CN114601175A - Preparation method of enteric slow-release high-activity glutathione nanospheres - Google Patents
Preparation method of enteric slow-release high-activity glutathione nanospheres Download PDFInfo
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- 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
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- 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)
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Abstract
The invention relates to the technical field of glutathione nano-microspheres, and discloses a preparation method of an enteric sustained-release high-activity glutathione nano-microsphere, which comprises the following steps: (1) adding glutathione GSH into the solution for embedding the protein, and uniformly mixing to obtain a mixed solution; (2) spraying the mixed solution by electrostatic spraying equipment, and receiving to obtain glutathione nano-microspheres; the embedded protein solution is ethanol water solution of zein, wheat gliadin or coconut gliadin; the mass concentration of the embedded protein is more than or equal to 14 percent. The glutathione nanospheres prepared by the method have small particle size, good dispersibility, high glutathione coating rate and high stability in the storage process, simultaneously have higher release efficiency in intestinal tracts, and have good application prospects in the fields of functional foods, special medical application formula foods, special dietary foods and the like.
Description
Technical Field
The invention relates to the technical field of glutathione nano-microspheres, in particular to a preparation method of an enteric sustained-release high-activity glutathione nano-microsphere.
Background
The nano-microsphere is a new material obtained by embedding a core material such as solid, liquid or gas in a wall material by using a biocompatible or biodegradable polymer matrix as the wall material. The nano microspheres have small particle size, large specific surface area, high solubility, high bioactivity and good biocompatibility, have better sustained release effect and targeting property than the traditional embedding, and greatly improve the stability and bioavailability of the functional components. The nano-microspheres are applied to the fields of food and medicine, can obviously improve the biological activity of functional components such as heat sensitivity or easy oxidation and the like in the processing process, and enter cells under the action of cellular uptake through the combination of targeting molecules and cell surface specific receptors, thereby realizing safe and effective targeted functional component delivery and gene therapy, and further enhancing the health-care and treatment functions of the functional components.
Glutathione is a natural active peptide consisting of glutamic acid, cysteine and glycine, and is mainly used for whitening, detoxifying and resisting aging, curing tumors, protecting liver, detoxifying, relieving oxygen poisoning, improving endocrine disorders and the like in clinic. Glutathione has two forms of reduced Glutathione (GSH) and oxidized glutathione (GSSG), but only GSH has biological activity, because the sulfhydryl on the cysteine side chain group in GSH can protect important enzyme protein from oxidation, thus ensuring the physiological activity of enzyme. However, the sulfhydryl group of GSH has poor stability in aqueous solution and air, is easily oxidized to form GSSH, and further loses its physiological activity. In addition, GSH is an active factor with extremely strong whitening, toxin expelling and anti-aging functions, is mainly and completely absorbed in the small intestine of a human body, is easy to inactivate in the digestive process of the stomach, is not easy to permeate cell membranes, and has low bioavailability.
Chinese patent CN104338112A discloses a preparation method of a glutathione nano sustained-release capsule, which combines a nano microsphere technology and glutathione, converts corn starch into short straight chain starch through pullulanase after gelatinization treatment, and then coats glutathione to prepare the glutathione nano sustained-release capsule. The prepared nano glutathione capsule is easier to absorb in intestines than in stomach, which provides a new idea for the application of glutathione. However, the coating rate of glutathione in the prepared nanocapsule is 50.5%, the embedding rate needs to be improved, and the particle size ratio of the obtained sustained-release capsule is larger. Therefore, there is an urgent need for an embedding technique for improving the efficacy of GSH without affecting its activity, so as to achieve its application in functional foods and medicines and improve its efficacy.
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 as to improve the embedding rate of glutathione GSH in the prepared glutathione nano-microspheres, and the prepared glutathione nano-microspheres have higher bioactivity compared with GSH subjected to other embedding treatment.
The invention provides the following technical scheme:
a preparation method of enteric slow-release high-activity glutathione nano-microspheres takes protein as an embedding wall material, and comprises the following steps:
(1) adding glutathione GSH into the solution for embedding the protein, and uniformly mixing to obtain a mixed solution;
(2) spraying the mixed solution by electrostatic spraying equipment, and receiving to obtain enteric-coated slow-release high-activity glutathione nano-microspheres; the solution of the embedded protein is ethanol aqueous solution of zein, wheat prolamin or coconut prolamin;
the mass concentration of the embedded protein is more than or equal to 14 percent.
