CN114601175B - Preparation method of enteric sustained-release high-activity glutathione nano-microsphere - Google Patents
Preparation method of enteric sustained-release high-activity glutathione nano-microsphere Download PDFInfo
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 345
- 229960003180 glutathione Drugs 0.000 title claims abstract description 172
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 94
- 239000004005 microsphere Substances 0.000 title claims abstract description 66
- 230000000694 effects Effects 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 238000013268 sustained release Methods 0.000 title claims description 10
- 239000012730 sustained-release form Substances 0.000 title claims description 10
- 238000000034 method Methods 0.000 claims abstract description 61
- 239000000243 solution Substances 0.000 claims abstract description 52
- 241000209140 Triticum Species 0.000 claims abstract description 47
- 235000021307 Triticum Nutrition 0.000 claims abstract description 47
- 229920002494 Zein Polymers 0.000 claims abstract description 42
- 239000005019 zein Substances 0.000 claims abstract description 42
- 229940093612 zein Drugs 0.000 claims abstract description 42
- 108010061711 Gliadin Proteins 0.000 claims abstract description 31
- 239000011259 mixed solution Substances 0.000 claims abstract description 23
- 238000007590 electrostatic spraying Methods 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 16
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 238000005507 spraying Methods 0.000 claims abstract description 7
- 239000012460 protein solution Substances 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 57
- 108010068370 Glutens Proteins 0.000 claims description 27
- 235000021312 gluten Nutrition 0.000 claims description 25
- 239000002105 nanoparticle Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 235000018102 proteins Nutrition 0.000 claims description 15
- 108010050792 glutenin Proteins 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 12
- 239000008055 phosphate buffer solution Substances 0.000 claims description 12
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- 239000007788 liquid Substances 0.000 claims description 7
- 235000013372 meat Nutrition 0.000 claims description 7
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- 108010055615 Zein Proteins 0.000 description 36
- 238000002835 absorbance Methods 0.000 description 18
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
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- 235000011389 fruit/vegetable juice Nutrition 0.000 description 4
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
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- 108010053070 Glutathione Disulfide Proteins 0.000 description 3
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- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 3
- 238000007602 hot air drying Methods 0.000 description 3
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- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- 210000004051 gastric juice Anatomy 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 239000002102 nanobead Substances 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 230000002087 whitening effect Effects 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
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- 238000000265 homogenisation Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000002653 sulfanylmethyl group Chemical group [H]SC([H])([H])[*] 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/045—Organic compounds containing nitrogen as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
- B01J13/043—Drying and spraying
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Dispersion Chemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to the technical field of glutathione nano-microsphere preparation, and discloses a preparation method of enteric slow-release high-activity glutathione nano-microsphere, which comprises the following steps: (1) Adding glutathione GSH into the protein-embedded solution, and uniformly mixing to obtain a mixed solution; (2) Spraying the mixed solution through electrostatic spraying equipment, and receiving to obtain glutathione nano-microspheres; the embedded protein solution is ethanol water solution of zein, wheat gliadin or coco gliadin; the mass concentration of the embedded protein is more than or equal to 14 percent. The glutathione nano-microsphere prepared by the method has the advantages of small particle size, good dispersibility, high glutathione coating rate, high glutathione stability in the storage process, higher release efficiency in intestinal tracts, and good application prospects in the fields of functional foods, special medical use formula foods, special meal foods and the like.
Description
Technical Field
The invention relates to the technical field of glutathione nano-microsphere preparation, in particular to a preparation method of enteric slow-release type high-activity glutathione nano-microsphere.
Background
The nanometer microsphere is a new material which is obtained by taking a biocompatible or biodegradable polymer matrix as a wall material and embedding solid, liquid or gas core materials and the like in the wall material. The nanometer microsphere has small particle size, large specific surface area, high solubility, high biological activity and better biocompatibility, has better slow release effect and targeting performance than the traditional embedding, and greatly improves the stability and bioavailability of the functional components. The nanometer microsphere is applied to the fields of food and medicine, can obviously improve the biological activity of functional components such as thermosensitive or easily oxidized in the processing process, and enters cells under the action of cell uptake by combining targeting molecules with cell surface specific receptors, so that safe and effective targeted functional component delivery and gene therapy are realized, and the health care and treatment functions of the functional components are enhanced.
Glutathione is a natural active peptide composed of glutamic acid, cysteine and glycine, and is mainly used for whitening, expelling toxin, resisting aging, curing tumors, protecting liver, expelling toxin, relieving oxygen poisoning, improving endocrine disturbance and the like clinically. Glutathione has two existing forms of reduced Glutathione (GSH) and oxidized glutathione (GSSG), but only GSH has biological activity, because sulfhydryl on cysteine side chain group in GSH can protect important enzyme protein from oxidation, thereby ensuring the physiological activity of enzyme. However, the thiol group of GSH has poor stability in aqueous solution and air, and is easily oxidized to GSSH, thereby losing its physiological activity. In addition, GSH is taken as an active factor with extremely strong whitening, toxin expelling and anti-aging functions, is mainly absorbed completely in the small intestine of a human body, is easy to inactivate in the digestion process of the stomach, is not easy to permeate cell membranes, and has low bioavailability.
