CN113397032A - Nutrient delivery system based on burdock glycoprotein and preparation method and application thereof - Google Patents
Nutrient delivery system based on burdock glycoprotein and preparation method and application thereof Download PDFInfo
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- CN113397032A CN113397032A CN202110562425.1A CN202110562425A CN113397032A CN 113397032 A CN113397032 A CN 113397032A CN 202110562425 A CN202110562425 A CN 202110562425A CN 113397032 A CN113397032 A CN 113397032A
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- burdock
- glycoprotein
- delivery system
- solution
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Links
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/14—Vegetable proteins
-
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/66—Proteins
-
- 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
-
- 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
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
-
- 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
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
- A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
-
- 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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a nutrient delivery system based on burdock glycoprotein and a preparation method and application thereof, wherein the preparation method comprises the following steps: step 1: separating to obtain burdock glycoprotein. Step 2: and (3) carrying out ultrasonic crushing treatment on the burdock glycoprotein. And step 3: and loading nutrients. And 4, step 4: and (3) assembling burdock glycoprotein nanoparticles. The nutrient delivery system of the present invention is obtained. The chemical composition is simple, the cost is controllable, the effective regulation and control of the size of a delivery system can be realized by changing the parameters of ultrasonic crushing treatment in the early stage according to the product requirements, the nanoparticle with the core-shell structure is formed by combining the acidic ethanol treatment and the ultrahigh pressure treatment, the use of toxic chemicals is not involved, and the environment is friendly. The whole preparation process is carried out at normal temperature or low temperature, so that flavor change and nutrient activity reduction caused by heating are effectively inhibited.
Description
Technical Field
The invention relates to a nutrient delivery system based on burdock glycoprotein and a preparation method and application thereof, belonging to the technical field of controlled release of drugs.
Background
The steady-state delivery of food-derived nutritionally active factors and their use in functional foods have become a research hotspot in the field of food processing today. In order to improve the safety, biocompatibility and degradability of the delivery preparation, researchers mostly adopt oil and fat, surfactants, proteins, polysaccharides and the like extracted and separated from natural foods as wall materials. The most commonly used forms of carriers are emulsions, microcapsules, nanoparticles, complex coacervates, etc., depending on the requirements of the application environment. At present, in order to further improve the product stability and improve the preparation efficiency, a novel physical field processing means is widely applied to the construction of a delivery system. For example, patent 201710411623.1 discloses a swept-frequency ultrasonic preparation method of rapeseed protein-chitosan nanoparticles, wherein swept-frequency ultrasonic treatment effectively promotes protein and polysaccharide to form aggregates by crosslinking, and each aggregate is further assembled under the condition of hydrophobic interaction to obtain nanoparticles, thereby realizing encapsulation of active ingredients such as curcumin and lutein; in patent 201110142629.6, food materials with a fat content of less than 12% are mixed with nutrients in a certain ratio, and the mixture is ground by a ball or rod mill to achieve nanocrystallization and core loading. However, the physical field processing method has high energy consumption, and easily generates heat during the preparation of the nano-carrier, which adversely affects the flavor of the product and the stability of the core material, and thus has limited application in actual production.
The existing ultrahigh pressure technology is also called high static pressure technology, and refers to a processing method for effectively changing the physical properties of food by sealing the food in a high pressure resistant elastic container, using fluid as a pressure transmission medium, applying high static pressure (100-. As a non-thermal processing technology, ultrahigh pressure has been widely applied in the fields of sterilization, food preservation and the like. In addition, during the ultrahigh pressure treatment process, non-covalent bonds such as hydrogen bonds, hydrophobic interactions, ionic bonds and the like among biological macromolecules are changed, so that the denaturation and aggregation of proteins, polysaccharides, starch and the like are initiated. If the ultrahigh pressure technology is used for constructing the carrier, the activity loss of the thermosensitive core material is greatly inhibited. However, the ultrahigh pressure technology has unique characteristics, and has high requirements on the edible wall material, and the effective assembly of the carrier can be realized only by accurately regulating and controlling the properties of the edible wall material in advance. However, there are few reports on this aspect, which greatly limits the application of ultra-high pressure technology to the construction of delivery systems.
