average diameter of these systems can be measured by standard method well known by persons skilled in the art, and In experimental section, such as it is described (see below).The nano-particle of the present invention, it is characterised in that have less than 1 μm Particle mean size, typically ranges between 1 to 999nm, preferably 10 to 900nm, more preferably 50 to 500nm, the most more preferably 100 to 200nm.In the specific embodiment of one, the particle mean size that the nano-particle of the present invention has is 50 to 200nm, is preferably About 140nm.

Nano-particle

On the one hand, the present invention relates to the nano-particle of a kind of nano-particle, the hereinafter referred to as present invention, including casein base Matter, basic amino acid and the metal selected from divalent metal, trivalent metal and combinations thereof.

In the specific embodiment of one, described basic amino acid selects free arginine, lysine, histidine and mixes In the group of compound composition.

In the specific embodiment of another kind, described metal is preferably the divalent metal of food stage, its free calcium of choosing, In the group of magnesium, zinc, ferrum (with the form of bivalence) and combinations thereof composition.

In the specific embodiment of another kind, described metal is the ferrum of the trivalent metal of food stage, such as trivalent form.

The nano-particle of the present invention can be used as processing additive, such as fat substitute etc..The nanometer of the present invention Granule has the ability of encapsulating bioactive compound (BAC) further.

Thus, in the specific embodiment of another kind, the nano-particle of the present invention farther includes biological activity chemical combination Thing (BAC).Described BAC can be water solublity BAC or fat-soluble BAC；In this case, the nano-particle of the present invention is at this Description is equal to " the load nano-particle of the present invention " sometimes.

In the specific embodiment of one, described BAC is water solublity BAC, preferably acidic water-soluble BAC.In one More particularly in embodiment, the following group constituted of described water solublity BAC choosing:

A) vitamin B group or C vitamin；

B) according to vitamin derivative a)；

C) selected from hyaluronic acid, chondroitin sulfate and the compound of thioctic acid；

D) arbitrary aforesaid compound a) is to salt c) or ester；And

E) a combination thereof.

In the specific embodiment of one, described water solublity BAC select free folic acid, PABA, nicotinic acid or Vitamin B3, pantothenic acid or vitamin B5, monophosphate thiamine, diphosphothiamine, thiamine triphosphate, ascorbic acid, butterfly Acyl polyglutamic acid (folic acid derivatives: folic acid poly-glutamic acid salt；Poly-glutamic acid folate), folic acid, nicotinic acid, hyaluronic acid, sulfur Octanoic acid or alpha lipoic acid, p-coumaric acid, caffeic acid, it is pharmaceutically or cosmetics are acceptable or the derivant of food stage, In ester or salt, and the group of mixture composition.

In the specific embodiment of one, described BAC is acidic water-soluble BAC, such as folic acid, pantothenic acid, ascorbic acid Deng.

It is not intended to be limited by any theory, it is believed that in the case of there is metal, such as divalent metal (such as calcium), When the phosphoserine combined with radical being present in its structure is to cationic moiety, their hydrophilic and surface charge Lose, α and β caseins aggregate.This water solublity BAC, (the such as folic acid) of preferred acidic, also with described metal electrostatic phase interaction With, thus it will be trapped in by hydrophobic matrix produced by the casein of these types.In turn, κ casein not with Metal (such as calcium) reacts, and is therefore incorporated into this granule by its hydrophobic part, its water-soluble portion and external water Property medium contacts.In addition to a high proportion of carbonyl (acidic group of aminoacid, such as glutamic acid or aspartic acid), described water-soluble Property part also there is polar group, it is corresponding to being bound to trisaccharide and the seryl of tetrose and threonyl residues.It is thus regarded that After forming this nano-particle, owing to the electrostatic interaction of its carboxyl with described part is [such as, at them through spray dried After during dry device (if Shi Dang) heating it, they may covalency keyed jointing], the alkalescence existed in solution Aminoacid (such as lysine) will be attached to the surface of these nano-particle.Fig. 1 shows the load nanometer describing the present invention The schematic diagram of grain, this load nano-particle includes casein substrate, lysine (basic amino acid) and calcium (divalent metal).

In the specific embodiment of another kind, described BAC is fat-soluble BAC, although will need excellent in this case Form slection becomes BAC unit for uniform suspension in an aqueous medium, or is more preferably dissolved in organic solution by BAC, lentamente by institute State waterborne suspension or described organic solution adds to comprising (such as caseinate) in caseic solution, and it is mixed to hatch this Compound.

This catch mechanism is by different, because fat-soluble BAC will be trapped in nanometer with described water solublity BAC In the inner hydrophobic part of granule, reason is the affinity between the two part, and do not consider they whether have with It is somebody's turn to do the ability of (bivalence or trivalent) metal interaction.

In the specific embodiment of one, described BAC is fat-soluble BAC, and it is selected from vitamin, such as A race, D race, E Race, K vitamin and derivant thereof, phospholipid, carotenoid (carotene, lycopene, phylloxanthin, capsanthin, maize Matter etc.), omega-3 fatty acid (such as DHA, EPA etc.), aminoacid (such as isoleucine, leucine, methionine, phenylalanine, color Propylhomoserin and valine), plant sterol and phytosterol (such as sitosterol, campesterol, stigmasterol etc.), polyphenol (such as Cortex querci dentatae Glycosides, rutin, resveratrol, nimbecetin, myricetin, isorhamnetin etc.) and derivant.

The BAC of the load nano-particle of the present invention: caseic weight ratio can have wide in range scope；In a kind of non-limit In the illustrative mode of property processed, BAC: the caseic weight ratio of the load nano-particle of the present invention can be 1:1 to 1: 200, preferably 1:10 to 1:80, more preferably from about 1:15 to 1:35.In the specific embodiment of one, this BAC is water solublity BAC, and the present invention load nano-particle (water solublity) BAC: caseic weight ratio be 1:1 to 1:50, preferably 1:10 To 1:30, more preferably from about 1:15 to 1:20.In the specific embodiment of another kind, this BAC is fat-soluble BAC, and this (fat-soluble) BAC: the caseic weight ratio of bright load nano-particle is 1:1 to 1:200, preferably 1:10 to 1:80, more excellent Elect about 1:20 to 1:35 as.

It addition, if necessary, the nano-particle of the present invention, including those and the unsupported BAC that are loaded with BAC Those, all can introduce antioxidant in their preparation, and such as ascorbic acid (vitamin C) etc., are used for increasing them to temperature Degree and the stability of oxidation.In the specific embodiment of one, this BAC is folic acid and this antioxidant is ascorbic acid, its Seemingly avoid, by protection folic acid, the degraded that caused by ultraviolet radiation, pH value change, heat, oxygen etc. and work, and Further provide for the nutritional support of ascorbic acid.Described antioxidant can be by common encapsulating or be introduced into this together with BAC In the coating of bright nano-particle.

The method obtaining nano-particle

On the other hand, the present invention relates to a kind of method manufacturing nano-particle, this nano-particle includes casein substrate, alkali Acidic amino acid and the metal (nano-particle of the present invention) selected from divalent metal, trivalent metal and combinations thereof, hereinafter referred to as " this The method [1] of invention ", comprising:

A) preparation comprises casein raw material and basic amine group aqueous acid；With

B) in the solution of step a), add the aqueous solution of the metal selected from divalent metal, trivalent metal and combinations thereof.

On the other hand, the invention still further relates to a kind of method manufacturing nano-particle, this nano-particle includes casein substrate, Basic amino acid, selected from the metal of divalent metal, trivalent metal and combinations thereof, and bioactive compound (present invention's is negative Carry nano-particle), hereinafter referred to as " method [2] of the present invention ", comprising:

A) mixing (i) comprises casein raw material and the first basic amine group aqueous acid and (ii) comprises biological activity chemical combination The solution of thing；With

B) in the mixture that step a) is obtained, add the water of the metal selected from divalent metal, trivalent metal and combinations thereof Solution.

In the step a) of the inventive method [1], comprise casein raw material and basic amine group aqueous acid passes through ability Prepared by conventional method known to field technique personnel, such as by adding described casein raw material and basic amino acid to aqueous Mode in medium.

In the step a) of the inventive method [2], will comprise casein raw material and basic amine group aqueous acid (i) with bag Solution containing BAC mixes mutually.Comprise the described solution of BAC character (ii) and component type based on BAC and character and Different.Thus, in the specific embodiment of one, when BAC is water solublity BAC, comprise the described solution of BAC (ii) For aqueous solution；In the specific embodiment of another kind, when this BAC is acidic water-soluble BAC, comprise the described solution of BAC (ii) for farther including the second basic amine group aqueous acid；And in the specific embodiment of another kind, when BAC is fat During dissolubility BAC, comprise the described solution of BAC (ii) for suspension in an aqueous medium or preferably organic solution, more excellent Elect the organic solution of water miscible agent, such as alcohol, such as ethanol as.

Can be used for implement both approaches [method [1] of the present invention and [2]] casein can essentially be from In the casein in any source, such as milk, beans etc..Be present in the casein in described solution can be acid casein or The form of person's caseinate.In the specific embodiment of one, described casein raw material includes the cheese egg of caseinate form In vain, preferred sodium caseinate.Although calcium caseinate and calcium phosphate (phosphocalcium) can also be used, but they Practice has less advantage, because calcium is used to form nanometer after being mixed mutually with this active component by caseinate Grain, therefore, if caseinate solution has had calcium in media as well, then the enforcement of described method may give a discount greatly Button.

In the aqueous solution formed in the step a) of the inventive method [1] and in the step a) of the inventive method [2] Use aqueous solution (i) [comprise casein raw material and the first basic amino acid] included in caseic consumption can have Wide in range scope；But, in the specific embodiment of one, the caseic consumption being included in described aqueous solution is 0.1% To 10%(w/v), preferably 0.5% to 5%, more preferably 1% to 3%.