Chinese patent CN104338112A discloses a preparation method of glutathione nanometer slow-release capsules, which combines a nanometer microsphere technology and glutathione, and after corn starch gelatinization treatment, the corn starch is converted into short amylose by pullulanase, and then glutathione is coated to prepare the glutathione nanometer slow-release capsules. The prepared nano glutathione capsules are easier to absorb in the intestines than the stomach, which provides a new idea for the application of glutathione. However, the coating rate of glutathione in the prepared nanocapsules is 50.5%, the embedding rate is to be improved, and the particle size of the prepared sustained-release capsules is relatively large. Therefore, there is a strong need for an embedding technique that can achieve the effects of GSH in functional foods and medicines and improve the effects thereof, without affecting the GSH activity.
Disclosure of Invention
In order to improve the embedding rate of glutathione, the invention aims to provide a preparation method of glutathione nano-microspheres, so that the embedding rate of glutathione GSH in the prepared glutathione nano-microspheres is improved, and the prepared glutathione nano-microspheres have higher biological activity compared with GSH subjected to other embedding treatments.
The invention provides the following technical scheme:
the preparation method of the enteric sustained-release high-activity glutathione nanoparticle takes protein as an embedding wall material and comprises the following steps:
(1) Adding glutathione GSH into the protein-embedded solution, and uniformly mixing to obtain a mixed solution;
(2) Spraying the mixed solution through electrostatic spraying equipment, and receiving to obtain enteric slow-release high-activity glutathione nano microspheres; the embedded protein solution is an ethanol water solution of zein, wheat gliadin or coco gliadin;
the mass concentration of the embedded protein is more than or equal to 14 percent.
The embedded proteins such as zein, wheat gliadin or coco-gliadin are different from gelatinized starch, have amphipathy, can autonomously form microsphere and nanoparticle structures in an alcohol aqueous solution or an aqueous solution, realize the coating of glutathione and have high coating rate. Meanwhile, an electrostatic spraying method is selected in the preparation of the glutathione nanoparticle, the particle size and the morphology of the prepared polymer particles are controllable, and the method is combined with the trend of forming microsphere and nanoparticle structures of the embedded protein, so that the GSH embedding rate of the prepared nanoparticle is high, the particle size distribution range is narrow, the particle size is small, meanwhile, the biological activity of GSH is kept, the whole biological activity of the GSH nanoparticle, such as reducing capacity, free radical removing capacity and the like, is improved, the slow release effect is good, and the release effect in the intestines is better than that in the stomach.
Preferably, the ethanol volume concentration in the ethanol aqueous solution in the step (1) is 75-85%. The prolamin has good solubility in this range.
Preferably, the solution of the embedded protein in the step (1) is an ethanol aqueous solution of coco-prolamin, and the mass concentration of coco-prolamin is 14-32%. The cocoyl zein has low cost, oxidation resistance, biodegradability, strong ductility and no risk of transgenosis, and little research on the cocoyl zein is developed at present and is not researched as a carrier of nano microspheres. The inventor applies coco zein as a wall material to the embedding of GSH for the first time, and the embedding rate of GSH in the prepared nano microsphere is obviously higher than that of other proteins.
As a preferred embodiment of the method of the present invention, the coco prolamin is prepared by the following steps:
dispersing defatted coconut meat powder in ethanol with the volume fraction of 95% or more, controlling the solid-liquid ratio to be 1:14-20, carrying out water bath for 30-60 min at 50-60 ℃, adjusting the pH value to 8.0-10, adding water to dilute to the ethanol until the volume fraction is 50-70%, extracting for 30-60 min, centrifuging to obtain supernatant, salting out, standing, centrifuging to separate precipitate, washing to be neutral, and drying to obtain the coco zein.
As a preferred aspect of the method of the present invention, the mass ratio of GSH to the solution or dispersion of embedded proteins used in step (1) is from 1:400 to 450.
As a preferred embodiment of the method of the present invention, GSH is used in the form of a phosphate buffer solution having a pH of 5.5 to 6.5 and a molar concentration of 0.04 to 0.1mol/L. The phosphoric acid buffer solution improves the stability of the mixed solution system during preparation, and the storage stability of the obtained nano microsphere is improved.