Disclosure of Invention
The purpose of the invention is as follows: in view of the existing problems and disadvantages, the present invention aims to provide a burdock glycoprotein based nutrient delivery system, a preparation method and an application thereof, and an assembly method for treating burdock glycoprotein nanoparticles at a normal temperature by using an ultrahigh pressure, so as to effectively inhibit the activity loss of a core material in an encapsulation process and improve the encapsulation efficiency of the core material.
The technical scheme is as follows: in order to realize the purpose, the invention adopts the following technical scheme:
a preparation method of a nutrient delivery system based on burdock glycoprotein comprises the following steps:
step 1: separating to obtain the burdock glycoprotein,
taking fresh burdock roots, cleaning, slicing, putting into boiling water for boiling and enzyme inactivation, taking out the burdock root slices subjected to enzyme inactivation, putting into an oven for drying, crushing by using an ultrafine crusher, weighing the obtained burdock powder, adding water according to a material-liquid ratio of 1: 15-1: 25 for uniform dispersion, adjusting the pH of the mixed solution to 5.0-7.0, adding alpha-amylase for hydrolysis, and then heating in a boiling water bath for enzyme inactivation; adjusting the pH of the mixed solution to 3.0-6.0, adding glucoamylase for hydrolysis, carrying out boiling water bath treatment for enzyme deactivation, centrifugally separating supernatant, concentrating to 1/3-1/4 of the volume of the supernatant, adding a mixed solvent of chloroform and n-butyl alcohol, stirring, centrifugally separating, removing an organic solvent layer and a denatured protein layer, repeating the operation for a plurality of times, adding absolute ethyl alcohol, standing at the temperature of 2-6 ℃, centrifugally separating to obtain a precipitate, and freeze-drying to obtain burdock glycoprotein;
step 2: carrying out ultrasonic crushing treatment on the burdock glycoprotein,
dissolving the burdock glycoprotein obtained in the step 1 in water to reach the concentration of 2-8 mg/mL, and carrying out ultrasonic crushing treatment under the condition of an external ice bath to obtain a burdock glycoprotein solution;
and step 3: loading nutrients, namely dissolving the water-soluble nutrients in distilled water to reach the concentration of 1-1.5%; dissolving fat-soluble nutrients in ethyl acetate to reach a concentration of 1-1.5%; continuously dropwise adding a nutrient-containing solution into the burdock glycoprotein solution under the condition of continuous magnetic stirring, and regulating and controlling the volume of the dropwise added nutrient solution according to the load amount to be not more than 2% of the volume of the burdock glycoprotein solution to obtain a mixed solution;
and 4, step 4: the assembly of the burdock glycoprotein nano-particles,
under the condition of magnetic stirring, continuously dropwise adding absolute ethyl alcohol into the mixed solution in the step 3 to enable the volume fraction of the ethyl alcohol to reach 5-50%, adjusting the pH of the mixed solution to 1.0-6.0 by using HCl, stirring at the temperature of 2-6 ℃, sealing in a polyethylene self-sealing bag, forming a nanoparticle solution with core-shell structure stable nanoparticles under the condition of ultrahigh pressure of 400-600 Mpa, transferring the nanoparticle solution into a dialysis bag with the molecular weight cutoff of 8,000-14000 kDa, and dialyzing to remove solvents such as ethyl alcohol and ethyl acetate and unencapsulated nutrients.
Further, the burdock root slices in the step 1 are ground by an ultra-micro grinder, and are sieved by a 80-mesh sieve, the obtained burdock powder is weighed, water is added according to the material-liquid ratio of 1:20, the mixture is uniformly dispersed, the pH value of the mixture is adjusted to be 6.0, low-temperature alpha-amylase accounting for 0.6% of the mass of the burdock powder is added, hydrolysis is carried out at the temperature of 60 ℃ and the speed of 440rpm, and then the enzymes are heated in a boiling water bath to be inactivated; adjusting the pH of the mixed solution to 4.5, adding 1% saccharifying enzyme by mass of burdock powder, hydrolyzing at 60 deg.C and 440rpm, and inactivating enzyme by boiling water bath treatment; centrifuging to separate supernatant, concentrating at 40 deg.C under reduced pressure to 1/4, adding mixed solvent (4: 1 of chloroform and n-butanol) 1/3 volume of supernatant, stirring, centrifuging to remove organic solvent layer and denatured protein layer, and repeating for 5 times; adding anhydrous ethanol with 4 times volume of the aqueous phase, standing at 4 deg.C, centrifuging to obtain precipitate, and lyophilizing to obtain burdock glycoprotein.