The embedded protein used in the invention, such as zein, wheat gliadin or coconut gliadin, is different from gelatinized starch, has amphipathy, can independently form a microsphere and nanoparticle structure in an alcohol-water solution or a water solution, realizes coating of glutathione, and has high coating rate. Meanwhile, an electrostatic spraying method is selected in the preparation of the glutathione nano-microspheres, the particle size and the form of the prepared high-molecular particles are controllable, and the particle size and the form of the prepared high-molecular particles are combined with the tendency of forming microsphere and nanoparticle structures of embedded protein, so that the GSH embedding rate of the prepared nano-microspheres is high, the particle size distribution range is narrow, the particle size is small, the bioactivity of the GSH is maintained, the overall bioactivity of the GSH nano-microspheres, such as reducing capability, free radical scavenging capability and the like, is improved relative to the GSH, the slow release effect is good, and the release effect in intestines is better than the release effect in stomach.
Preferably, in the method of the present invention, the ethanol volume concentration in the ethanol aqueous solution in the step (1) is 75 to 85%. Prolamines have good solubility in this range.
Preferably, in the method of the present invention, the solution for embedding the protein in the step (1) is an ethanol aqueous solution of coco prolamin, and the mass concentration of the coco prolamin is 14-32%. The coconut prolamin has the advantages of low cost, oxidation resistance, biodegradability, strong ductility and no risk of transgenosis, and few research on development of the coconut prolamin is carried out at present, and the coconut prolamin is not researched as a carrier of nano microspheres. The inventor applies coconut gliadin as a wall material to the embedding of GSH for the first time, and the embedding rate of the GSH in the prepared nano-microsphere is obviously higher than that of other proteins.
Preferably, the coconut prolamin is prepared by the following steps:
dispersing the degreased coconut meat powder in ethanol with volume fraction of 95% or more, controlling the solid-liquid ratio to be 1: 14-20, carrying out water bath at 50-60 ℃ for 30-60 min, adjusting the pH value to 8.0-10, adding water to dilute the degreased coconut meat powder to 50-70% of the volume fraction of the ethanol, extracting the solution for 30-60 min, carrying out centrifugal separation to obtain a supernatant, salting out, standing, carrying out centrifugal separation and precipitation, washing the precipitate with water to be neutral, and drying the precipitate to obtain the coconut meat alcohol-soluble protein.
Preferably, the mass ratio of the GSH used in step (1) to the solution or dispersion of the encapsulating protein is 1: 400-450.
Preferably, the GSH is used in the form of a phosphate buffer solution, the pH of the phosphate buffer solution is 5.5-6.5, and the molar concentration of the GSH is 0.04-0.1 mol/L. The phosphoric acid buffer solution improves the stability of the mixed solution system during preparation, and the storage stability of the obtained nano microspheres is improved.
Preferably, the step (1) further comprises adding wheat gliadin to the solution of the embedded protein before adding the GSH, mixing uniformly, and then adding the wheat gluten. In the further research of using coconut prolamin as embedding protein, the inventor combines wheat prolamin and wheat gluten into the step (1) for use, the wheat prolamin is of a single-chain structure, and is aggregated with disulfide bonds in the gluten to form a three-dimensional network structure to be inserted into a microsphere structure of the coconut prolamin under the action of non-covalent bond force, so that the storage stability of the nano microspheres can be improved, and the GSH loss rate is slowed. Since gliadin and wheat gluten are the main components of gluten and wheat gluten, the inventors also tried to use gluten and wheat gluten directly instead of a combination of gliadin and wheat gluten in their research. When the gluten protein or the wheat gluten powder is directly used, the viscosity of the mixed solution is increased to cause difficult spraying, the particle size of the prepared small ball is increased, the spherical structure has defects, the encapsulation rate and the storage stability of GSH are reduced to a certain degree, wherein the reduction of the wheat gluten powder is more obvious, the inventor thinks that the reason is related to the dosage of the wheat gliadin and the wheat gluten, and the interaction between the wheat gluten and the wheat gliadin is fast caused to appear as a whole, thereby reducing the possibility of participation of the coconut protein; thirdly, the wheat gluten powder also comprises components such as albumin and the like, which influences the stability of the nano-microsphere.
Preferably, 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 viscosity of the system is increased after the wheat gluten content is too high and the wheat gluten is matched with the wheat gliadin, and the expected effect cannot be achieved. Meanwhile, the glutathione nano-microspheres are more expected to be released and absorbed in intestinal tracts, the decomposition and digestion performance of the wheat gluten in the intestinal tracts is poor, the release of GSH is influenced, and the excessive accumulation of the wheat gluten can cause anaphylactic reaction, so that the reasonable dosage needs to be controlled.
Preferably, the step (1) further comprises homogenizing the prepared mixed solution at 2-3 MPa for 30-60 min. The homogeneity of a mixed liquid system is promoted through low-pressure homogenization treatment, and the stability of the prepared microspheres is favorably improved.