As a preferable mode of the method, the step (1) further comprises adding wheat gliadin to the solution of embedded protein before adding GSH, and adding wheat glutenin after adding GSH and mixing uniformly. In the further study of using coco zein as embedding protein, the inventor combines the wheat zein and the wheat glutelin into the step (1) for use, the wheat zein is of a single-chain structure, and is penetrated in a microsphere structure of the coco zein by a three-dimensional network structure formed by aggregation of non-covalent bond acting force and disulfide bonds in the glutelin, so that the storage stability of the nano microsphere can be improved, and the GSH loss rate is reduced. Since gliadin and wheat gluten are the main components of gluten and wheat gluten, the inventors have also tried to directly replace the combination of gliadin and wheat gluten with gluten and wheat gluten in research. When gluten or wheat gluten is directly used, the viscosity of the mixed solution is increased to cause difficult spraying, the particle size of the prepared pellets is increased, the spherical structure is defective, the encapsulation rate and the storage stability of GSH are reduced to a certain extent, the reduction of the gluten is more obvious, the inventor considers that the reasons are related to the dosage of the wheat gliadin and the wheat glutenin, and the wheat gliadin are integrally formed, the interaction between the wheat gliadin and the wheat gliadin is rapid, and the possibility of participation of the coco gliadin is reduced; thirdly, the wheat gluten powder also comprises albumin and other components, which influence the stability of the nano microsphere.
As a preferable mode of the method, the mass ratio of the embedded protein to the wheat gliadin and the wheat glutelin is 1:0.1-0.2:0.01-0.03. The content of the wheat gluten is too high, so that the viscosity of the system is increased after the wheat gluten is matched with the wheat gliadin, and the expected effect cannot be achieved. Meanwhile, glutathione nano-microspheres are more expected to be released and absorbed in the intestinal tract, and the decomposition and digestion performance of wheat gluten in the intestinal tract are poor, so that GSH release is influenced, and anaphylactic reaction can be caused by excessive accumulation of the wheat gluten, so that reasonable dosage needs to be controlled.
As a preferable mode of the method, the step (1) further comprises homogenizing the prepared mixed solution for 30-60 min under 2-3 MPa. The uniformity of the mixed liquid system is promoted by low-pressure homogenization treatment, which is beneficial to improving the stability of the prepared microsphere.
As the swordsman of the method of the invention, the conditions of electrostatic spraying in the step (2) are as follows:
the flow rate of the polymer solution is 15-25 mu L/min;
the voltage is 10-20 kV;
the receiving distance is 10-20 cm. The inventors found in the study that the flow rate, voltage and receiving distance of the mixed solution all affect the embedding effect of GSH in the prepared nano-microsphere.
The beneficial effects of the invention are as follows:
in the glutathione nano-microsphere prepared by the method, the coating rate of GSH is high, and the biological activity of the released GSH can be normally exerted; the antioxidation capability of the nano-microsphere is enhanced relative to GSH; the GSH has high stability and low loss in the storage process, and meanwhile, the nano microsphere has narrow particle size distribution range, small particle size, good dispersibility, good GSH slow release effect, higher release efficiency in intestinal tracts, and good application prospect in the fields of functional foods, special medical use formula foods, special meal foods and the like.
Drawings
FIG. 1 is an SEM characterization map of glutathione nanospheres prepared in accordance with the present invention.
FIG. 2 is a graph showing the particle size distribution of glutathione nanomicrospheres prepared in accordance with the present invention.
FIG. 3 is a graph showing the potential distribution of glutathione nanomicrospheres prepared in accordance with the present invention.
FIG. 4 is a graph showing the simulated intestinal release profile of glutathione nanomicrospheres prepared in accordance with the present invention.
FIG. 5 is a graph showing the simulated stomach release profile of glutathione nanomicrospheres prepared according to the present invention.
FIG. 6 shows the DPPH scavenging effect of glutathione nanomicrospheres obtained by different preparation processes.
FIG. 7 shows the pair of superoxide anions O of glutathione nanomicrospheres obtained by different preparation processes 2 - Cleaning effect.
FIG. 8 shows the OH scavenging effect of glutathione nanomicrospheres obtained by different preparation processes.
Fig. 9 is the effect of total reducing power of glutathione nanomicrospheres obtained by different preparation processes.
Detailed Description
The following is a further description of embodiments of the invention.
Unless otherwise indicated, all starting materials used in the present invention are commercially available or are commonly used in the art, and unless otherwise indicated, the methods in the examples below are all conventional in the art.