Basic amino acid facilitate caseic dissolving and, suitably, facilitate BAC, particularly acidic water-soluble BAC Dissolving, therefore it plays in the present invention loads the manufacture of the nano-particle of BAC and the nano-particle of those unsupported BAC Very important role.It is true that seem to increase the pH value of solution, this basic amino acid allows need not use inorganic salt In the case of dissolve this caseinate, and its be further used as maintaining the hydrophilic end of this caseic κ part be in cloudy from The base of sub-form, so that this granule with negative surface charge is maintained in suspension and due to Coulomb repulsion not Condense.

The basic amino acid that can be used for implementing the two method [method [1] of the present invention and [2]] selects free arginine, relies In the group of propylhomoserin, histidine and mixture composition thereof, it is preferably selected from the group being made up of arginine, lysine and mixture thereof. This basic amino acid may be located at nano-particle of the present invention either internally or externally, and it plays important technique role, because Before forming nano-particle, it promotes component to dissolve, and it is (interior in the both sides of nano-particle after obtaining nano-particle Portion and outside) all maintain suitable pH value.Illustrating, folic acid is slightly soluble in water and is soluble in subalkaline aqueous solution In, therefore the existence of basic amino acid helps the dissolving of folic acid.

In a kind of particularly embodiment of the inventive method [2], when BAC is acidic water-soluble BAC, comprise BAC Described solution be (ii) aqueous solution, it farther includes the second basic amino acid (in order to prevent BAC to precipitate).Although that Situation may consider use two kinds of different basic amino acids, but in the specific embodiment of one, be used for preparing bag The aqueous solution of casein containing protein raw material (the first basic amino acid) and comprise BAC(the second basic amino acid for preparation) water-soluble The basic amino acid of liquid is identical, and it selects in the group that free arginine, lysine, histidine and mixture thereof form, It is preferably selected from the group being made up of arginine, lysine and mixture thereof.

In the solution formed in the step a) of the inventive method [1] and step a) molten of the inventive method [2] Liquid (i) included in the amount of basic amino acid can have scope and generally depend on used basic amine group widely Acid.Therefore, although this basic amino acid: caseic weight ratio may be widely different, but at the specific embodiment of one In, in the step a) of the inventive method [1] formed solution in or the inventive method [2] solution (i) in this alkali Acidic amino acid: caseic weight ratio is 1:1 to 1:50, preferably 1:10 to 1:40, more preferably when the basic amine group used When acid is lysine, weight ratio about 1:12 or when the basic amino acid used is arginine, weight ratio is about 1:25.

When BAC is acidic water-soluble BAC, the solution of the step a) of the inventive method [2] comprising described BAC (ii) enters One step includes above-mentioned second basic amino acid, and it can be identical or different with described first basic amino acid；In this case, In the inventive method [2], i.e. basic amino acid after the solution (i) and (ii) mixing of the step a) of described method: cheese egg White ratio is 1:1 to 1:50, preferably 1:5 to 1:20, more preferably when the basic amino acid used is lysine, and ratio It is about 1:6, or when the basic amino acid used is arginine, ratio is about 1:9.

The method [1] of the present invention and the method [2] of the present invention all include by a) selected from divalent metal, trivalent metal and The aqueous solution [step b)] of the metal of combination adds the step to the solution of this step.It is not intended to be limited by any theory, It is believed that described metal, such as divalent metal (such as calcium), it is allowed to the present invention load nano-particle be internally formed bridge joint, its This BAC is stablized in help, particularly when BAC is water solublity BAC, and preferably acidic water-soluble BAC, or can be with described metal The water solublity BAC that (such as calcium) interacts, such as folic acid, pantothenic acid or B race or C vitamin or derivatives thereof；This In situation, described metal, the most described divalent metal (such as calcium) seem by casein (with the form of caseinate) and BAC, it is preferably water solublity BAC, more preferably acidic water-soluble BAC or can be with the water solublity of described metal interaction Bridge joint between BAC and work, make described BAC be captured by the hydrophobic part of the load nano-particle of the present invention.

In the specific embodiment of one, described metal is divalent metal, selected from calcium, magnesium, zinc, ferrous iron and group thereof Close, preferably calcium.In the specific embodiment of another kind, described metal is trivalent metal, such as ferric iron.

Although the solution of the actually arbitrary aqueous solution of calcium, advantageously food stage [sees " Codex General Standard for Food Additives " GSFA is online, the calcium salt ratio used in food microcapsule] can be used for reality Execute the method for the invention [1] and [2], but in the specific embodiment of one, the aqueous solution of described calcium salt selects free chlorine Change calcium, calcium acetate, calcium gluconate, calcium lactate, calcium sorbate, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate and In the group of mixture composition, preferably calcium chloride.In practice, do not recommend calcium carbonate or calcium alginate, because they are in water Insoluble salt or very slightly soluble salt.Similarly, any magnesium, zinc or bivalence or ferric food stage aqueous solution can be used for Implement the method for the invention [1] and [2].

Metal: caseic weight ratio, wherein " metal " is related to the institute selected from divalent metal, trivalent metal and combinations thereof State metal, can have wide in range scope；But, in the specific embodiment of one, this metal: caseic weight ratio is 1:5 to 1:15, preferably 1:7 to 1:10, more preferably 1:8.5.In the specific embodiment of one, described metal is bivalence Metal.

The method [2] of the present invention obtains the load nano-particle of the present invention, to this end, step a) includes (i) comprising cheese egg White raw material and the first basic amine group aqueous acid and the molten liquid-phase mixing (ii) comprising BAC.The feature of described BAC is upper Literary composition is mentioned.In the specific embodiment of one, described BAC is water solublity BAC, preferably acidic water-soluble BAC, such as leaf Acid, PABA, nicotinic acid or vitamin B3, pantothenic acid or vitamin B5, monophosphate thiamine, diphosphothiamine, three phosphorus Allithiamine element, ascorbic acid, pteroyl polyglutamic acid (folic acid derivatives: folic acid poly-glutamic acid salt；Poly-glutamic acid folate), leaf Acid, nicotinic acid, hyaluronic acid, thioctic acid, p-coumaric acid, caffeic acid, it is pharmaceutically or cosmetics are acceptable or food stage Derivant, ester or salt, and mixture.In the specific embodiment of another kind, described BAC is fat-soluble BAC, such as A Race, D race, E race, K vitamin and derivant thereof, phospholipid, carotenoid is (such as carotene, lycopene, phylloxanthin, peppery Green pepper flavin, cryptoxanthin etc.), omega-3 fatty acid (such as DHA, EPA etc.), aminoacid (such as isoleucine, leucine, egg ammonia Acid, phenylalanine, tryptophan and valine), plant sterol or phytosterol (such as sitosterol, campesterol, stigmasterol etc.), Polyphenol (Quercitroside, rutin, resveratrol, nimbecetin, myricetin, isorhamnetin etc.) or derivatives thereof.

The BAC of the load nano-particle of the present invention: caseic weight ratio can have wide in range scope；In a kind of non-limit Property processed illustratively in, the present invention load nano-particle BAC: caseic weight ratio can be 1:1 to 1:200, It is preferably 1:10 to 1:80, more preferably from about 1:15 to 1:35.In the specific embodiment of one, this BAC is water solublity BAC, And the present invention load nano-particle (water solublity) BAC: caseic weight ratio be 1:1 to 1:50, preferably 1:10 to 1: 30, even more preferably about 1:15 to 1:20.In the specific embodiment of another kind, this BAC is fat-soluble BAC, and the present invention Load nano-particle (fat-soluble) BAC: caseic weight ratio be 1:1 to 1:200, preferably 1:10 to 1:80, more preferably About 1:20 to 1:35.

Similarly, the weight ratio of this basic amino acid: BAC is (corresponding to comprising acidic water-soluble BAC and the inventive method [2] the second basic amine group aqueous acid used in step a) is (ii)) can have wide in range scope；But, one In kind of specific embodiment, described solution (ii) in this basic amino acid: the weight ratio of (acidic water-soluble) BAC is 1: 0.1 to 1:3, preferably 1:0.5 to 1:1, even more preferably about 1:0.75.

As it has been described above, the nano-particle of the present invention, it is loaded with those of those and unsupported BAC of BAC, all can be Their preparation combines antioxidant, such as ascorbic acid (vitamin C) etc., for increasing them, temperature and oxidation are stablized Property.In this case, described antioxidant can with BAC(suitably) together with by common encapsulating or be introduced into the present invention and receive In the coating of rice grain；To this end, the method for the invention [1] and [2] will be suitably adapted with the preparation at this nano-particle This antioxidant of middle combination, such as by adding antioxidant to comprising in described BAC and basic amine group aqueous acid.

In the specific embodiment of one, this BAC is folic acid and this antioxidant is ascorbic acid, and it seemingly passes through Protection folic acid avoids the degraded that caused by ultraviolet radiation, pH value change, heat, oxygen etc. and works, and further provides for The nutritional support of ascorbic acid.Described antioxidant can be by common encapsulating or be introduced into nano-particle of the present invention together with BAC Coating in.

It addition, if necessary, the method [1] of the present invention and method [2] may each comprise one or more additionally By using different processing methods to stablize the step of obtained nano-particle.

In the specific embodiment of one, described stabilized treatment includes making the nano-particle comprising the present invention and being formed, bearing The suspension of those or unsupported those nano-particle that are loaded with BAC carries out the pressure of HIGH PRESSURE TREATMENT, such as 100 to 800MPa Power, typically 350 to 600MPa.In the specific embodiment of one, described process includes the suspension making nano-particle Under the pressure of 100MPa to 800MPa, preferably 350 to 600MPa, experience the time cycle of 3 to 5 minutes,；It practice, The pressure of 400MPa provides good result.

In the specific embodiment of another kind, described stabilized treatment include making comprise the present invention and formed nano-particle, The suspension of those or unsupported those nano-particle that are loaded with BAC carries out UHT(ultra-high temperature) process, such as exist 2 to 5 seconds are continued, the most quickly cooling at a temperature of 130 DEG C to 140 DEG C.