The conditions for electrostatic spraying in step (2) of the swordsman as the method of the present invention are:
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 inventor finds in research that the mixed solution flow rate, the voltage and the receiving distance all influence the embedding effect of GSH in the prepared nano-microspheres.
The invention has the following beneficial effects:
in the glutathione nanospheres prepared by the method, the GSH coating rate is high, and the biological activity of the released GSH can be normally exerted; the oxidation resistance of the nano-microspheres is enhanced relative to GSH; the GSH has high stability and low loss in the storage process, meanwhile, the nano microspheres have narrow particle size distribution range, small particle size, good dispersibility and good GSH slow release effect, and simultaneously have higher release efficiency in intestinal tracts, thereby having good application prospect in the fields of functional foods, special medical purpose formula foods, special dietary foods and the like.
Drawings
FIG. 1 is an SEM characterization map of glutathione nano-microspheres prepared by the invention.
FIG. 2 is a particle size distribution diagram of the glutathione nano-microspheres prepared by the invention.
FIG. 3 is a potential distribution diagram of the glutathione nano-microsphere prepared by the invention.
FIG. 4 is a release curve of the glutathione nano-microspheres prepared by the invention in simulated intestinal tracts.
FIG. 5 is a simulated gastric release curve of glutathione nanospheres prepared according to the present invention.
FIG. 6 shows the DPPH removing effect of glutathione nano-microspheres obtained by different preparation processes.
FIG. 7 shows the glutathione nano-microsphere pair superoxide anion O obtained by different preparation processes2 -The effect of removal of (1).
FIG. 8 shows the effect of glutathione nano-microspheres obtained by different preparation processes on removing OH.
FIG. 9 shows the effect of total reducing power of glutathione nano-microspheres obtained by different preparation processes.
Detailed Description
The following further describes embodiments of the present invention.
The starting materials used in the present invention are commercially available or commonly used in the art, unless otherwise specified, and the methods in the following examples are conventional in the art, unless otherwise specified.
Example 1
A preparation method of enteric slow-release high-activity glutathione nano-microspheres comprises the following steps:
(1) uniformly dissolving coco coir prolamin in 80% (v/v) ethanol water solution, wherein the mass concentration of the coco coir prolamin is 26%, then adding 0.05mol/L phosphoric acid buffer solution of GSH (pH is 6) into the coco coir prolamin/ethanol water solution, wherein the mass ratio of the GSH to the coco coir prolamin/ethanol water solution is 1:425, and continuously stirring until the mixture is uniformly mixed to ensure that the polymer is completely hydrated to obtain mixed solution;
(2) sucking the mixed solution obtained in the step (1) into a 10mL injection pump, setting the voltage to be 15kV, setting the propelling speed to be 20 mu L/min, starting electrostatic spraying equipment for spraying, collecting at a collecting distance of 15cm to obtain enteric-coated slow-release high-activity glutathione nano microspheres, and placing the collected sample in a sealed bag filled with a drying agent for storage and standby;
the preparation process of the coconut prolamin comprises the following steps:
sieving defatted coconut meat powder with a 80-mesh sieve, dispersing the defatted coconut meat powder in 95% ethanol by volume, controlling the solid-liquid ratio to be 1:15, carrying out water bath at 55 ℃ for 40min, adjusting the pH value to 9.0, adding water to dilute the defatted coconut meat powder until the ethanol volume fraction is 60%, extracting the defatted coconut meat powder for 40min, carrying out centrifugal separation at 4000r/min, taking supernatant, salting out the supernatant by using 1:1 mass percent of sodium chloride, standing the mixture for 12h, carrying out centrifugal separation and precipitation to obtain wet CG, repeatedly washing the wet CG to be neutral, and carrying out hot air drying at 40 ℃ for 4h to obtain the coconut prolamin.