Example 1
The preparation method of the enteric sustained-release high-activity glutathione nanoparticle comprises the following steps:
(1) Uniformly dissolving coco zein in 80% (v/v) ethanol water solution, wherein the mass concentration of coco zein is 26%, then adding 0.05mol/L GSH phosphate buffer solution (pH=6) into the coco zein, wherein the mass ratio of GSH to coco zein to ethanol water solution is 1:425, and continuously stirring until the coco zein and the ethanol water solution are uniformly mixed so as to ensure that the polymer is completely hydrated to obtain a mixed solution;
(2) Sucking the mixed solution obtained in the step (1) into a 10mL injection pump, setting a voltage of 15kV, a propulsion rate of 20 mu L/min, starting an electrostatic spraying device for spraying, collecting at a collecting distance of 15cm to obtain enteric slow-release high-activity glutathione nano-microspheres, and placing the collected sample in a sealed bag filled with a drying agent for storage for later use;
the coco prolamin used was prepared as follows:
sieving defatted coconut meat powder with 80 mesh sieve, dispersing in 95% ethanol, controlling solid-liquid ratio at 1:15, water-bathing at 55deg.C for 40min, adjusting pH to 9.0, diluting with water to 60% ethanol volume fraction, extracting for 40min, centrifuging for 4,000r/min to obtain supernatant, salting out with 1% sodium chloride by mass fraction 1:1, standing for 12 hr, centrifuging to obtain wet CG, repeatedly washing with water to neutrality, and hot air drying at 40deg.C for 4 hr to obtain coco prolamin.
Example 2
The preparation method of the enteric sustained-release high-activity glutathione nanoparticle comprises the following steps:
(1) Uniformly dissolving coco zein in 75% (v/v) ethanol water solution, wherein the mass concentration of coco zein is 14%, then adding 0.04mol/L GSH phosphate buffer solution (pH 5.5) into the coco zein, wherein the mass ratio of GSH to coco zein to ethanol water solution is 1:400, and continuously stirring until the coco zein and the ethanol water solution are uniformly mixed so as to ensure that the polymer is completely hydrated to obtain a mixed solution;
(2) Sucking the mixed solution obtained in the step (1) into a 10mL injection pump, setting a voltage of 10kV, a propulsion rate of 15 mu L/min, starting an electrostatic spraying device for spraying, collecting at a collecting distance of 10cm to obtain enteric slow-release high-activity glutathione nano-microspheres, and placing the collected sample in a sealed bag filled with a drying agent for storage for later use;
the coco prolamin used was prepared as follows:
sieving defatted coconut meat powder with 80 mesh sieve, dispersing in 95% ethanol with solid-liquid ratio of 1:14, water-bathing at 50deg.C for 60min, adjusting pH to 10, diluting with water to 50% ethanol volume fraction, extracting for 60min, centrifugating for 4000r/min to obtain supernatant, salting out with 1:1 mass fraction of sodium chloride, standing for 12 hr, centrifugating to obtain wet CG, repeatedly washing with water to neutrality, and hot air drying at 40deg.C for 4 hr to obtain coconut meat prolamin.
Example 3
A preparation method of glutathione nano-microspheres comprises the following steps:
(1) Uniformly dissolving coco zein in 85% (v/v) ethanol water solution, wherein the mass concentration of coco zein is 32%, then adding 0.1mol/L GSH phosphate buffer solution (pH 6.5) into the coco zein, and continuously stirring until the coco zein and the coco zein water solution are uniformly mixed, so as to ensure that the polymer is completely hydrated to obtain a mixed solution;
(2) Sucking the mixed solution obtained in the step (1) into a 10mL injection pump, setting a voltage of 20kV, a propulsion rate of 20 mu L/min, starting an electrostatic spraying device for spraying, collecting at a collecting distance of 20cm to obtain enteric slow-release high-activity glutathione nano-microspheres, and placing the collected sample in a sealed bag filled with a drying agent for storage for later use;
the coco prolamin used was prepared as follows:
sieving defatted coconut meat powder with 60 mesh sieve, dispersing in 96% ethanol, controlling solid-liquid ratio at 1:20, water-bathing at 60deg.C for 30min, adjusting pH to 8.0, diluting with water to 70% ethanol volume fraction, extracting for 30min, centrifuging to obtain supernatant, salting out with 1% sodium chloride (mass fraction 1:1), standing for 12 hr, centrifuging to obtain wet CG, repeatedly washing with water to neutrality, and hot air drying at 40deg.C for 4 hr to obtain coconut meat prolamin.
Example 4
A method for preparing glutathione nano-microspheres, which is different from example 1 in that GSH is directly added into coco-zein/ethanol aqueous solution in step (1).
Example 5
A method for preparing glutathione nano-microspheres, which is different from example 1 in that zein is used for replacing coco zein.
Example 6
A method for preparing glutathione nano-microspheres, which is different from example 1 in that the gliadin is used for replacing the coco-prolamin.