Similarly, if necessary, the method [1] of the present invention and the method [2] of the present invention may each comprise and be dried step Suddenly, it is for being dried the suspension comprising formed nano-particle, thus obtains the nano-particle of the present invention of powder type, Including those and those nano-particle of unsupported BAC of being loaded with BAC.The appearance of this form of described nano-particle promotees Entered their stability and for them at food, such as in flour, bread, cake goods, frumentum, milk powder with And the possible application in cosmetics and/or pharmaceutical preparation is also particularly advantageous.

May be used to it is true that any suitable in the routine techniques or method that are dried the suspension comprising nano-particle Perform this drying steps；But, in the specific embodiment of one, it is dried by spray drying method or by lyophilization Comprise the suspension of nano-particle.This process is generally by the protective agent by suitable described nano-particle, such as saccharide, example Such as lactose, trehalose, mannitol, sucrose, maltodextrin, glucose, Sorbitol, maltose etc. and mixture thereof, add extremely The suspension of this nano-particle is implemented.The nano-particle of the described protective agent protection present invention resists the heat in dry run Degraded and oxidation.

This casein: the weight ratio of sugar can have wide in range scope；But, in the specific embodiment of one, should Casein: the weight ratio of sugar is 1:1 to 1:4, preferably from about 1:2.

Similarly, in the specific embodiment of one, the solution comprising saccharide can comprise antioxidant, such as further Ascorbic acid (vitamin C) etc.；In this case, this casein: sugar: the weight ratio of antioxidant (such as vitamin C) is permissible It is 1:0.75-2.5:0.01-1.5, preferably 1:2.0:0.10.

The nano-particle of the present invention that the method according to the invention [1] obtains, i.e. includes casein substrate, basic amino acid With the nano-particle of the metal selected from divalent metal, trivalent metal and combinations thereof, prepared by the method comprised the steps: A) preparation comprises casein raw material and basic amine group aqueous acid；And b) will be selected from divalent metal, trivalent metal and combinations thereof The aqueous solution of metal add in the solution of step a), form another aspect of the present invention.

Similarly, the load nano-particle of the present invention that the method according to the invention [2] obtains, i.e. include casein base Matter, basic amino acid and selected from the metal of divalent metal, trivalent metal and combinations thereof and the nano-particle of BAC, by including Prepared by the method for following steps: a) mixing (i) comprises casein raw material and the first basic amine group aqueous acid and (ii) wraps Solution containing BAC；And in the mixture that step a) is obtained, b) add the gold selected from divalent metal, trivalent metal and combinations thereof The aqueous solution belonged to, forms another aspect of the present invention.

Application

The nano-particle of the present invention can be used as processing additive, such as fat substitute etc..They also have encapsulating BAC Ability, such as water solublity BAC or fat-soluble BAC.

In the specific embodiment of one, the nano-particle of the present invention can encapsulate BAC, preferably water solublity BAC, more It is preferably acidic water-soluble BAC, and it can be combined in medicine, cosmetics and food composition, because other component is Non-natural polymer (avoids the toxicity relevant to synthetic polymer), and the preparation not being used in the present invention of nonfood grade And in end article (nano-particle).Described nano-particle avoids BAC due to external factor (change of light, pH value, oxidation etc.) And degrade.

Advantageously, the particle mean size that the nano-particle of the present invention has is less than 1 μm, preferably 50 to 200nm, more preferably About 140nm, thus prevent the change (taste characteristics) of organoleptic attribute.

Similarly, the nano-particle of the present invention improves the bioavailability of the BAC of enteral, protects described BAC to avoid stomach The infringement of portion's acid condition and promote BAC to dissolve and release at enteral.

The nano-particle of the present invention can suspend in an aqueous medium so that BAC avoids degrading in dissolving.They are also BAC can be maintained to be in stable condition and can store for a long time (especially presented in dried powder Ground, is incorporated in solid food product).

It addition, the nano-particle of the present invention applies also for preparing the cosmetics for local application and pharmaceutical composition.

Therefore, on the other hand, the present invention relates to a kind of compositions, hereinafter referred to as " compositions of the present invention ", including at least A kind of nano-particle of the present invention；In the specific embodiment of one, the nano-particle of the present invention for include casein substrate, Basic amino acid and the nano-particle of the metal selected from divalent metal, trivalent metal and combinations thereof；Specifically implement at another kind In mode, the nano-particle of the present invention is the load nano-particle of the present invention, i.e. includes casein substrate, basic amino acid, choosing From the metal of divalent metal, trivalent metal and combinations thereof with have nutritious, treatment and/or cosmetic activity BAC, and medicine On or cosmetically acceptable carrier or be applicable to the nano-particle of carrier of food.

In the specific embodiment of one, described BAC select free amino acid, antimicrobial, aromatizer, preservative, Sweeting agent, steroid, medicine, hormone, lipid, peptide, polynucleotide, polysaccharide, protein, Dan Baiduotang proteoglycan PG, aromatic, vitamin and In the group of its mixture composition.

In the specific embodiment of one, described BAC is water solublity BAC, preferably acidic water-soluble BAC.Water solublity The nonrestrictive illustrative example of BAC includes vitamin, such as vitamin B group or C vitamin and derivant thereof, salt or Ester；Hyaluronic acid, chondroitin sulfate, thioctic acid, its salt or ester, etc..In the specific embodiment of one, described water solublity BAC select free folic acid, PABA, nicotinic acid, pantothenic acid, monophosphate thiamine, diphosphothiamine, thiamine triphosphate, Ascorbic acid, pteroyl polyglutamic acid (folic acid derivatives: folic acid poly-glutamic acid salt；Poly-glutamic acid folate), folic acid, nicotinic acid, Hyaluronic acid, thioctic acid, p-coumaric acid, caffeic acid, it is pharmaceutically or cosmetics are acceptable or the derivant of food stage, In ester or salt, and the group of mixture composition.

In the specific embodiment of another kind, described BAC is fat-soluble BAC.The nonrestrictive explanation of fat-soluble BAC The example of property includes vitamin, such as A race, D race, E race, K vitamin and derivant thereof, phospholipid, carotenoid (Radix Dauci Sativae Element, lycopene, phylloxanthin, capsanthin, cryptoxanthin etc.), omega-3 fatty acid (such as DHA, EPA etc.), aminoacid is (such as Isoleucine, leucine, methionine, phenylalanine, tryptophan and valine), plant sterol and phytosterol (such as paddy steroid Alcohol, campesterol, stigmasterol etc.), polyphenol (such as Quercitroside, rutin, resveratrol, nimbecetin, myricetin, isorhamnetin Deng) and derivant.

In the specific embodiment of one, the compositions of the present invention is a kind of medicine being applicable to and using with topic route Compositions；To this end, described compositions includes pharmaceutically acceptable carrier, it includes that one or more are applicable to topic route The excipient used, such as with the form of gel, ointment, Emulsion etc..About being applicable to the medicine that is intended to use with topic route The information of the excipient of composite preparation and the manufacture of described pharmaceutical composition can be found in books " Tratado de Farmacia Gal é nica ", C.Faul í i Trillo, the 10th edition, 1993, Luz á n 5, S.A.de Ediciones.This The application dosage of bright nano-particle can have wide in range scope, such as, comprise 0.1% to 30%, preferably 0.5% to 5% this The compositions of the present invention of bright nano-particle is about the area that 0.5(is pending, g/cm²) to area pending for about 2(, g/ cm²).

In the specific embodiment of another kind, the compositions of the present invention is the cosmetics being applicable to use with topic route Compositions；To this end, described compositions includes acceptable carrier used for cosmetic, it includes that one or more are applicable to way, local The excipient that footpath is used, such as with the form of gel, Emulsion, shampoo, washing liquid etc..About being applicable to be intended to execute with topic route The excipient of cosmetic composition preparation and the information of manufacture of described cosmetic composition see books " Manual De Cosmetolog í a ", Octavio D í ez Sales, the 1st edition, 1998, Editorial Videocinco, S.A..

In the specific embodiment of another kind, the compositions of the present invention is food compositions, such as solid, liquid or Semi-solid food products goods.

In the specific embodiment of one, the compositions of the present invention includes:

Casein, 10wt% to 50wt%；

Folic acid, 0.9wt% to 2.5wt%；

Calcium, 1wt% to 6wt%；With

Basic amino acid, 1wt% to 7wt%；With

Saccharide, 30wt% to 80wt%,

The most all of percentage by weight is all in terms of the gross weight of said composition.

In the specific embodiment of another kind, the compositions of the present invention includes:

Casein, 10wt% to 50wt%；

Folic acid, 0.9wt% to 2.5wt%；

Calcium, 1wt% to 6wt%；With

Basic amino acid, 1wt% to 7wt%；

Saccharide, 20wt% to 55wt%；And

Ascorbic acid, 1wt% to 25wt%,

The most all of percentage by weight is all in terms of the gross weight of said composition.

Alternatively, the compositions of the present invention can be combined in food；Therefore, on the other hand, the present invention relates to one Food including the present composition.Described food can be presented in liquid, semisolid or solid.Advantageously, in order to keep away Exempting from or minimize all or part of stability dissolved and thus improve them of nano-particle of the present invention, described food has There is acid ph value, i.e. less than 7, preferably lower than or equal to 6, more preferably less than or equal to 5.Rich in the present composition or utilization The illustrative example of food of present composition strengthening includes milk and derivant (Yoghurt, cheese, curdled milk etc.) thereof, really Juice, fruit jam, baked goods and cake goods, fermented meat, flavouring agent etc..Being similar to, the compositions of the present invention can be incorporated into In animal food, such as in feedstuff.

Specific embodiment

Following embodiment describes the manufacture of casein particle, and this casein particle can binding bioactive wherein Compound, particularly folic acid.Casein particle can protect compound to avoid to experience in food and depend on carrying above And the degraded of many factors.Described embodiment also illustrates that these nano-particle are absorbed protection folic acid afterwards at it and avoid stomach Environmental effect and the ability discharged to intestines medium.