Example 2
A preparation method of enteric slow-release high-activity glutathione nano-microspheres comprises the following steps:
(1) uniformly dissolving coconut meat alcohol-soluble protein in 75% (v/v) ethanol water solution, wherein the mass concentration of the coconut meat alcohol-soluble protein is 14%, then adding 0.04mol/L phosphoric acid buffer solution (pH5.5) of GSH, wherein the mass ratio of the GSH to the coconut meat alcohol-soluble protein/ethanol water solution is 1:400, and continuously stirring until the mixture is uniformly mixed to ensure that the polymer is completely hydrated to obtain mixed solution;
(2) sucking the mixed solution obtained in the step (1) into a 10mL injection pump, setting the voltage to be 10kV, setting the propelling speed to be 15 mu L/min, starting electrostatic spraying equipment for spraying, collecting at a collecting distance of 10cm to obtain enteric-coated slow-release high-activity glutathione nano microspheres, and placing the collected sample in a sealed bag filled with a drying agent for storage and standby;
the preparation process of the coconut prolamin comprises the following steps:
sieving defatted coconut meat powder with a 80-mesh sieve, dispersing the defatted coconut meat powder in 95% ethanol by volume, controlling the solid-liquid ratio to be 1:14, carrying out water bath at 50 ℃ for 60min, adjusting the pH value to 10, adding water to dilute the defatted coconut meat powder until the ethanol volume fraction is 50%, extracting the defatted coconut meat powder for 60min, carrying out centrifugal separation at 4000r/min to obtain supernatant, salting out the supernatant by using 1:1 mass percent sodium chloride, standing the mixture for 12h, carrying out centrifugal separation and precipitation to obtain wet CG, repeatedly washing the wet CG to be neutral, and carrying out hot air drying at 40 ℃ for 4h to obtain the coconut protein prolamin.
Example 3
A preparation method of glutathione nano-microspheres comprises the following steps:
(1) uniformly dissolving coconut prolamin in 85% (v/v) ethanol water solution, wherein the mass concentration of the coconut prolamin is 32%, then adding 0.1mol/L phosphoric acid buffer solution (pH6.5) of GSH, and continuously stirring until the mixture is uniformly mixed, wherein the mass ratio of the GSH to the coconut prolamin/ethanol water solution is 1:450, so as to ensure that the polymer is completely hydrated to obtain mixed solution;
(2) sucking the mixed solution obtained in the step (1) into a 10mL injection pump, setting the voltage to be 20kV, setting the propelling speed to be 20 mu L/min, starting electrostatic spraying equipment for spraying, collecting at a collecting distance of 20cm to obtain enteric-coated slow-release high-activity glutathione nano microspheres, and placing the collected sample in a sealed bag filled with a drying agent for storage and standby;
the preparation process of the coconut prolamin comprises the following steps:
sieving defatted coconut meat powder with a 60-mesh sieve, dispersing the defatted coconut meat powder in 96% ethanol by volume, controlling the solid-liquid ratio to be 1:20, carrying out water bath at 60 ℃ for 30min, adjusting the pH value to 8.0, adding water to dilute the defatted coconut meat powder until the ethanol volume fraction is 70%, extracting the defatted coconut meat powder for 30min, carrying out centrifugal separation at 4000r/min to obtain supernatant, salting out the supernatant by using 1:1 mass percent of sodium chloride, standing the mixture for 12h, carrying out centrifugal separation and precipitation to obtain wet CG, repeatedly washing the wet CG to be neutral, and carrying out hot air drying at 40 ℃ for 4h to obtain the coconut prolamin.
Example 4
The difference between the preparation method of the glutathione nanospheres and the embodiment 1 is that GSH is directly added into a coconut prolamin/ethanol aqueous solution in the step (1).
Example 5
The preparation method of the glutathione nanospheres is different from the preparation method in the embodiment 1 in that the zein is used for replacing the coconut prolamin.
Example 6
The preparation method of the glutathione nano-microspheres is different from the preparation method in the embodiment 1 in that the coconut prolamin is replaced by the wheat prolamin.
Example 7
The difference between the preparation method of the glutathione nano-microspheres and the embodiment 1 is that the preparation process of the mixed solution in the step (1) is as follows: uniformly dissolving coconut prolamin and wheat prolamin in an 80% (v/v) ethanol water solution, wherein the mass concentration of the coconut prolamin is 26%, adding 0.05mol/L phosphoric acid buffer solution of GSH (pH is 6), continuously stirring until the mixture is uniformly mixed, adding wheat glutelin, and continuously stirring until the mixture is uniformly mixed to obtain a mixed solution, wherein the mass ratio of the GSH to the coconut prolamin/ethanol water solution is 1:425, and the mass ratio of the coconut prolamin to the wheat glutelin is 1:0.1: 0.01.
Example 8
The difference between the preparation method of the glutathione nano-microspheres and the embodiment 1 is that the preparation process of the mixed solution in the step (1) is as follows: uniformly dissolving coconut prolamin and wheat prolamin in an 80% (v/v) ethanol water solution, wherein the mass concentration of the coconut prolamin is 26%, adding 0.05mol/L phosphoric acid buffer solution of GSH (pH is 6), continuously stirring until the mixture is uniformly mixed, adding wheat glutelin, and continuously stirring until the mixture is uniformly mixed to obtain a mixed solution, wherein the mass ratio of the GSH to the coconut prolamin/ethanol water solution is 1:425, and the mass ratio of the coconut prolamin to the wheat glutelin is 1:0.2: 0.03.