Example 7
The preparation method of the glutathione nanoparticle is different from that of the embodiment 1 in that the preparation process of the mixed solution in the step (1) is as follows: uniformly dissolving coco prolamin and wheat gliadin in an 80% (v/v) ethanol aqueous solution, wherein the mass concentration of coco prolamin is 26%, then adding a phosphate buffer solution (pH=6) of GSH with the concentration of 0.05mol/L, continuously stirring until uniformly mixing, then adding wheat glutelin, continuously stirring uniformly to obtain a mixed solution, wherein the mass ratio of GSH to coco prolamin/ethanol aqueous solution is 1:425, and the mass ratio of coco prolamin to wheat gliadin to wheat glutelin is 1:0.1:0.01.
Example 8
The preparation method of the glutathione nanoparticle is different from that of the embodiment 1 in that the preparation process of the mixed solution in the step (1) is as follows: uniformly dissolving coco prolamin and wheat gliadin in an 80% (v/v) ethanol aqueous solution, wherein the mass concentration of coco prolamin is 26%, then adding a phosphate buffer solution (pH=6) of GSH with the concentration of 0.05mol/L, continuously stirring until uniformly mixing, then adding wheat glutelin, continuously stirring uniformly to obtain a mixed solution, wherein the mass ratio of GSH to coco prolamin/ethanol aqueous solution is 1:425, and the mass ratio of coco prolamin to wheat gliadin to wheat glutelin is 1:0.2:0.03.
Example 9
A preparation method of glutathione nanospheres is different from example 7 in that the obtained mixed solution is homogenized for 60min under 2 MPa.
Example 10
A preparation method of glutathione nanospheres is different from example 7 in that the obtained mixed solution is homogenized for 30min under 3 MPa.
Example 11
A preparation method of glutathione nano-microspheres is different from example 7 in that zein is used for replacing the zein in step (1) and the addition of the zein is omitted.
Example 12
A method for preparing glutathione nano-microspheres, which is different from example 7 in that the addition of wheat gluten is omitted in step (1).
Comparative example 1
The difference from example 7 is that wheat gliadin and wheat gluten in step (1) are mixed uniformly in an aqueous ethanol solution and then added to the aqueous ethanol solution of coco gliadin.
Comparative example 2
The difference from example 7 is that the mass ratio of gliadin to wheat gluten in step (1) is 1:1.
Comparative example 3
The preparation method of the glutathione nanoparticle is different from that of the example 7 in that in the step (1): uniformly dissolving and dispersing coco-prolamin and wheat gluten in an 80% (v/v) ethanol water solution, wherein the mass concentration of coco-prolamin is 26%, then adding a phosphate buffer solution (pH=6) of GSH (GSH) with the concentration of 0.05mol/L, and continuously stirring until the coco-prolamin and the wheat gluten are uniformly mixed, wherein the mass ratio of coco-prolamin to the wheat gluten is 1:0.2.
Comparative example 4
The preparation method of the glutathione nanoparticle is different from that of the example 7 in that in the step (1): uniformly dissolving and dispersing coco-prolamin and wheat gluten in an 80% (v/v) ethanol aqueous solution, wherein the mass concentration of coco-prolamin is 26%, then adding a 0.05mol/L GSH phosphate buffer solution (pH=6) into the coco-prolamin aqueous solution, and continuously stirring until the coco-prolamin and the wheat gluten are uniformly mixed, wherein the mass ratio of coco-prolamin to the wheat gluten is 1:0.3.
Performance test of each microsphere
1. The shape and particle size distribution of the nano microsphere prepared by the method of the invention
The glutathione nanoparticle prepared in example 1 was subjected to SEM electron microscope scanning and fluorescence particle size measurement, and the results are shown in FIGS. 1 to 3. As can be seen from FIG. 1, the glutathione nanospheres are good in shape and smooth in surface, and all the glutathione nanospheres are in a monodisperse state. As can be seen from FIG. 2, the average particle size of the glutathione nanobeads is about 100nm, and the glutathione nanobeads have good dispersibility and narrow particle size distribution. From FIG. 3, it can be seen that the average value of Zeta potential of the glutathione nano-microsphere is-47.7 mV, the surface of the glutathione nano-microsphere shows negative charge, further aggregation can be avoided, and the monodisperse stability is good.
2. Stability test of glutathione nano-microsphere in-vitro simulated digestion process
The functional food or drug delivery system is absorbed by human body and then enters into blood circulation of human body after passing through oral cavity and stomach. For an ideal functional food or drug delivery system, it should be able to remain relatively stable in the strong acid environment of the stomach, yet sufficiently release the functional factor upon entry into the intestinal environment to enhance the bioavailability of the functional factor. Digestive juices simulating gastric juice (SGF) and simulated intestinal juice (SIF) were prepared by reference to Dupont et al (Comparative resistance of food proteins to adult and infant in vitro digestion models).
2.1 Simulated Gastric Fluid (SGF): 7mL of concentrated hydrochloric acid solution is dissolved in 20% (m/m) sodium chloride aqueous solution, after uniform stirring, pH is regulated to 1.2 by 0.1mol/L hydrochloric acid solution, the solution is fixed to a 1L volumetric flask, pepsin solution with the mass concentration of 3.2mg/mL is prepared, and after the preparation is finished, the solution is stored at the temperature of 4 ℃ for standby, wherein pepsin is added at the beginning of a digestion experiment.