Further, dissolving the burdock glycoprotein in the step 2 in water to reach the concentration of 5mg/mL, carrying out ultrasonic crushing treatment under the condition of an external ice bath, and adjusting the power of ultrasonic crushing to 200-800W and the time to 2-10 min.
Further, the power of the ultrasonic crushing is adjusted to 200W, 400W or 600W during the ultrasonic crushing treatment in the step 2.
Further, in step 2, the power and time of ultrasonication are adjusted to control the molecular mass and chain length of the glycoprotein.
Further, in the step 4, under the condition of magnetic stirring, the volume fraction of the anhydrous ethanol continuously dropwise added into the mixed solution in the step 3 reaches 25%, then the mixed solution is adjusted to pH 4.5 by using 1mol/L HCl, stirred for 12-15 h at 4 ℃, sealed in a polyethylene self-sealing bag, kept for 10-15 min under the condition of 400-600 MPa under the condition of ultrahigh pressure to form core-shell structured nanoparticles, subjected to ultrahigh pressure treatment, transferred into a dialysis bag with the molecular weight cutoff of 8,000-14000 kDa, and dialyzed for 5-10h to remove solvents such as ethanol and ethyl acetate and unencapsulated nutrients.
A burdock glycoprotein based nutrient delivery system made by the method of any one of claims 1-6.
Use of a burdock glycoprotein based nutrient delivery system, wherein nutrients or drugs are loaded in the burdock glycoprotein based nutrient delivery system of claims 1-6, and the burdock glycoprotein based nutrient delivery system is assembled to be prepared into liquid or freeze-dried powder and then added into food or beverage.
Has the advantages that: compared with the prior art, the invention has the following advantages: the method is a novel assembling method for preparing the burdock glycoprotein nano-particles by using ultrahigh pressure treatment at normal temperature, can effectively inhibit the activity loss of the core material in the encapsulating process, and improves the encapsulating rate of the core material.
Drawings
FIG. 1 is a flow chart of a method of the present invention;
figure 2 is a TEM image of example 1 of the present invention,
in the figure, A is burdock glycoprotein, B is ultrasonic crushing treatment, C is acid ethanol induction, and D is ultrahigh pressure treatment.
Detailed Description
The present invention is further illustrated by the following figures and specific examples, which are to be understood as illustrative only and not as limiting the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalent modifications thereof which may occur to those skilled in the art upon reading the present specification.
Referring to the steps shown in fig. 1, a burdock glycoprotein-based nutrient delivery system and a method for preparing the same are illustrated below by referring to two examples and a control group.
Example 1
1. Separation of burdock glycoprotein
Cleaning fresh radix Arctii, slicing, decocting in boiling water for 10s to inactivate enzyme, oven drying at 45 deg.C in oven, pulverizing with superfine pulverizer, and sieving with 80 mesh sieve; weighing burdock powder, adding water according to the material-liquid ratio of 1:20, uniformly dispersing, adjusting the pH value of the mixed solution to 6.0, and keeping the subsequent enzyme activity of the alpha-amylase. Adding low temperature alpha-amylase 0.6% of burdock powder, hydrolyzing at 60 deg.C and 440rpm for 40min, heating in boiling water bath for 5min, and inactivating enzyme; the pH of the mixture was adjusted to 4.5 to ensure the enzymatic activity of the subsequent saccharifying enzyme. Adding saccharifying enzyme 1% of burdock powder, hydrolyzing at 60 deg.C and 440rpm for 40min, and treating with boiling water bath for 5min to inactivate enzyme; centrifuging to separate supernatant, concentrating at 40 deg.C under reduced pressure to 1/4, adding mixed solvent (4: 1 of chloroform and n-butanol) 1/3 volume of supernatant, stirring for 30min, centrifuging to remove organic solvent layer and denatured protein layer, and repeating for 5 times; adding anhydrous ethanol with 4 times volume of the water phase, standing at 4 deg.C, centrifuging to obtain precipitate, and lyophilizing to obtain burdock glycoprotein.