Example 9
A preparation method of glutathione nano-microspheres is different from that in example 7 in that the obtained mixed solution is homogenized for 60min under 2 MPa.
Example 10
A preparation method of glutathione nano-microspheres is different from that in example 7 in that the obtained mixed solution is homogenized for 30min under 3 MPa.
Example 11
A method for preparing glutathione nano-microspheres, which is different from the method in the embodiment 7 in that zein is used for replacing wheat gliadin in the step (1), and the addition of wheat gluten is omitted.
Example 12
The preparation method of the glutathione nano-microspheres is different from the preparation method of the embodiment 7 in that the addition of wheat gluten is omitted in the step (1).
Comparative example 1
The difference from the embodiment 7 is that the small gliadin and the wheat gluten in the step (1) are added into the ethanol water solution of the coconut prolamin after being uniformly mixed in the ethanol water solution.
Comparative example 2
The difference from example 7 is that the mass ratio of the small gliadin to the wheat gluten in step (1) is 1: 1.
Comparative example 3
The preparation method of the glutathione nanospheres is different from that of the example 7 in that the step (1): uniformly dissolving and dispersing coconut prolamin and wheat gluten in an 80% (v/v) ethanol water solution, wherein the mass concentration of the coconut prolamin is 26%, adding a phosphoric acid buffer solution (pH is 6) of GSH (glutathione) at 0.05mol/L, and continuously stirring until the coconut prolamin and the wheat gluten are uniformly mixed, wherein the mass ratio of the coconut prolamin to the wheat gluten is 1: 0.2.
Comparative example 4
The preparation method of the glutathione nanospheres is different from that of the example 7 in that the step (1): uniformly dissolving and dispersing coconut prolamin and wheat gluten powder in an 80% (v/v) ethanol water solution, wherein the mass concentration of the coconut prolamin is 26%, adding a phosphoric acid buffer solution (pH is 6) of GSH (glutathione) of 0.05mol/L, and continuously stirring until the coconut prolamin and the wheat gluten are uniformly mixed, wherein the mass ratio of the coconut prolamin to the wheat gluten is 1: 0.3.
Performance testing of the microspheres
1. The shape and the particle size distribution of the nano-microspheres prepared by the method
SEM scanning and fluorescence particle size measurement are carried out on the glutathione nano-microspheres prepared in the example 1, and the results are shown in figures 1-3. As can be seen from FIG. 1, the glutathione nanospheres have good shapes and smooth surfaces, and are all in a monodisperse state. As can be seen from FIG. 2, the average particle size of the glutathione nanospheres is about 100nm, and the glutathione nanospheres have good dispersibility and narrow particle size distribution. As can be seen from FIG. 3, the average value of the Zeta potential of the glutathione nano-microsphere is-47.7 mV, the surface shows negative charges, further aggregation can be avoided, and the monodispersion stability is good.
2. Stability test of glutathione nano-microspheres in-vitro simulated digestion process
The functional food or drug delivery system is absorbed by the small intestine and enters the blood circulation of the human body after being taken by the human body. For an ideal functional food or drug delivery system, the functional factor can be kept relatively stable in the strong acid environment of the stomach, and the functional factor can be fully released after entering the intestinal environment, so that the bioavailability of the functional factor is improved. Reference is made to Dupont et al (synthetic resistance of food proteins to additives and additives in vitro diagnostic models) for the preparation of Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) digestion stocks.
2.1 Simulated Gastric Fluid (SGF): dissolving 7mL of concentrated hydrochloric acid solution in 20% (m/m) of sodium chloride aqueous solution, stirring uniformly, adjusting the pH to 1.2 by using 0.1mol/L hydrochloric acid solution, metering to a 1L volumetric flask, preparing a pepsin solution with the mass concentration of 3.2mg/mL, and storing at 4 ℃ for later use after the preparation is completed, wherein the 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 taken, the two are uniformly mixed, the pH value is adjusted to 7.4 by using 0.01mol/L of sodium hydroxide solution, and the volume is fixed to 1L. Then, a trypsin solution with a mass concentration of 3.2mg/mL and a bile solution with a mass concentration of 0.2mg/mL were prepared for future use, and the prepared solutions were stored at 4 ℃. Wherein pancreatin and bile are added at the beginning of the digestion experiment.