2.2 Simulated Intestinal Fluid (SIF): 190mL of sodium hydroxide solution with the concentration of 0.1mol/L is measured, 6.8g of aqueous solution of dipotassium hydrogen phosphate is further taken, the two solutions are uniformly mixed, the pH value is regulated to 7.4 by using 0.01mol/L sodium hydroxide solution, and the volume is fixed to 1L. Then preparing trypsin solution with the mass concentration of 3.2mg/mL and bile solution with the mass concentration of 0.2mg/mL for standby, and storing the prepared solutions at the temperature of 4 ℃. Wherein pancreatin and bile are added at the beginning of the digestion experiment.
2.3 in vitro simulated gastric and intestinal digestion alone: the glutathione nanomicrospheres prepared in example 1 were taken, 300mg was mixed uniformly with 5mL of SGF and 10mLSIF solution, respectively, and pH was adjusted to 2 with 6mol/L HCl and 7 with 1mol/L NaOH, respectively, and the mixture was shaken in a constant temperature water bath at 37 ℃ and incubated in a 100r/min shaker to simulate digestion reaction. Sampling and analyzing at regular digestion time intervals, wherein the sampling time is as follows: 0. the GSH content in the samples was measured at 10, 20, 30, 60, 120, 240min, and the release rate was calculated according to the following formula, three times per sample, averaged:
release rate (%) = (Z-Y)/z×100;
wherein: z is the content of free GSH measured before digestion in milligrams (mg);
y is the content of free GSH measured in digestive juice, and is expressed in milligrams (mg).
Wherein the intestinal simulated digestion results are shown in FIG. 4 and the gastric simulated digestion results are shown in FIG. 5. The result shows that the glutathione nano-microsphere can slowly release GSH in intestines and stomach, wherein the release rate in intestinal juice is larger than that in gastric juice, and has good intestinal absorbability.
3. Biological activity of nano microsphere prepared by different preparation processes
The nanoparticle prepared in example 1 and the nanoparticle prepared by spray drying method and solvent method were tested for 1, 1-diphenyl-2-picrylhydrazine radical (DPPH), superoxide anion (O) 2 - The scavenging ability of hydroxyl radicals (OH), and the total reducing ability.
3.1DPPH clearance determination: preparing 1.00, 2.00, 3.00, 4.00 and 5.00mg/mL of non-embedded GSH and nano microspheres prepared by an electrostatic spraying method, a spray drying method and a solvent method as sample solutions, respectively taking 2.0mL of each solution, adding 2.0mL 0.2mmol DPPH solution (prepared by 95% ethanol), standing for 30min at room temperature, zeroing by 95% ethanol, and measuring absorbance (Am) by an ultraviolet-visible spectrophotometer; the absorbance of 2.0mL of the sample solution and the absorbance of 2.0mL of the solution of LDPPH were measured at 517nm, respectively, in the same manner (A n ) And absorbance (A) of a mixture of 2.0m of the LDPPH solution and 2.0m of ethanol 0 ). DPPH clearance was calculated as follows:
DPPH clearance% = (a m -A n )/A 0 ×100；
Wherein: a is that n Absorbance values for the sample set (2.0 mL sample solution and 2.0mL ethanol solution); a is that m Absorbance values for the control group (2.0 mL of 0.5mol/L DPPH solution and 2.0mL of sample solution); a is that 0 Absorbance values for the blank (2.0 mL of pph solution with 2.0mL of ethanol solution).
The results of different GSH embedding processes on DPPH clearance are shown in fig. 6, and within the experimental range, different embedding processes increase with increasing concentration on DPPH-clearance, with a certain dose effect. Compared with other embedding processes, the clearance rate of the electrostatic spraying process to DPPH is much higher under different concentration conditions. Compared with non-embedded GSH, the clearance rate of the glutathione nano microsphere prepared by electrostatic spraying to DPPH is higher.
3.2O 2 - Clearance determination: the nano microspheres prepared by adopting a pyrogallol method, an unencapsulated GSH, an electrostatic spraying method, a spray drying method and a solvent method are respectively prepared into sample solutions of 2.00, 3.00, 4.00, 5.00 and 6.00 mg/mL. 6mL of 0.05mol/L Tris-HCl buffer (pH 8.2) was added to each of the above solutions, 0.5mL was added to each of the above solutions, the mixture was subjected to water bath at 37℃for 10 minutes, 7mmol/L of the pyrogallol hydrochloric acid solution (1 mL) preheated at 37℃was added thereto, and the mixture was uniformly mixed, reacted at 37℃for 4 minutes, and the reaction was terminated with 0.5mL of concentrated hydrochloric acid. Determination of the absorbance A at 325nm 1 . The absorbance A was measured using an equal volume of Tris-HCl buffer pH 8.2% as a blank 0 . Calculation of O as follows 2 - Clearance rate:
O 2 - clearance% = (a) 0 -A 1 )/A 0 ×100；
Wherein: a is that 1 Absorbance values for the sample set; a is that 0 Absorbance values for the control group.