2. Burdock glycoprotein ultrasonication treatment
Dissolving the obtained glycoprotein in water to reach a concentration of 5mg/mL, adding ice water bath, and performing ultrasonic disruption treatment at 200W, 400W and 600W for 5 min. Ultrasonic crushing treatment for 5min under 200W condition is ultrasonic crushing treatment a1400W ultrasonic crushing treatment for 5min and ultrasonic crushing treatment b1Ultrasonic crushing treatment for 5min under the condition of 600W is ultrasonic crushing treatment c1。
3. Nutrient loading
Preparing a beta-carotene solution with the mass concentration of 1.0% by using ethyl acetate, slowly dropwise adding the solution into the burdock glycoprotein solution after ultrasonic treatment under the condition of magnetic stirring, and finally dropwise adding the solution with the volume of 1.0% of the burdock glycoprotein;
4. assembly of burdock glycoprotein nanoparticles
Under magnetic stirring, anhydrous ethanol was continuously added dropwise to the above mixed solution so that the final volume fraction of ethanol reached 25%, and then the mixed solution was adjusted to pH 4.5 with 1M HCl so that protein groups on the polysaccharide aggregated. Stirring for 12h at 4 ℃; sealing the solution in a polyethylene self-sealing bag, and keeping the solution under the conditions of ultrahigh pressure and 400MPa for 15 min; after ultrahigh pressure treatment, transferring the nanoparticle solution into a dialysis bag with the molecular weight cutoff of 8,000-14000 kDa, and dialyzing for 5-10h to remove solvents such as ethanol, ethyl acetate and the like and unencapsulated nutrients.
Efficacy testing
The burdock glycoprotein extracted by the scheme has the polysaccharide content of 65.21 percent, and the monosaccharide composition of: fucose 0.50%, rhamnose 8.06%, Ara20.29 percent of primary sugar, 12.34 percent of galactose, 3.46 percent of glucose, 1.28 percent of xylose, 0.45 percent of mannose, 1.90 percent of fructose, 50.80 percent of galacturonic acid and 0.93 percent of glucuronic acid; the protein content was 19.43%. Number average relative molecular mass (M) of burdock glycoproteinn) 33.9kDa, weight average relative molecular mass (M)w) 168.6kDa, and a dispersion coefficient of 4.976.
As in a in fig. 2, before ultrasonic treatment, the hydration kinetics of the DLS detection burdock glycoprotein is over 4000nm, and the fiber length cannot be distinguished under the TEM condition; as shown in B in figure 2, after ultrasonic treatment for 5min, D of burdock glycoproteinZThe size is greatly reduced, and a short rod-shaped structure can be seen under TEM conditions and is easy to gather together. According to the treatment process, DZBetween 432.98nm to 543.21 nm;
as shown in FIG. 2C, under ethanol condition, the conformation of glycoprotein changes, the hydrophobic residues of protein groups are exposed, the interaction between different molecules is enhanced, and thus the protein aggregation is generated, and when the ethanol condition is adjusted to be under acidic condition, the protein aggregation is further intensified to form an elliptical loose structure;
in fig. 2D, under the condition of ultra-high pressure, the denaturation degree of burdock glycoprotein is strengthened, and aggregation further occurs, so that a circular nano delivery system with clear boundaries is formed, and the nano delivery system has a typical 'core-shell' structure. The sizes of the three nanoparticles are 224.22, 267.32 and 343.22nm, respectively, depending on the power of the sonication. Thus, the sonication parameters can be manipulated to manipulate the nanoparticle size.
Taking out 0.5mL of nanoparticle solution from the dialyzed sample, adding 3mL of n-hexane, extracting for 5min by vortex oscillation, continuously extracting for 3 times, combining n-hexane phases, detecting the content of beta-carotene, and calculating the encapsulation rate to be 65.34% according to the following formula.