2.3 simulation of gastric, intestinal independent digestion in vitro: 300mg of glutathione nanospheres prepared in example 1 were mixed with 5mL of SGF and 10mL of SIFIF solution, respectively, and adjusted to pH 2 with 6mol/L HCl and pH 7 with 1mol/L NaOH, respectively, and the mixture was incubated in a shaker at 100r/min with shaking in a thermostatic water bath at 37 ℃ to simulate the digestion reaction. Sampling and analyzing at regular intervals of digestion time, wherein the sampling time is as follows: 0. the GSH content of the samples was measured at 10, 20, 30, 60, 120, 240min and the release rate was calculated according to the following formula, with each sample measured in triplicate and averaged:
release rate (%) - (Z-Y)/zx 100;
in the formula: z is the free GSH content, measured in milligrams (mg), prior to digestion;
y is the free GSH content determined in the digestive juices, in milligrams (mg).
Wherein the results of simulated digestion of the intestine are shown in FIG. 4 and the results of simulated digestion of the stomach are shown in FIG. 5. The results show that the glutathione nano-microsphere can slowly release GSH in intestines and stomachs, wherein the release rate in intestinal juice is larger than that in gastric juice, and the glutathione nano-microsphere has good intestinal absorptivity.
3. Biological activity of nano microsphere prepared by different preparation processes
The nanospheres prepared in example 1 and the nanospheres prepared by spray drying and solvent method were tested for 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) and superoxide anion (O) without changing the composition of the mixed solution2 -H), the scavenging capacity of hydroxyl radicals (OH), and the total reducing capacity.
3.1DPPH clearance assay: respectively preparing the non-embedded GSH and nano microspheres prepared by an electrostatic spraying method, a spray drying method and a solvent method into 1.00,2.00, 3.00, 4.00 and 5.00mg/mL of the above solutions are taken as sample solutions, 2.0mL of each solution is taken, 2.0mL of 0.2mmol of DPPH solution (prepared by 95% ethanol) is added, the mixture is placed at room temperature for 30min, the solution is adjusted to zero by 95% ethanol, and the absorbance (Am) of the solution is measured by an ultraviolet-visible spectrophotometer; the absorbance of 2.0mL of the sample solution and 2.0mL of the LDPPH solution were measured at 517nm by the same method (A)n) And absorbance of a mixture of 2.0ml of the PPH solution and 2.0m of ethanol (A)0). DPPH-clearance was calculated as follows:
DPPH clearance%m-An)/A0×100；
In the formula: a. thenThe absorbance values for the sample sets (2.0mL sample solution versus 2.0mL ethanol solution); a. themAbsorbance values for the control (2.0mL of 0.5mol/L DPPH solution and 2.0mL of sample solution); a. the0The absorbance values for the blanks (2.0mL of the LDPPH solution in 2.0mL of ethanol).
The results of different GSH embedding processes on DPPH clearance are shown in fig. 6, and within the experimental range, the DPPH-clearance of different embedding processes increases with increasing concentration within a certain range, and a certain dose effect exists. Compared with other embedding processes, the electrostatic spraying process has a much higher DPPH clearance rate under different concentration conditions. Compared with non-embedded GSH, the glutathione nano-microsphere prepared by electrostatic spraying has higher DPPH clearance rate.
3.2O2 -Clearance assay: by adopting a pyrogallol method, the non-embedded GSH and the nano microspheres prepared by an electrostatic spray method, a spray drying method and a solvent method are prepared into sample solutions of 2.00, 3.00, 4.00, 5.00 and 6.00mg/mL respectively. Taking 6mL of 0.05mol/L Tris-HCl buffer solution (pH 8.2), respectively adding 0.5mL of the solution, carrying out water bath at 37 ℃ for 10min, adding 1mL of 7mmol/L pyrogallol hydrochloric acid solution preheated at 37 ℃, uniformly mixing, reacting at 37 ℃ for 4min, and stopping the reaction by using 0.5mL of concentrated hydrochloric acid. The absorbance value A is determined at 325nm1. The absorbance value A of the Tris-HCl buffer solution with the same volume as the pH value of 8.2 percent is used as a blank0. Calculating O as follows2 -Clearance rate:
O2 -clearance%0-A1)/A0×100；
In the formula: a. the1Is the absorbance value of the sample set; a. the0Absorbance values for the control group.
Different GSH embedding process pairs O2 -The clearance results are shown in FIG. 7, where different embedding processes were performed on O over the range tested2 -Clearance rate increases with increasing concentration over a range and there is a dose effect. Electrostatic spray process on O compared to other encapsulation processes2 -The clearance rate is much higher under different concentration conditions. In addition, compared with GSH which is not embedded, glutathione nano-microsphere pair O prepared by electrostatic spraying2 -The clearance rate is higher, which shows that the coconut protein serving as a wall material not only embeds GSH, but also enhances the glutathione nano-microsphere to O2 -The clearance capability of the nano-microspheres embodies the O-pair of the glutathione nano-microspheres2 -Stronger scavenging ability and the advantage of electrostatic embedding with cocoanut prolamin as wall material.