Manufacture the basic skills of empty casein nano-particle

The method manufacturing casein nano-particle includes sodium caseinate (ANVISA, Madrid, Spain) and makes a reservation for The basic amino acid of amount dissolves in an aqueous medium together, next adds scheduled volume under conditions of magnetic agitation and continuous stream Calcium solution, formed and there is the nano-particle of milk-white coloured suspension outward appearance.

The Physico-Chemical Characterization of nano-particle

Various researchs needed for obtaining whole Physico-Chemical Characterizations of this nano-particle are as described below.

The granularity of nano-particle and surface charge are detected by physical chemistry and measure, and the latter is come by the measurement of electrokinetic potential Determine.First parameter is obtained by Photon Correlation Spectroscopy, and it uses Zetasizer nano Z-S(Malvern Instruments/Optilas, Spain), wherein this electrokinetic potential uses Zeta potential analyser (Brookhaven Instruments Corporation, New York, the U.S.) measure.

The method forms the productivity of nano-particle by remaining free caseic the most next after obtaining nano-particle Calculating, it is collected in the supernatant being centrifuged said preparation and obtain (17,000x g, 20 minutes).Thus, estimate in system The caseic amount forming granule in agent is, initial addition and the difference of the amount in the supernatant of collection in purification step Value.It is described that quantitatively by ultraviolet (UV) spectrometry at 282nm, (Agilent 8453, the visible spectrum of ultraviolet light is surveyed Determine system) carry out.Productivity estimation is as follows:

Productivity (%)=[(total milligram number of caseinate-caseinate milligram number) in the supernatant/caseinate Total milligram number] × 100

[equation 1]

In order to carry out different calculating, employ 150 to 1, the standard curve (R between 500 μ g/mL²=0.9992；LD =36μg/mL；LQ=119 μ g/mL).

Additionally, the bead obtained after quantitative study is centrifugal, to determine the total casein obtained and to be included in supernatant Casein between the result of difference.In this case, use the NaOH of 0.05M to degrade this granule, this be with for Prepare the medium that this standard curve is identical.Therefore, in this case, productivity estimation is as follows:

Productivity (%)=[total milligram number of (the milligram number of the caseinate in bead)/caseinate] × 100

[equation 2]

The absorption maximum of the caseinate prepared in the medium is positioned at 300nm.For building the concentration model of normal line Enclose and can also is that 150 to 1,500 μ g/mL (R²=0.9996；LD=26μg/mL；LQ=85μg/mL).

The form of this nano-particle is observed by scanning electron microscope (Zeiss, DSM 940A Germany).For This, the nano-particle of this lyophilizing is coated with the golden molecular layer (Emitech K550Team, Sputter-Coater, Britain) of 9nm, And utilize Zeiss DMS 940A microscope (U.S.) that it is taken a picture.

Manufacture the basic skills of the casein nano-particle comprising folic acid

The manufacture method of the casein nano-particle comprising folic acid includes the basic amine group of sodium caseinate Yu scheduled volume Acid is dissolved in an aqueous medium together, the most under magnetic stirring by the folic acid solution of scheduled volume prepared before and predetermined The basic amino acid of amount adds to aqueous medium.After hatching this mixture a few minutes, last step includes adding calcium Salt, produces the nano-particle with slightly yellow cream outward appearance.

Optionally, the nano-particle formed can carry out high hydrostatic pressure treatment (Stansted Fluid Power, ISOLAB Model FPG11500B110;Series No.:7844), within the time cycle of 1 to 5 minute 100 to Under the pressure of 800MPa, so that they are stable.

Afterwards, and after carrying out 3 minutes homogenizing to it by stirring, add in the case of not stopping stirring The sugar juice (lactose, trehalose, mannitol, glucose, Sorbitol, maltodextrin or maltose) of scheduled volume.Finally, This suspension carries out lyophilizing or at spray dryer (B ü chi Mini Spray Drier B-191, B ü chi Labortechnik AG, Switzerland) inherent following under conditions of spray:

-air inlet temperature: 60-100 DEG C

-air outlet temperature: 30-90 DEG C

-air pressure: 2-10bar [2-10x 10⁵Pa]

-sample pump transmission rate: 2-9mL/min

-suck: 30-100%

-air velocity: 200-900L/h

It is optionally possible to utilize the mode of lyophilizing rather than the mode of spray drying that said preparation is carried out after adding sugar It is dried.

The mensuration of the folate content relevant to casein particle

The folate content relevant to this nano-particle is according to Faye [Faye Russell, L., Quantitative Determination of Water-Soluble Vitamins.In Food Analysis by HPLC,Nollet, L.M.L. (Ed.), Marcel Dekker, Inc., New York, second edition, the 10th chapter (2000) page number 444-445] describe Method is determined by high performance liquid chromatography (HPLC).This analysis is being connected to the model of diode array UV detecting system 1100LC palette spectrometer (Agilent, Waldbornn, Germany) performs.Profit in Hewlett-Packard computer Data analysis is carried out with Chem-Station G2171 software.In order to separate folic acid, it is heated to the Alltech of 40 ° of C C18Alltima^TMPost (5 μm, 150mm x 2.1mm) and compatibilityC18AJO-7596 post is used together.Flowing phase By H₃PO₄The gradient mixture (table 1) of (33mM, pH 2.3)/acetonitrile forms, and it pumps under the speed of 0.25mL/min. Examinations under 290nm.Sample feeding volume is 10 μ L.Folic acid retention time is 22.6 ± 0.5 minutes.

Table 1

Gradient condition (the A:H of flowing phase₃PO₄33mM, B: acetonitrile)

Before this sample of quantitative analysis, preparation is between the normal line of the variable concentrations of 2 to 400 μ g/mL, it is thus achieved that be more than Accurately and the accurately result of 95%, and confirm that the casein in solution and/or amino acid whose existence are correct without interference with folic acid Quantitative analysis.

For the analysis (before being dried them) of fresh sample, the supernatant obtained after filtering the preparation of predetermined Liquid passes through300,000MWCO dialysis tube (VIVASPIN 2, Sartorius stedim Biotech, Germany) comes Quantitative analysis.Then during bead is dissolved in the NaOH of 0.05M thus degradation particles, and maintain casein and folic acid and aminoacid Carry out quantitative analysis in the solution and thus to it.The total content of the folic acid in two parts (supernatant and bead) is always Consistent with the total amount being initially added.Furthermore, it is also possible to quantitatively divide by dissolving the preparation of 1mL in the 0.05MNaOH of 1mL Analysis folic acid total amount.This has determined in the case of all, and the amount of the folic acid of interpolation is determined with the chromatography described by utilization Component analysis thus difference between the amount of folic acid that obtains are all more than 10%.

Additionally, use the nano-particle of 10mg that this powder sample is carried out quantitative analysis；They are re-suspended at 2mL Water in and be centrifuged, obtain fresh sample the most in the same way.

From this nano-particle, the release dynamics research of folic acid is discharged in Gl tract medium

The release dynamics of folic acid is discharged by disperseing about 10mL in the simulation stomach medium of 2mL from this nano-particle (0 to 2h) (USP XXIII) nano-particle, measures at 37 ± 1 DEG C.This nano granule suspension is centrifuged certain time (17, 000x g, 20 minutes), and carry out the folate content in quantitative analysis supernatant by aforesaid HPLC method.From stomach After medium removes this supernatant, at 37 ± 1 DEG C, add intestines medium (2 to 24h) (USP XXIII) of simulation, afterwards Proceed in the way of identical with case above.

Calculating the percentage composition of the folic acid of release of all of time period, each research is all in view of being present in said preparation Vitamin total amount.

The bioavailability of the folic acid in casein nano-particle was studied-was encapsulated in dynamic medicine generation

Regulation according to Ethics Committee of medical institutions (Institution Ethics Committee) and about reality The European Law (86/609/EU) testing animal carries out the dynamic research of medicine generation.To this end, average weight is 200g 25 male DABAI Mus experiences in normal bright-dark condition (12 hours-12 hours), and the week age before research, on-demand nursing folic acid Not enough diet (Folic Acid Deficient Diet.TD.95247.Harlan, USA) and water.Before using said preparation 12 hours, rat was isolated in metabolic cage, does not carries out feeding fasting to it, but can freely drink water.

These animals are divided into 5 process groups (often 5 rats of group).First group, only use 1mL's by oral route PBS(phosphate buffered solution, pH is 7.4).Ensuing three groups merely with the 1mg/kg(200 every Mus of μ g) be combined under arbitrarily The oral dose stating the folic acid in preparation processes: (i) the folic acid that dissociates (encapsulating) (Aditio, Panreac Qu í mica, Barcelona, Spain)；(ii) it is encapsulated with the casein nano-particle of folic acid；(iii) with the folic acid of being encapsulated with of HIGH PRESSURE TREATMENT Casein nano-particle.Every kind of different preparation of the 1mL being dispersed in water is intubated by stomach esophagus to be used.Finally, dissolve The free folic acid (1mg/kg) of the same dose in normal saline serum (0.5mL) is applied to the 5th group, and it passes through vein Injection enters saphena.

Before using said preparation, extract blood from the saphena of afterbody thus check the basic vitamin water of every rat Flat.After using, utilize serum separator tube (SARSTEDT Microtube 1.1mLZ-Gel) at different decimation in time about 500 μ The blood of L volume.In the case of all, suction-type anesthesia (isoflurane: oxygen) is utilized to extract blood again after making animal sleep Liquid, to prevent rat from experiencing pain, checks the state of rat at any time.

It follows that blood volume is by the 500 μ L normal saline blood serum substitutings used to intraperitoneal, it is heated to move before it The temperature of thing.In the meantime, check the situation (mobility, aggressivity, allergy and body temperature) of this animal, do not observe aobvious The change write.