Different GSH embedding process pairs O 2 - The clearance results are shown in FIG. 7, and in the experimental range, the different embedding processes are used for the treatment of O 2 - The clearance increases with increasing concentration over a range, with a certain dose effect. Compared with other embedding processes, the electrostatic spraying process is used for O 2 - The clearance is much higher at different concentrations. In addition, compared with GSH which is not embedded, the glutathione nanoparticle pair O prepared by electrostatic spraying 2 - The clearance rate is higher, which indicates that the wall material cocoyl zein not only embeds GSH, but also enhances the O of the glutathione nano microsphere 2 - The scavenging ability of the glutathione nanoparticle pair O is reflected 2 - Stronger scavenging ability and the advantage of electrostatic embedding with coco-prolamin as wall material.
3.3 determination of OH-scavenging Rate of hydroxyl radical
By using phenanthroline-Fe 2+ The oxidation method is to prepare glutathione nano-microspheres prepared by an unencapsulated GSH, an electrostatic spraying method, a spray drying method and a solvent method into sample solutions of 2.00, 4.00, 6.00, 8.00 and 10.00mg/mL respectively. 1.5mL of 5mmol/L phenanthroline solution is measured, and 0.5mol/L phosphate buffer solution (pH 7.4) 4.0mL and 7mmol/L FeSO are sequentially added 4 1.0mL of solution, 1.0mL of sample solution and 0.1% H 2 O 2 1.0mL was added with deionized water to a volume of 10.0mL and water-bath was performed at 37deg.C for 90min, and the absorbance at 510nm was measured (A m ). With samples and H 2 O 2 All were replaced with deionized water, and absorbance was measured by the same procedure (A n ) By H 2 O 2 The solution was replaced with deionized water as a blank, and absorbance was measured by the same procedure (A 0 ). Hydroxyl radical (·oh) clearance was calculated as follows:
OH clearance% = (a m -A 0 )/(A n- A 0 )×100；
Wherein: a is that m To add samples (antioxidants) and H 2 O 2 Absorbance values of (2); a is that 0 Pure water is used for replacing samples and H 2 O 2 Absorbance values of (2); a is that n For sample and H 2 O 2 Absorbance values each replaced with pure water.
The results of different GSH embedding processes on OH clearance are shown in figure 8, and in the experimental range, the OH clearance of different embedding processes increases along with the increase of concentration within a certain range, and a certain dosage effect exists. Compared with other embedding processes, the electrostatic spraying process has much higher OH clearance rate under different concentration conditions. In addition, compared with GSH which is not embedded, the glutathione nanospheres prepared by electrostatic spraying have higher OH clearance, which means that the wall material cocoyl zein not only embeds GSH, but also enhances the OH clearance of the glutathione nanospheres, and the advantages of the glutathione nanospheres that the OH clearance is stronger and the cocoyl zein is used as the wall material are reflected.
3.4 determination of GSH reducing Capacity
The nano-microspheres prepared by the electrostatic spraying method, the spray drying method and the solvent method are respectively prepared into 2.00, 4.00, 6.00, 8.00 and 10.00mg/mL serving as sample solutions. 1.0mL of the mixture is taken, 2.5mL of lwt% potassium ferricyanide solution and 2.5mL of 0.2mol/L phosphate buffer solution (pH 6.6) are added, the mixture is uniformly mixed, the mixture is placed in a water bath at 50 ℃ for reaction for 30min, the mixture is rapidly cooled, and 10% trichloroacetic acid 2.5mL and 4000r/min are added for centrifugation for 10min. 2.5mL of supernatant is taken, 2.5mL of distilled water and 2.5mL of 0.1wt% ferric trichloride solution are added, the mixture is uniformly mixed, and then the mixture is kept stand for 10min, and the absorbance value of the sample at 700nm is respectively measured.
The results of different GSH embedding processes on GSH reducing power are shown in fig. 9, and in the experimental range, the influence of different embedding processes on GSH reducing power increases with increasing concentration within a certain range, and a certain dosage effect exists. Compared with other embedding processes, the electrostatic spraying process has much higher GSH reducing capability under different concentration conditions. In addition, compared with the GSH which is not embedded, the reduction capacity of the glutathione nanospheres prepared by electrostatic spraying is higher, which indicates that the wall material cocoyl alcohol soluble protein not only embeds the GSH, but also enhances the reduction capacity of the glutathione nanospheres, and the advantages of stronger reduction capacity of the glutathione nanospheres and electrostatic embedding by taking cocoyl alcohol soluble protein as the wall material are reflected.