3.3 determination of hydroxyl radical OH. clearance
Using phenanthroline-Fe2+And (2) an oxidation method, namely preparing 2.00, 4.00, 6.00, 8.00 and 10.00mg/mL sample solutions from non-embedded GSH and glutathione nano-microspheres prepared by an electrostatic spray method, a spray drying method and a solvent method respectively. Measuring 1.5mL of 5mmol/L phenanthroline solution, and sequentially adding 4.0mL of 0.5mol/L phosphate buffer solution (pH 7.4) and 7mmol/L FeSO41.0mL of solution, 1.0mL of sample solution and 0.1% H2O21.0mL, adding deionized water to make up the volume to 10.0mL, bathing in water at 37 deg.C for 90min, and measuring the absorbance (A) at 510nmm). With the sample and H2O2All are replaced by deionized water, and the absorbance (A) is measured by the same methodn) With H2O2The solution was replaced with deionized water as a blank and the absorbance (A) was measured by the same procedure0). Hydroxyl radical (. OH) clearance was calculated as follows:
OH-clearance%m-A0)/(An-A0)×100；
In the formula: a. themFor adding the sample (antioxidant) and H2O2The absorbance value of (a); a. the0Pure water is used to replace the sample and H2O2The absorbance value of (a); a. thenIs a sample and H2O2Absorbance values were all replaced with pure water.
The results of different GSH embedding processes on OH-clearance are shown in fig. 8, and within the test range, the OH-clearance of different embedding processes 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 OH & clearance rate under different concentration conditions. In addition, compared with GSH which is not embedded, the glutathione nano-microspheres prepared by electrostatic spraying have higher OH & removing rate, which shows that the wall material coconut meat alcohol-soluble protein not only embeds the GSH, but also enhances the OH & removing capacity of the glutathione nano-microspheres, and embodies the stronger OH & removing capacity of the glutathione nano-microspheres and the advantage of electrostatic embedding by taking the coconut meat alcohol-soluble protein as the wall material.
3.4 measurement of GSH reducing ability
The non-embedded GSH and the nano microspheres prepared by the electrostatic spray method, the spray drying method and the solvent method are respectively prepared into sample solutions of 2.00, 4.00, 6.00, 8.00 and 10.00 mg/mL. Taking 1.0mL of the solution, adding 2.5mL of lwt% potassium ferricyanide solution and 2.5mL of 0.2mol/L phosphate buffer solution (pH 6.6), mixing uniformly, placing in a water bath at 50 ℃ for reaction for 30min, rapidly cooling, adding 2.5mL of 10% trichloroacetic acid, and centrifuging at 4000r/min for 10 min. Taking 2.5mL of supernatant, adding 2.5mL of distilled water and 2.5mL of 0.1 wt% ferric trichloride solution, mixing uniformly, standing for 10min, and measuring the light absorption value of the sample at 700nm respectively.
The results of different GSH embedding processes on the GSH reduction capacity are shown in fig. 9, and in the test range, the influence of different embedding processes on the GSH reduction capacity is increased along with the increase of the concentration in a certain range, and a certain dosage effect exists. Compared with other embedding processes, the GSH reducing capability of the electrostatic spraying process is much higher under different concentration conditions. In addition, compared with the non-embedded GSH, the glutathione nano-microsphere prepared by electrostatic spraying has higher reduction capability, which shows that the wall material coconut protein not only embeds GSH, but also enhances the reduction capability of the glutathione nano-microsphere, and embodies the stronger reduction capability of the glutathione nano-microsphere and the advantage of electrostatic embedding by taking coconut protein as the wall material.
4 embedding rate and storage stability of each nano microsphere
4.1 storage stability testing of glutathione Nanopalls
The glutathione nano-microspheres prepared in each example and each comparative example are subjected to embedding rate tests on 0 day, 30 days, 60 days, 90 days and 120 days at 25 ℃ and 45 ℃ respectively. The research of the prior literature indicates that the non-embedded GSH is unstable and becomes GSSG after being easily oxidized, so the test takes the GSH content as an index, the GSH is separated from glutathione nano-microspheres, the amount of free GSH is measured, and then the calculation is carried out according to the following formula:
GSH embedding rate: EE ═ C (1-C)f/Ct)×100％；
EE in the formula: the GSH embedding rate; cf: free GSH content; ct: total content of GSH.