The folic acid pretreatment of blood serum sample and quantitative analysis

Folic acid in the blood serum sample obtained after the centrifugal test tube (6,000rpm, 20min, 20 ° of C) with blood Quantitative analysis is performed by the method for Enzyme Immunoassay.To this end, use by FDA ratify for quantitative determining in food Enzyme-linked immunologic detecting kit (Diagnostic automation, INC.Calabasas, California, U.S. of folic acid State).Process this blood serum sample the most in advance and carry out quantitative analysis according to the description of manufacturer.

Because this test kit is to design for food, so carrying out a series of formerly analyzing, thus confirm its energy It is enough in the quantitative analysis of vitamin in blood serum sample.Described analysis include implementing for the result that obtained by this test kit with Carry out detailed comparison between those results obtained by high performance liquid chromatography being described above, have following before Phase set-up procedure: the folic acid of the most commensurability (0-300 μ L) is dissolved in the tetraboric acid of preparation in 1% (w/v) sodium ascorbate of 50mM In sodium solution, it is added into the serum of 50 μ L.The sodium tetraborate solution of the solution and this 50mM that obtain forms 350 μ L Final volume (serum-dilution 1:7).Every kind of mixture is all heated to be boiling for 30 minutes, and is next cooled to 2 ° of C And preserve at said temperatures overnight.

Make the sample of acquisition 20, after being centrifuged 20 minutes and filtered by 20 μm filters under 000rpm, pass through Aforesaid high performance liquid chromatography is used to carry out quantitative analysis folate content.In this case, and due to the vitamin of serum Concentration is low, uses Standard entertion technology minimize the error in quantitative analysis and remove arbitrary matrix interference.

In the case of all researchs, the difference of the Serum Folic Acid Concentration of both approaches is less than 10%.Therefore, enzyme is selected The sample that immuno analytical method quantitative analysis is whole because its need a small amount of serum for analyzing and its be one the simplest Just and the most efficiently technology, detection limit (2ng/mL) is more much smaller than chromatographic technique.

Manufacture the basic skills of the casein nano-particle comprising fat-soluble active substance Quercitroside

The manufacture method of the casein nano-particle comprising Quercitroside includes the alkaline ammonia by sodium caseinate and scheduled volume Base acid is dissolved in an aqueous medium together, adds the ascorbic acid solution of scheduled volume the most under magnetic stirring and adds subsequently Enter the Quercitroside dissolved in advance in ethanol.After hatching this mixture a few minutes, last step includes adding calcium salt, shape Become there is the nano-particle of faint yellow cream outward appearance.

Optionally, the nano-particle of this formation can carry out high hydrostatic pressure treatment (Stansted Fluid Power, ISOLAB Model FPG11500B110;Series No.:7844), within the time cycle of 1 to 5 minute 100 to Under the pressure of 800MPa, so that they are stable.

Afterwards, and after carrying out 3 minutes homogenizing to it by stirring, add in the case of not stopping stirring The sugar juice (lactose, trehalose, mannitol, glucose, Sorbitol, maltodextrin or maltose) of scheduled volume.Finally, This suspension is lyophilized or at spray dryer (B ü chi Mini Spray Drier B-191, B ü chi Labortechnik AG, Switzerland) inherent following under conditions of spray:

-air inlet temperature: 60-100 DEG C

-air outlet temperature: 30-90 DEG C

-air pressure: 2-10bar [2-10x 10⁵Pa]

-sample pump transmission rate: 2-9mL/min

-suck: 30-100%

-air velocity: 200-900L/h

It is optionally possible to utilize the mode of lyophilizing rather than the mode of spray drying that said preparation is carried out after adding sugar It is dried.

The mensuration of the Determination of Quercitrin relevant to casein particle

The Determination of Quercitrin relevant to this nano-particle is according to Lacopini (Lacopini et al., J Food Comp Anal 2008;Method 21:589-598) described carrys out quantitative analysis, although having by high performance liquid chromatography (HPLC) Different.This analysis be connected to the model 1100LC palette spectrometer of diode array UV detecting system (Agilent, Waldbornn, Germany) middle execution.Chem-Station G2171 software is utilized to carry out in Hewlett-Packard computer Analytical data.In order to separate folic acid, it is heated to the Alltech C18Alltima of 40 ° of C^TMPost (5 μm, 150mm x 2.1mm) with CompatibleThe gradient mixture (seeing table 2) of C18AJO-7596 post and water/methanol/glacial acetic acid is used together, mixed Compound pumps under the speed of 0.25mL/min as flowing.Detect under 260nm, sample feeding volume be 10 μ L also And Quercitroside retention time is 24.2 ± 0.2 minutes.

Table 2

The gradient condition (A: water, B: methanol, C: glacial acetic acid) of flowing phase

Before this sample of quantitative analysis, in water alcohol medium, (75% ethanol) preparation is between the variable concentrations of 1 to 100 μ g/mL Normal line, it is thus achieved that the accurately and accurately result more than 95%.

For the analysis (before being dried them) of fresh sample, receive purifying this by filtration (17000rpm, 20min) The supernatant of acquisition is diluted, until obtaining 50%(w/v after the process of rice grain) aqueous solution of ethanol content.

Finally, relevant to this nano-particle Determination of Quercitrin [envelop rate (E.E.)] is by the initial Quercitroside amount added (Q) difference between the amount of quantitative analysis calculates according to following equalities and in supernatant:

Embodiment 1

The preparation of empty casein nano-particle and qualification: obtaining the productivity of their method, used is amino acid whose The type stability for nano-particle and the impact of physicochemical characteristics

The sodium caseinate of 1g is dissolved in the water of 75mL together with the lysine of 90mg.It follows that in magnetic agitation Flow down the CaCl of 40mL 0.8% continuously₂Add to this solution.This process is in triplicate.

Fig. 2 (A and B) is shown through the transmission electron microscope image of the casein particle that the method obtains.

Additionally, there is not aminoacid, or by using the arginine of 50mg to carry out in the case of replacing lysine Identical research, thus understand the impact for the physicochemical characteristics of granule of the amino acid whose type.

Table 3 summarises the Main physical chemical characteristic of the nano-particle of acquisition.

Table 3

The physicochemical characteristics (meansigma methods ± standard deviation, n=10) of casein nano-particle: aminoacid, lysine or

Arginine is respectively 1:11 and 1:20 with the weight ratio of this protein

^aPDI: polydispersity；

^bProductivity: change into the percentage composition of the protein of nano-particle.

Performed statistical research (nonparametric independent sample is checked: rank test) display does not has on statistical significance Significantly evidence proves to there are differences between the physical-chemical parameters of said preparation.It was therefore concluded that, amino acid whose class Type can't affect the described characteristic of the nano-particle of sky.

This identical analysis is performed, it can be deduced that similar knot by the ratio of change aminoacid addition to said preparation Opinion, the most amino acid whose ratio and type all do not interfere with the final response of the granule of this sky.

In order to recognize the stability of said preparation, determine the nano-particle of this three types physics as time goes by Chemical parameters.The result obtained includes in table 4.

Table 4

The physicochemical characteristics of casein nano-particle (meansigma methods ± standard deviation, n=6) as time goes by: ammonia Base acid, lysine or arginine are respectively 1:11 and 1:20 with the weight ratio of this protein

PDI: polydispersity.

When obtaining them, the nano-particle of this three types has the granularity of same order and relatively low polydispersion Property (in view of PDI value less than 0.3, this particle size distribution is uniform).Utilizing in the case of aminoacid preparation nano-particle, passing through Wear these these granularities of whole research and dispersion value does not demonstrate significant change.But, after obtaining both hour, The nano-particle not using aminoacid to prepare all occurs in that in their particle mean size and can not neglect in their polydispersity Depending on increase (for the polydispersion numerical value more than 0.3, this size value is the most representative, and it is only used as a kind of index, Represent that its diameter is the most uneven), terminate to reach the most much higher value afterwards in this analysis.Described increase to The instruction of clustering phenomena is there is between Li.These phenomenons even can be identified on a macroscopic scale, because ought be the most right When these three preparation is observed, have confirmed that not comprise and precipitated by amino acid whose nano-particle, cause the formation of emulsus layer, And the nano-particle utilizing aminoacid to prepare forms uniform suspension.In view of these results, it is believed that amino acid whose existence for Obtain the most stable granule it is critical that.

Additionally, the preparation of this three types is again prepared and is being done it by the method for spray drying technology After dry, the physicochemical characteristics to them is analyzed.The condition of the method is:

-air inlet temperature: 90 DEG C

-air outlet temperature: 49 DEG C

-air pressure: 6bar [6x 10⁵Pa]

-sample pump transmission rate: 4.5mL/min

-suck: 100%

-air velocity: 600L/h

For the purpose of the amino acid whose impact of understanding, perform when this nano-particle is i.e. dried obtaining them when This analysis, because scribing agent does not exist the phenomenon of gathering for the moment at that.The result obtained includes in table 5.

Table 5

By the physical chemistry of the casein nano-particle (meansigma methods ± standard deviation, n=3) that spray dried form is dried Characteristic: aminoacid, lysine or arginine are respectively 1:11 and 1:20 with the weight ratio of protein

By be dried for powder include amino acid whose nano-particle be again suspended in aqueous medium time, it was observed that particle diameter Distribution continue as monodispersity and they particle diameter less times greater than they by be spray-dried and before being dried right The particle diameter answered.But, do not utilize the nano-particle of amino acid ligand to have bigger particle diameter and polydispersity numerical value shows it Be dried during may experience clustering phenomena.Thus, when being dried this granule by the way of spray drying, ammonia The existence of base acid is also required.

In consideration of it, it may be concluded that include the physicochemical characteristics of amino acid whose nano-particle and do not comprise amino The nano-particle of acid is different；They have low gathering tendency and thus said preparation be selected for use in encapsulating bioactive process Compound.