The data of the results of the final tests are shown in the following table 1:
TABLE 1
Example 2
1. Separation of burdock glycoprotein
Cleaning fresh radix Arctii, slicing, decocting in boiling water for 6s to inactivate enzyme, oven drying at 50 deg.C, pulverizing with superfine pulverizer, and sieving with 80 mesh sieve; weighing burdock powder according to a material-liquid ratio of 1:20, adding water for uniform dispersion, adjusting the pH of the mixed solution to 6.0, adding low-temperature alpha-amylase accounting for 0.6% of the mass of the burdock powder, hydrolyzing at 60 ℃ and 440rpm for 40min, and then heating in a boiling water bath for 5min to inactivate enzyme; adjusting pH of the mixed solution to 4.5, adding 1% diastase, hydrolyzing at 60 deg.C and 440rpm for 40min, and treating with boiling water bath for 5min to inactivate enzyme; centrifuging to separate supernatant, concentrating at 40 deg.C under reduced pressure to 1/4, adding mixed solvent (4: 1 of chloroform and n-butanol) 1/3 volume of supernatant, stirring for 30min, centrifuging to remove organic solvent layer and denatured protein layer, and repeating for 5 times; adding anhydrous ethanol with 4 times volume of the water phase, standing at 4 deg.C, centrifuging to obtain precipitate, and lyophilizing to obtain burdock glycoprotein.
2. Burdock glycoprotein ultrasonication treatment
Dissolving the obtained glycoprotein in water to reach the concentration of 5mg/mL, adding an ice bath, and treating for 8min under the condition of 400W;
3. nutrient loading
Preparing epigallocatechin gallate (EGCG) solution with mass concentration of 1.5% with distilled water, slowly dropwise adding the solution into the ultrasonically-treated burdock glycoprotein solution under the condition of magnetic stirring, and finally dropwise adding the solution with volume of 0.5% of the burdock glycoprotein;
4. assembly of burdock glycoprotein nanoparticles
Under the condition of magnetic stirring, continuously dropwise adding absolute ethyl alcohol into the mixed solution to enable the final volume fraction of the ethyl alcohol to reach 40%, then adjusting the pH of the mixed solution to 4.5 by using 1M HCl, and stirring for 15 hours at the temperature of 4 ℃; sealing the solution in a polyethylene self-sealing bag, and keeping the solution under the conditions of ultrahigh pressure and 500MPa for 12 min; after ultrahigh pressure treatment, transferring the nanoparticle solution into a dialysis bag with the molecular weight cutoff of 8,000-14000 kDa, and dialyzing for 5-10h to remove solvents such as ethanol, ethyl acetate and the like and unencapsulated nutrients.
Detecting to obtain EGCG-loaded burdock glycoprotein nanoparticle DZ286.44 nm; and adding 3mL of ethyl acetate into 0.5mL of nanoparticle solution, performing vortex extraction for 15min, continuously extracting for 3 times, combining ethyl acetate phases, detecting the content of the EGCG by using HPLC (high performance liquid chromatography), and calculating the encapsulation rate of the EGCG to be 56.95%.
Control group
The conventional common heating treatment (keeping at 90 ℃ for 10min) is adopted for replacing the ultrahigh pressure treatment (keeping the pressure at 400-600 MPa for 10-15 min) in the examples 1 and 2, and the rest conditions are not changed, namely the control group 1 is used as the control of the example 1, and the control group 2 is used as the control of the example 2.
(1) Control group 1
When the burdock glycoprotein is subjected to ultrasonic crushing treatment, the ultrasonic crushing treatment is carried out for 5min under the condition of 200W, namely ultrasonic crushing treatment a2400W ultrasonic crushing treatment for 5min and ultrasonic crushing treatment b2Ultrasonic crushing treatment for 5min under the condition of 600W is ultrasonic crushing treatment c2The results in the control group 1 and the example 1 are shown in the following table 2:
TABLE 2
The encapsulation efficiency of beta-carotene detected in control group 1 was 42.23%, which is significantly lower than the encapsulation efficiency of 65.34% in example 1, and this is related to the degradation of beta-carotene caused by heating, and also indicates that the existing delivery system is easy to affect the delivery effect due to different environments.
The sizes of the nanoparticles after the ultrasonic crushing pretreatment for 5min under the conditions of 200W, 400W and 600W are 425.18 nm, 304.39 nm and 261.03nm respectively, and are higher than those of the nanoparticles prepared by adopting ultrahigh pressure treatment (pressure maintaining for 15min under 400 MPa) under the same ultrasonic crushing condition, which indicates that the structure of the nanoparticles obtained by a heating method is not compact by the ultrahigh pressure method.