4 embedding rate and storage stability of each nanometer microsphere
4.1 storage stability test of glutathione nanomicrospheres
The glutathione nano-microspheres prepared in each example and comparative example are respectively subjected to embedding rate tests on day 0, day 30, day 60, day 90 and day 120 at 25 ℃ and 45 ℃. The prior literature research shows that the GSH which is not embedded is unstable and is easy to oxidize to become GSSG, so the test takes the GSH content as an index, separates the GSH from the glutathione nano microsphere, determines the free GSH content, and then calculates according to the following formula:
GSH embedding rate: ee= (1-C) f /C t )×100％；
Wherein EE: GSH embedding rate; c (C) f : free GSH content; c (C) t : total GSH content.
4.2 storage stability of GSH nanomicrospheres obtained in this example are shown in Table 1.
TABLE 1 embedding Effect and storage stability of the nanomicrospheres
From the above results, the embedding rate of the GSH nano-microspheres prepared by the method is high, the embedding rate of the GSH nano-microspheres does not change greatly after the GSH nano-microspheres are stored at the low temperature of 25 ℃ and the low temperature of 45 ℃ for a period of time, and the GSH nano-microspheres have good storage stability, wherein the cocoyl zein has the best effect. The result of the compounded use of zein or wheat gliadin and coco-gliadin is lower than that of the single use of coco-gliadin, which indicates that the zein or the wheat gliadin and coco-gliadin cannot play a synergistic enhancement role. The embedding rate of GSH is not obviously improved by introducing the wheat gliadin and the wheat glutelin into the nano microsphere of the coco gliadin, which is probably that the coating effect of the coco gliadin is good, the lifting space is limited, but the storage stability is improved. However, the wheat gliadin and the wheat glutenin are added after being mixed, or the dosage of the wheat glutenin is obviously improved, and both the embedding effect and the stability are reduced. At the same time, gluten or gluten protein is substituted, and the stability is lowered, which may be related to the influence of the spherical structure of the prepared microspheres. Meanwhile, the inventors confirmed in the enteric effect test of the nano-microspheres prepared in examples 7 and 8 that the GSH release effect in the intestines is similar to that of the nano-microsphere of example 1, and no significant drop occurs, which is caused by the difference between the factors affecting the storage stability and the factors causing the release of the nano-microsphere in the intestines.
Claims (4)
1. The preparation method of the enteric sustained-release high-activity glutathione nanoparticle is characterized by taking protein as an embedding wall material and comprising the following steps of:
(1) Adding glutathione into the protein-embedded solution, and uniformly mixing to obtain a mixed solution;
the embedded protein solution is an ethanol water solution of cocoyl zein, the volume concentration of ethanol in the ethanol water solution is 75-85%, and the mass concentration of cocoyl zein is 14-32%;
the glutathione is used in the form of phosphate buffer solution, the pH value of the phosphate buffer solution is 5.5-6.5, and the molar concentration of the glutathione is 0.04-0.1 mol/L;
the mass ratio of the glutathione to the embedded protein solution is 1:400-450;
(2) Spraying the mixed solution through electrostatic spraying equipment to obtain enteric slow-release high-activity glutathione nano microspheres;
the step (1) further comprises adding wheat gliadin into the solution of the embedded protein before adding glutathione, adding the glutathione, uniformly mixing, and then adding wheat gluten;
the mass ratio of the embedded protein to the wheat gliadin to the wheat glutelin is 1:0.1 to 0.2:0.01 to 0.03.
2. The method for preparing the enteric sustained-release high-activity glutathione nanoparticle according to claim 1, wherein the cocoyl zein is prepared by the following steps:
dispersing defatted coconut meat powder in ethanol with the volume fraction of more than or equal to 95%, controlling the solid-liquid ratio to be 1:14-20, carrying out water bath for 30-60 min at 50-60 ℃, adjusting the pH value to 8.0-10, adding water to dilute to the ethanol until the volume fraction is 50% -70%, extracting for 30-60 min, centrifuging to obtain supernatant, salting out, standing, centrifuging to separate precipitate, washing to be neutral, and drying to obtain the coco zein.
3. The method for preparing enteric sustained-release high-activity glutathione nano-microspheres according to claim 1, wherein the step (1) further comprises homogenizing the prepared mixed solution for 30-60 min under 2-3 MPa.
4. The method for preparing enteric sustained-release high-activity glutathione nanoparticle according to claim 1, wherein the electrostatic spraying conditions in the step (2) are as follows:
the flow rate of the polymer solution is 15-25 mu L/min;
the voltage is 10-20 kV;
the receiving distance is 10-20 cm.
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