Embodiment 2

Comprise preparation and the qualification of the casein nano-particle of folic acid: lysine and the folate content impact on envelop rate

In the water that final volume is 7.5mL, preparation comprises sodium caseinate and the most commensurability lysine (0-8.5mg) of 100mg Different solutions.

Additionally, the folic acid of 300mg is dissolved in the water of 50mL together with the lysine of 400mg.

It follows that the folic acid solution of 1mL is added to this caseinate solution.After hatching 5 minutes, in magnetic agitation and company By the CaCl of 4mL 0.8% under afterflow₂Add to this mixture.For each type of preparation, this process in triplicate.

Fig. 3 shows the transmission electron microscope figure of the casein particle being encapsulated with folic acid obtained by this method Picture.

The physicochemical characteristics obtained in each case is included in table 6:

Table 6

Include the physicochemical characteristics (meansigma methods ± standard of the casein nano-particle of folic acid and different amounts of lysine Partially

Difference, n=6): this folic acid is 1:17 with the weight ratio of protein

^aBefore adding folic acid solution

FA: folic acid；NP: nano-particle

Performed statistical research (nonparametric independent sample is checked: rank test) display does not has on statistical significance Significantly evidence thinks the physics of these final be included in table three preparations (lysine content is 3.9,4.5 and 8.5mg) Chemical characteristic there are differences.In the first case, although confirming that this folic acid solution has lysine, but at initial casein In saline solution, lack amino acid promotes to include the folic acid moieties precipitation of calcium, which results in the mistake in vitamin quantitative analysis Difference, because the folic acid in not all bead is the most encapsulated.

But when vitamin solution include aminoacid caseinate solution do not comprise aminoacid time, determining additionally The maximum of the folic acid that precipitation does not occurs can being bonded in said preparation is 4mg, is similar in table 6 result (25.5 obtained ± 1 μ gFA/mg NP and envelop rate: 68.7 ± 0.5).It is thus identified that amino acid whose existence can't affect encapsulated dimension life The amount of element.But, because the nano-particle not utilizing aminoacid to prepare is more unstable and has bigger gathering tendency and (sees Embodiment 1), thus exist these amino acid whose in the case of prepare.

Add to the folic acid amount in said preparation for the impact of the physicochemical characteristics of this granule to understand, by only The addition changing folic acid solution carries out identical research, the aminoacid in the case of all, in initial casein solution Content perseverance is: 8.5mg.

Fig. 4 shows the folic acid amount function relative to the vitamin amount added to said preparation of encapsulating.

In all situations of the preparation of research, particle size range is between 132 to 140nm, and polydispersity is less than 0.2. In this embodiment, the numerical value of this envelop rate cannot be carried out comparing, because the folic acid amount added to each preparation is different. For casein: when the weight ratio of folic acid is 13.5:1, maximum is 73.1 ± 7.5.

According to this research as a result, it is possible to reach a conclusion, along with the fall of the milligram casein/milligram F/A ratio in preparation Low (i.e. along with the increase of the initial content added to the folic acid in said preparation), is encapsulated in the amount of folic acid within this nano-particle Will increase.But, when the caseic amount (representing with mg) being present in the said preparation folic acid relative to every mg is less than experiment During value, will observe and such as not deposit the preparation of precipitation and the instability occurred in the case of lysine.

Embodiment 3

By the preparation of the casein nano-particle comprising folic acid being dried and qualification: dry run is to The impact of whole preparation

In the water that final volume is 75mL, preparation comprises the sodium caseinate of 1,000mg and the two of the lysine of 90mg Plant solution.

Additionally, the folic acid of 600mg is dissolved in the water of 100mL together with the lysine of 800mg.

Subsequently, the folic acid solution of 7.5mL is added to each caseinate solution.After hatching 5 minutes, in magnetic agitation Flow down the CaCl of 40mL 0.8% continuously₂Add to this mixture.

Finally, one of centrifugal said preparation, with quantitative analysis folic acid in supernatant and bead, is using spray dried simultaneously Before dry device is dried, by 1, the lactose of 900mg adds to another preparation.The condition of this process is:

-air inlet temperature: 90 DEG C

-air outlet temperature: 45 DEG C

-air pressure: 6bar [6x 10⁵Pa]

-sample pump transmission rate: 4.5mL/min

-suck: 95%

-air velocity: 600L/h

The most viewed physicochemical characteristics includes in table 7.

Table 7

In fresh nano-particle or after being dried it by spray dryer, quantitative analysis has The physicochemical characteristics (meansigma methods ± standard deviation, n=6) of the casein nano-particle of folic acid: lysine in the final formulation With

The weight ratio of protein is 1:7, and this folic acid is 1:22 with caseic ratio

FA: folic acid；NP: nano-particle

Performed statistical research (nonparametric independent sample is checked: rank test) shows the bag that two kinds of preparations are obtained Significant difference (p < 0.05) on statistical significance is there is between envelope rate.This difference is likely due to by being spray-dried in institute The Partial digestion of this casein nano-particle that the method for dry said preparation is caused at a temperature of appointment, thus cause wrapping in advance The part release of the folic acid of envelope.

The explanation of these results needs to implement a kind of method and cross-links this granule, because if doing so, can improve them Stability, and the reduction of envelop rate during aforesaid centrifugal or dry said preparation can be stoped.

Embodiment 4

By high-voltage-stable and be dried by the method for spray drying technology, there is lysine, comprise leaf The preparation of acid casein nano-particle and qualification: this process is for the impact of nano-particle physicochemical characteristics

Preparing solution different in the water that final volume is 75mL, all of solution all comprises 1, the casein of 000mg Acid sodium and the lysine of 90mg.

Additionally, the folic acid of 600mg is dissolved in the water of 100mL together with the lysine of 800mg.

Next the folic acid solution of 7.5mL is added to caseinate solution.After hatching 5 minutes, stir at magnetic force Mix and stir and flow down the CaCl of 40mL 0.8% continuously₂Add to this mixture.

Once this granule is formed, and is just transferred to by said preparation in the plastic bag sealed and carries out high hydrostatic pressure treatment (0MPa; 100MPa, 5 minutes;200MPa, 5 minutes;400MPa, 5 minutes;600MPa, 5 minutes, or 800MPa, 5 minutes).

Once this process terminates, and is just dissolved in 1 in water, and the lactose of 900mg adds to each preparation and following Under conditions of use spray drying technology perform preparation is dried:

-air inlet temperature: 85 DEG C

-air outlet temperature: 45 DEG C

-air pressure: 6bar [6x 10⁵Pa]

-sample pump transmission rate: 4.5mL/min

-suck: 95%

-air velocity: 600L/h

Table 8 summarises the Main physical chemical characteristic of obtained nano-particle.

Table 8

Include folic acid and carry out different HIGH PRESSURE TREATMENT casein nano-particle physicochemical characteristics (meansigma methods ± Standard deviation, n=6): the final weight between lysine and casein is than for the weight between 1:7, and folic acid and casein Ratio is 1:22

FA: folic acid；NP: nano-particle

As in table 8 it is observed that, do not consider to apply the type of the process to said preparation, this nano-particle has closely As surface charge.But, this Data Detection increases to along with the processing pressure that applied, it is thus achieved that granularity become less, reach To the reduction of maximum 7%.But, the amount (and envelop rate thus) of the vitamin encapsulated reaches along with executing stressed increase To bigger numerical value, the increase obtaining 65% relative to the preparation being not handled by (carries out, using 800MPa, the sample processed In).

Do not carry out the preparation of HIGH PRESSURE TREATMENT additionally, Fig. 5 shows and utilize at the pressure of 100,400 and 800MPa The microscopical microgram of Scanning Electron that the preparation of reason is obtained.They demonstrate the nano-particle not carrying out high hydrostatic pressure treatment How being by different process changes, these processes process of experience after being to obtain them (is carried out by spray drying It is dried, is centrifuged, carries out microphotograph at high temperature), and the nano-particle experiencing different HIGH PRESSURE TREATMENT is more stable.

The high hydrostatic pressure treatment that the explanation of these results is applied has cross-linked this nano-particle so that they are more stable, and And thus prevent them to degrade after being centrifuged, be dried and taking a picture.All these explains and why is being processed Sample in obtain bigger envelop rate, because using the part of the nano-particle being dried or in centrifugal method at some Degraded will cause the release of folic acid and be derived from relatively low envelop rate.

Embodiment 5

Use high pressure, be dried by spray drying, there is arginine, include the casein nano-particle of folic acid Preparation and qualification: the aminoacid the used impact on final result

In the water that final volume is 210mL, preparation comprises the sodium caseinate of 3,065mg and the arginic molten of 123mg Liquid.

Additionally, the folic acid of 605mg is dissolved in the water of 100mL together with the arginine of 800mg.

Next the folic acid solution of 27mL is added to caseinate solution.After hatching 5 minutes, in magnetic agitation and company By the CaCl of 120mL 0.8% under afterflow₂Add to this mixture.

Once form granule, just said preparation is transferred in the plastic bag sealed and carries out the cycle time by 5 minutes and The high hydrostatic pressure treatment of the stress structure of 400MPa.

Once this process terminates, and is just dissolved in 5 in water, and the mannitol addition of 880mg passes through HIGH PRESSURE TREATMENT to 300mL Preparation and under conditions of following use spray drying technology perform it is dried:

-air inlet temperature: 85 DEG C

-air outlet temperature: 45 DEG C

-air pressure: 6bar [6x 10⁵Pa]

-sample pump transmission rate: 4.5mL/min

-suck: 95%

-air velocity: 600L/h

The Main physical chemical characteristic of the preparation of acquisition is summarised in table 9.

Table 9

Including arginine and folic acid, the casein that the method by HIGH PRESSURE TREATMENT and by spray drying is dried is received The physicochemical characteristics (meansigma methods ± standard deviation, n=6) of rice grain: the final weight ratio between arginine and protein is 1: 9,

And the weight ratio between folic acid and casein is 1:19

FA: folic acid；NP: nano-particle

Fig. 6 is shown under 400MPa and processes 5 minutes include folic acid and have arginic cheese in their preparation Scanning electron microscope (SEM) microgram of protein nano granule.