4.2 the storage stability of the GSH nanospheres obtained in this example is shown in Table 1.
TABLE 1 encapsulation effect and storage stability of the respective nanospheres
From the above results, it can be seen that the embedding rate of the GSH nanospheres obtained by the method of the present invention is high, the embedding rate of the GSH nanospheres after storage at a low temperature of 25 ℃ and a low temperature of 45 ℃ for a period of time is not changed greatly, the storage stability of the GSH nanospheres is good, and the effect of the coconut protein is the best. The results of the combination of zein or wheat gliadin and coconut gliadin are lower than the results of the single use of coconut gliadin, which indicates that the zein and the wheat gliadin cannot play a role in synergistic enhancement. And the wheat gliadin and wheat gluten are introduced into the coconut prolamin nanospheres, so that the embedding rate of GSH is not obviously improved, which may be that the coconut prolamin coating effect is good, the lifting space is limited, but the storage stability is improved. However, the wheat prolamin and the wheat gluten are mixed and then added, or the dosage of the wheat gluten is obviously increased, and the embedding effect and the stability are both reduced. At the same time, the replacement with gluten or mucedin decreases the stability, which may be related to the influence on the spherical structure of the prepared microspheres. Meanwhile, the inventors confirmed in the enteric effect test of the nanospheres prepared in examples 7 and 8 that the release effect of GSH in the intestine is similar to that of the nanosphere prepared in example 1, and no significant decrease occurs, which should be caused by different factors affecting the storage stability and factors causing the nanospheres to release in the intestine.
Claims (10)
1. A preparation method of enteric slow-release high-activity glutathione nanospheres is characterized in that protein is used as an embedding wall material, and comprises the following steps:
(1) adding glutathione GSH into the solution for embedding the protein, and uniformly mixing to obtain a mixed solution;
(2) spraying the mixed solution by electrostatic spraying equipment to obtain enteric-coated slow-release high-activity glutathione nano-microspheres;
the solution of the embedded protein is ethanol aqueous solution of zein, wheat gliadin or coconut gliadin;
the mass concentration of the embedded protein is more than or equal to 14 percent.
2. The preparation method of the enteric slow-release high-activity glutathione nanosphere as claimed in claim 1, wherein the volume concentration of ethanol in the ethanol aqueous solution in the step (1) is 75-85%.
3. The preparation method of the enteric slow-release high-activity glutathione nanosphere according to claim 1, wherein the solution for embedding the protein in the step (1) is an ethanol aqueous solution of coco prolamin, and the mass concentration of the coco prolamin is 14-32%.
4. The preparation method of the enteric slow-release high-activity glutathione nanosphere according to claim 1 or 3, wherein the coconut prolamin is prepared by the following steps:
dispersing the degreased coconut meat powder in ethanol with volume fraction of more than or equal to 95%, controlling the solid-liquid ratio to be 1: 14-20, carrying out water bath at 50-60 ℃ for 30-60 min, adjusting the pH value to 8.0-10, adding water to dilute the degreased coconut meat powder to 50-70% of the volume fraction of the ethanol, extracting the solution for 30-60 min, carrying out centrifugal separation to obtain supernatant, salting out, standing, carrying out centrifugal separation and precipitation, washing the precipitate with water to be neutral, and drying the precipitate to obtain the coconut meat alcohol-soluble protein.
5. The preparation method of the enteric slow-release high-activity glutathione nanosphere according to claim 1, wherein the mass ratio of the GSH used in the step (1) to the solution or dispersion of the embedded protein is 1: 400-450.
6. The preparation method of the enteric slow-release high-activity glutathione nanosphere according to claim 1 or 5, wherein GSH is used in the form of a phosphate buffer solution, the pH of the phosphate buffer solution is 5.5-6.5, and the molar concentration of GSH is 0.04-0.1 mol/L.
7. The preparation method of the enteric slow-release high-activity glutathione nano-microsphere according to claim 3, wherein the step (1) further comprises the steps of adding wheat gliadin into the solution of the pre-embedded protein before adding GSH, mixing uniformly and then adding wheat gluten.
8. The preparation method of the enteric slow-release high-activity glutathione nanosphere according to claim 7, wherein the mass ratio of the embedded protein to the wheat gliadin and wheat glutenin is 1: 0.1-0.2: 0.01 to 0.03.
9. The preparation method of the enteric slow-release high-activity glutathione nanosphere according to claim 7 or 8, wherein the step (1) further comprises homogenizing the prepared mixed solution at 2-3 MPa for 30-60 min.
10. The preparation method of the enteric slow-release high-activity glutathione nano-microsphere according to claim 1, characterized in that 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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