There it can be seen that the preparation obtained has the nano-particle being similar to use lysine for arginine to be obtained Characteristic.

Embodiment 6

Gl tract medium discharges from this nano-particle folic acid release dynamics research: HIGH PRESSURE TREATMENT for The impact of release dynamics

In order to carry out this releasing research, use describe in example 4 powder formulation (unused HIGH PRESSURE TREATMENT, Process under 100MPa and 400MPa).

Fig. 7 shows not by obtained release dynamics in the case of the sample of HIGH PRESSURE TREATMENT.Wherein, it can be seen that After hatching two hours in stomach medium, reach the maximum folic acid release value of 4%.But, in intestines environment, dissolving The percentage composition of the vitamin of casein particle release improves and increases (in this research within the 24th hour, reach 90%).And, at this In medium, hatching sample centrifugal afterwards and the most do not have casein bead, casein bead at the dissolved state of sample is It will be apparent that and thus discharge this vitamin.Therefore, it can be seen that designed preparation causes encapsulated folic acid to pass through stomach Portion, prevents stomach environment from reducing its bioavailability.Additionally, this nano-particle dissolves at enteral, promote the release of vitamin also Eliminate the toxicity problem that any existence being likely to be due to nano-particle causes.

In the case of by this sample of HIGH PRESSURE TREATMENT, Fig. 8 (A and B) shows the release dynamics of sample.Wherein, may be used To find out that its curve is similar with the sample not carrying out HIGH PRESSURE TREATMENT, maximum after 6 hours in simulation intestines medium Release percentage composition (70%) is slightly less than not handled by the sample content (80%) in this moment.

Thus, this casein nano-particle is applied high hydrostatic pressure not significantly change from this for their crosslinking The curve of the component discharged of casein nano-particle, although the total amount of the vitamin discharged after 6 hours reduces 10%。

Embodiment 7

Medicine generation dynamic learning of the folic acid being encapsulated in casein nano-particle is studied

Table 10 summarises in the medicine generation dynamic Main physical chemical characteristic learning the nano-particle tested in research: two kinds of Nano-particle (utilize or do not utilize HIGH PRESSURE TREATMENT) obtains according to the method described in embodiment 5.

Table 10

In the medicine generation dynamic physicochemical characteristics (meansigma methods learning the casein nano-particle including folic acid used in research ± standard deviation, n=6)

FA: folic acid；NP: nano-particle；Cas NP FA: be encapsulated with the casein nano-particle of folic acid；Cas NP FA The casein nano-particle being encapsulated with folic acid of HP: HIGH PRESSURE TREATMENT (400MPa, 5 minutes).

This medicine generation dynamic research is divided into three phases.First stage includes that what intravenous injection used 1mg/kg is dissolved in phosphorus Folic acid in hydrochlorate buffer solution；Second stage includes the phosphate buffered solution to 5 Orally administered 1mL of male rat group (in this group rat, research basic vitamin level over time).Finally, the phase III includes forming by 5 animals The folic acid being (i) dissolved in water of the Orally administered 1mg/kg of rat group, be (ii) encapsulated in the folic acid in casein nano-particle, (iii) utilize the folic acid being encapsulated in casein nano-particle of HIGH PRESSURE TREATMENT.

After administration, extract the blood of about 500 μ L volumes in the different time (0,1,2,3,8 and 24 hours) and be collected in blood Sorting, in pipe, next recovers the blood volume of this animal with the volume of the saline serum being equal to by lumbar injection.Execute Put down with the medicine generation of the data obtained after the folic acid dynamic analysis and utilization WiNNonlin 1.5 medicine generation dynamic non-chamber learning adjustment Program of learning The method of difference implements (Pharsight Corporation, Mountain View, the U.S.).

The result (after deducting this foundation level) obtained shown in Fig. 9.As being observed, the vein of folic acid Use (Fig. 9 A) and be shown in first sample the serum drug level peak value absorbed, the drastically fall of following serum level Low.When vitamin be the curve (Fig. 9 B) by obtaining time Orally administered be different because this concentration maxima is significantly lower； The time that they occur is longer and reduces with more progressive manner.But, (do not wrapped in a free form Orally administered Envelope) or it is encapsulated in casein nano-particle the vitamin water carried out after the folic acid of (carry out or do not carry out HIGH PRESSURE TREATMENT) Flat comparison, sets up the concentration curve at similar times point, but when using the vitamin of encapsulating, this maximum is bigger.

Include at the medicine generation dynamic numerical value learning parameter performing to be obtained after the non-compartment analysis of the experimental data of this research In table 11.

Table 11

The pharmacokinetic parameter (meansigma methods ± standard deviation, n=5) of different preparations to be tested

^*< 0.05vs. does not encapsulates folic acid to p.Graceful Whitney U checks.

^**< 0.01vs. does not encapsulates folic acid to p.Graceful Whitney U checks.

AUC: area under serum concentration profile

C_max: Cmax

T_max: reach C_maxTime

MRT: mean residence time

F_R: the relative bioavailability represented with percentage composition.

It is observed that AUC has significant change according to the preparation type used.When vitamin is encapsulated in Time in casein nano-particle, this AUC is significantly more than and and ties up the most further after using free folic acid Hold the AUC reaching 24 hours total time.It is observed that the mean residence time of blood plasma Folic Acid (MRT) is in both nanometers Granular preparation is similar, and if compared with free form (oral and intravenous injection), mean residence time more becomes.

According to these results, calculate the oral bioavailability of the casein nano-particle being encapsulated with folic acid in two kinds of preparations Degree is 52%, and it is more than 45% of the value by obtaining after Orally administered free folic acid.

Embodiment 8

Utilize the cosmetic formulations [1] of the casein nano-particle being encapsulated with folic acid

In the water that final volume is 15mL, preparation comprises the sodium caseinate of 200mg and the solution of the lysine of 18mg.

Additionally, the folic acid of 600mg is dissolved in the water of 100mL together with the lysine of 800mg.

Subsequently, this folic acid solution of 1.5mL is added to this caseinate solution.After hatching 5 minutes, in magnetic agitation Flow down the CaCl of 8mL 0.8% continuously₂Add to this mixture.

Finally, 17, centrifugal said preparation 20 minutes under 000x g.Abandon supernatant, and this bead is resuspended in the water of 25mL.

Additionally, preparation comprises 7g glycerol and the solution of 0.2g Sodium Paraben in the water of 42mL.By this solution at water Being heated to 50 DEG C in bath, and be subsequently added the aqueous solution of the casein nano-particle comprising folic acid, this cosmetic formulations will utilize Prepared by the final aqueous solution obtained.

Additionally, the Neo PCL O/W of 25g is also heated at 70 DEG C, until it is completely melt.Once this oil phase is melt Changing, just adding this aforesaid aqueous solution under constant stirring until obtaining O/W emulsion, it is appropriate over time and is steady Fixed.The sensory evaluation of the Emulsion obtained is positive, and it has the outward appearance of homogenizing and will not lump.

This identical research also utilizes by HIGH PRESSURE TREATMENT (400MPa, 5 minutes) and by institute the most in example 4 State the nanoparticle formulations that spray dryer is dried to perform.Measure the said preparation of 600mg and be resuspended in 25mL Water in, continue after this to carry out in the same way as described above.

The Emulsion obtained also has the outward appearance of homogenizing and will not lump.

Embodiment 9

Utilize the cosmetic formulations [2] of the casein nano-particle being encapsulated with folic acid

Preparation comprises the sodium caseinate of 200mg and the solution of the lysine of 18mg in the water that final volume is 15mL.

Additionally, the folic acid of 600mg is dissolved in the water of 100mL together with the lysine of 800mg.

Subsequently, this folic acid solution of 1.5mL is added to each caseinate solution.After hatching 5 minutes, stir at magnetic force Mix and stir and flow down the CaCl of 8mL 0.8% continuously₂Add to this mixture.

Finally, 17, centrifugal said preparation 20 minutes under 000x g.Abandon supernatant, and this bead is resuspended in the water of 25mL.

Additionally, the Carbopol Ultrez 10 of 0.5g is dissolved in the water of 75mL.By the suspension of this nano-particle Add to this solution.Once this mixture homogenizes, and just adds the trimethylamine of sufficient quantity until the pH value obtained is 10.This mixes Compound is homogenised until obtaining uniform and stable, slightly the 934 P gel of jaundice.

This identical test also utilizes by HIGH PRESSURE TREATMENT (400MPa, 5 minutes) and by institute the most in example 4 State the nanoparticle formulations that spray dryer is dried to perform.Measure the said preparation of 600mg and be resuspended in 25mL Water in, continue to state identical method above after this and carry out.The gel obtained also have slightly send out yellow with And homogenizing and stable outward appearance.

Embodiment 10

Utilize the cosmetic formulations [3] of the casein nano-particle being encapsulated with folic acid

The glyceryl monostearate of 3g is mixed with the isopropyl myristate of 5g and the spermol of 2g.The water-bath of 70 DEG C This mixture of middle heating.

Additionally, the 934 P the including as described in embodiment 8 of 87g with the casein nano-particle of folic acid coagulates Glue is heated to 50 DEG C in a water bath together with Pyrusussuriensis (sugared) the alcohol liquid of 3g.This solution is added to this aforementioned liquids, Gently stirring is until obtaining uniform emulsion.

Embodiment 11

Comprise preparation and the qualification of the casein nano-particle of Quercitroside

In the water of 7.5mL, preparation comprises lysine (or the smart ammonia of 5.5mg of the sodium caseinate of 100mg and 8.5mg Acid) solution.

Additionally, preparing concentration in water is the sodium ascorbate solution of 12mg/mL, this solution of 0.5mL is added to being somebody's turn to do In caseinate and lysine mixture.The reason using sodium caseinate is to prevent Quercitroside from obtaining this nano-particle process In oxidation.

Additionally, the Quercitroside of 50mg is dissolved in the ethanol of 5mL.

It follows that the Quercitroside solution of 0.15mL is added to this caseinate solution.After hatching 5 minutes, stir at magnetic force Mix and stir and flow down the CaCl of 4mL 0.8% continuously₂Add to this mixture.This process of each type of preparation is all repeated Three times.

Table 12 is included in the physicochemical characteristics obtained in every kind of situation.

Table 12

There is the physicochemical characteristics (meansigma methods ± mark of the casein nano-particle of Quercitroside, aminoacid and ascorbic acid Quasi-deviation, n=3): the weight ratio of Quercitroside and protein is 1:67；The weight ratio of Quercitroside and ascorbic acid is 1:34

Q: Quercitroside；NP: nano-particle

The nano-particle of the result display present invention obtained applies also for encapsulating the biological activity with fat-soluble characteristic Compound and high encapsulation rate percentage ratio can be obtained.

Additionally, one or another kind of amino acid whose existence of this results verification can't affect the life of obtained nano-particle Thing chemical characteristic.

In order to increase the Determination of Quercitrin of encapsulating, use lysine as this aminoacid and different amounts of Quercitroside (the ethanol Quercitroside solution of 0.05 to 0.50mL) repeats this research.Obtained result is included at table 13.

Table 13

The physicochemical characteristics with the casein nano-particle of lysine, ascorbic acid and the most commensurability Quercitroside is (average Value ± standard deviation, n=3): the weight ratio of ascorbic acid and protein (casein) is 1:17

Q: Quercitroside；NP: nano-particle

The result obtained shows that the Determination of Quercitrin of this encapsulating is with phase along with Determination of Quercitrin increase in said preparation Same ratio increases, and this envelop rate remains constant simultaneously.

It addition, carry out the test according to method as described above, but Quercetin is being added to this caseinate solution Before in, Quercetin is dispersed in water (substitute and dissolve in ethanol).The result display part Quercetin obtained is encapsulated in In this casein nano-particle, although this envelop rate is molten by it less than before being added to this caseinate solution by Quercetin Envelop rate in solution afore-mentioned in ethanol.

Claims (35)

1. a nano-particle, including casein substrate, basic amino acid and selected from divalent metal, trivalent metal and combinations thereof Metal, it is characterised in that basic amino acid is positioned at nano-particle interiorly or exteriorly, and it is attached to the surface of nano-particle, institute State nano-particle and also comprise water-soluble bioactive compound.

Nano-particle the most according to claim 1, wherein said basic amino acid choosing free arginine, lysine, group ammonia In the group of acid and mixture composition thereof.

Nano-particle the most according to claim 1, wherein said divalent metal selects free calcium, magnesium, zinc, ferrous iron and group thereof In the group being combined into.

Nano-particle the most according to claim 3, wherein said divalent metal is calcium.

Nano-particle the most according to claim 2, wherein said divalent metal selects free calcium, magnesium, zinc, ferrous iron and group thereof In the group being combined into.

Nano-particle the most according to claim 5, wherein said divalent metal is calcium.

Nano-particle the most according to claim 1, the following composition of wherein said water-soluble bioactive compound choosing Group in:

A) vitamin B group or C vitamin；

B) according to vitamin derivative a)；

C) selected from hyaluronic acid, chondroitin sulfate and the compound of thioctic acid；

D) arbitrary aforesaid compound a) is to salt c) or ester；And

E) a combination thereof.

Nano-particle the most according to claim 1, wherein said water-soluble bioactive compound choosing free folic acid, 4-ammonia Yl benzoic acid, pantothenic acid, monophosphate thiamine phosphoric acid, diphosphothiamine, thiamine triphosphate, ascorbic acid, pteroyl poly paddy Propylhomoserin, folinic acid, nicotinic acid, hyaluronic acid, thioctic acid, p-coumaric acid, caffeic acid, its pharmaceutically or cosmetics are acceptable or In the derivant of person's food stage, ester or salt, and the group of mixture composition.

9. the method manufacturing nano-particle, described nano-particle includes casein substrate, basic amino acid, water-soluble biological Reactive compound and the metal selected from divalent metal, trivalent metal and combinations thereof, described method includes:

A) preparation comprises casein raw material and basic amine group aqueous acid；With

B) aqueous solution of the metal selected from divalent metal, trivalent metal and combinations thereof is added to the solution of step a).

10. the method manufacturing nano-particle, described nano-particle includes casein substrate, basic amino acid, and water solublity is raw Thing reactive compound, selected from metal and the bioactive compound of divalent metal, trivalent metal and combinations thereof, described method bag Include:

A) mixing (i) contains casein raw material and the first basic amine group aqueous acid and (ii) contains bioactive compound Solution；With

B) in the mixture that step a) obtains, add the aqueous solution of the metal selected from divalent metal, trivalent metal and combinations thereof.

11. include sodium caseinate according to the method described in claim 9 or 10, wherein said casein raw material.

12. according to the method described in claim 9 or 10, wherein said basic amino acid choosing free arginine, lysine, group ammonia In the group of acid and combinations thereof composition.

13. according to the method described in claim 9 or 10, and wherein said metal is divalent metal, its select free calcium, magnesium, zinc, two In the group of valency ferrum and combinations thereof composition.

14. methods according to claim 13, wherein said metal is calcium.

15. according to the method described in claim 9 or 10, the wherein said gold selected from divalent metal, trivalent metal and combinations thereof The aqueous solution that aqueous solution is calcium salt belonged to, this calcium salt choosing free calcium chloride, calcium acetate, calcium gluconate, calcium lactate, sorbic acid In the group of calcium, calcium ascorbate, calcium citrate, calcium propionate, calcium sulfate and mixture composition thereof.

16. according to the method described in claim 9 or 10, and the choosing of wherein said water-soluble bioactive compound is following to be constituted Group:

A) vitamin B group or C vitamin；

B) according to vitamin derivative a)；

C) selected from hyaluronic acid, chondroitin sulfate and the compound of thioctic acid；

D) arbitrary aforesaid compound a) is to salt c) or ester；And

E) a combination thereof.

17. methods according to claim 16, wherein said water-soluble bioactive compound choosing free folic acid, 4-amino Benzoic acid, pantothenic acid, monophosphate thiamine, diphosphothiamine, thiamine triphosphate, ascorbic acid, pteroyl polyglutamic acid, Asia Folic acid, nicotinic acid, hyaluronic acid, thioctic acid, p-coumaric acid, caffeic acid, pharmaceutically or cosmetics are acceptable or food for it Derivant, ester or the salt of level, and the group that mixture is constituted.

18., according to the method described in claim 9 or 10, farther include the suspension experience making to comprise formed nano-particle At least one hydrostatic pressure circulates, and described pressure is 100 to 800MPa.

19., according to the method described in claim 9 or 10, farther include the suspension comprising formed nano-particle described in being dried Liquid.

20. methods according to claim 19, being dried protectant of the wherein said suspension comprising nano-particle In the presence of and optionally perform in the presence of antioxidant.

21. methods according to claim 20, wherein said protective agent is saccharide.

22. methods according to claim 20, wherein said antioxidant includes vitamin C.

23. 1 kinds of nano-particle, including casein substrate, basic amino acid, water-soluble bioactive compound and selected from bivalence The metal of metal, trivalent metal and combinations thereof, it is obtained by method according to claim 9.

24. 1 kinds of nano-particle, including casein substrate, basic amino acid, water-soluble bioactive compound, selected from bivalence gold The metal of genus, trivalent metal and combinations thereof and bioactive compound, it is obtained by method according to claim 10 ?.

25. 1 kinds of compositionss, including at least one according to the nano-particle described in any one of claim 1 to 8, or at least one Plant the nano-particle that the method according to any one of claim 9 to 24 obtains, and can connect in food, medicine or cosmetics The carrier being subject to.

26. compositionss according to claim 25, the mean diameter of wherein said nano-particle is 50 to 200nm.

27. compositionss according to claim 25, the mean diameter of wherein said nano-particle is 140nm.

28. compositionss according to claim 25, including:

Casein, 10wt% to 50wt%；

Folic acid, 0.9wt% to 2.5wt%；

Calcium, 1wt% to 6wt%；

Basic amino acid, 1wt% to 7wt%；With

Saccharide, 30wt% to 80wt%,

The most all of weight percentage is all in terms of the gross weight of said composition.

29. compositionss according to claim 26, including:

Casein, 10wt% to 50wt%；

Folic acid, 0.9wt% to 2.5wt%；

Calcium, 1wt% to 6wt%；

Basic amino acid, 1wt% to 7wt%；With

Saccharide, 30wt% to 80wt%,

The most all of weight percentage is all in terms of the gross weight of said composition.

30. compositionss according to claim 25, including:

Casein, 10wt% to 50wt%；

Folic acid, 0.9wt% to 2.5wt%；

Calcium, 1wt% to 6wt%；With

Basic amino acid, 1wt% to 7wt%；

Saccharide, 20wt% to 55wt%；With

Ascorbic acid, 1% to 25%,

The most all of weight percentage is all in terms of the gross weight of said composition.

31. compositionss according to claim 26, including:

Casein, 10wt% to 50wt%；

Folic acid, 0.9wt% to 2.5wt%；

Calcium, 1wt% to 6wt%；With

Basic amino acid, 1wt% to 7wt%；

Saccharide, 20wt% to 55wt%；With

Ascorbic acid, 1% to 25%,

The most all of weight percentage is all in terms of the gross weight of said composition.

32. compositionss according to claim 25, wherein said carrier includes medicine or cosmetic vehicle so that its It is acceptable for can being used by topic route.

33. according to the compositions described in any one of claim 25 to 32, and wherein said nano-particle is the form of dried powder.

34. 1 kinds of food, including according to the compositions described in claim 25 to 30 or 33 any one.

35. food according to claim 34, it is the form of liquid, semisolid or solid.