(2) Control group 2
In the control experiment of example 2, the conventional heating (10 min at 90 ℃) was also used instead of the ultrahigh pressure treatment (12 min at 500 MPa), and the rest conditions were not changed. The encapsulation efficiency of EGCG was detected to be 29.22%, which is significantly lower than that in example 2, which is related to degradation of EGCG by heating. The diameter of the nano-particles is 365.32nm, which is obviously higher than the diameter of the nano-particles prepared by the ultra-high pressure method.
The results show that the system can prepare the burdock glycoprotein nanoparticles by using ultra-high pressure treatment at normal temperature, effectively inhibit the activity loss of the core material in the encapsulating process and improve the encapsulating rate of the core material.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (8)
1. A preparation method of a nutrient delivery system based on burdock glycoprotein is characterized in that: the method comprises the following steps:
step 1: separating to obtain the burdock glycoprotein,
taking fresh burdock roots, cleaning, slicing, putting into boiling water for boiling and enzyme inactivation, taking out the burdock root slices subjected to enzyme inactivation, putting into an oven for drying, crushing by using an ultrafine crusher, weighing the obtained burdock powder, adding water according to a material-liquid ratio of 1: 15-1: 25 for uniform dispersion, adjusting the pH of the mixed solution to 5.0-7.0, adding alpha-amylase for hydrolysis, and then heating in a boiling water bath for enzyme inactivation; adjusting the pH of the mixed solution to 3.0-6.0, adding glucoamylase for hydrolysis, carrying out boiling water bath treatment for enzyme deactivation, centrifugally separating supernatant, concentrating to 1/3-1/4 of the volume of the supernatant, adding a mixed solvent of chloroform and n-butyl alcohol, stirring, centrifugally separating, removing an organic solvent layer and a denatured protein layer, repeating the operation for a plurality of times, adding absolute ethyl alcohol, standing at the temperature of 2-6 ℃, centrifugally separating to obtain a precipitate, and freeze-drying to obtain burdock glycoprotein;
step 2: carrying out ultrasonic crushing treatment on the burdock glycoprotein,
dissolving the burdock glycoprotein obtained in the step 1 in water to reach the concentration of 2-8 mg/mL, and carrying out ultrasonic crushing treatment under the condition of an external ice bath to obtain a burdock glycoprotein solution;
and step 3: the load of the nutrient is carried out,
dissolving water-soluble nutrients in distilled water to reach the concentration of 1-1.5%; dissolving fat-soluble nutrients in ethyl acetate to reach a concentration of 1-1.5%; continuously dropwise adding a nutrient-containing solution into the burdock glycoprotein solution under the condition of continuous magnetic stirring, and regulating and controlling the volume of the dropwise added nutrient solution according to the load amount to be not more than 2% of the volume of the burdock glycoprotein solution to obtain a mixed solution;
and 4, step 4: the assembly of the burdock glycoprotein nano-particles,
under the condition of magnetic stirring, continuously dropwise adding absolute ethyl alcohol into the mixed solution in the step 3 to enable the volume fraction of the ethyl alcohol to reach 5-50%, adjusting the pH of the mixed solution to 1.0-6.0 by using HCl, stirring at the temperature of 2-6 ℃, sealing in a polyethylene self-sealing bag, forming a nanoparticle solution with core-shell structure stable nanoparticles under the condition of ultrahigh pressure of 400-600 Mpa, transferring the nanoparticle solution into a dialysis bag with the molecular weight cutoff of 8,000-14000 kDa, and dialyzing to remove solvents such as ethyl alcohol and ethyl acetate and unencapsulated nutrients.
2. The method of preparing a burdock glycoprotein based nutrient delivery system according to claim 1, wherein: crushing the burdock root slices in the step 1 by using an ultrafine crusher, sieving by using a sieve of 80 meshes, weighing the obtained burdock powder, adding water according to the material-liquid ratio of 1:20, uniformly dispersing, adjusting the pH of the mixed solution to 6.0, adding low-temperature alpha-amylase accounting for 0.6% of the mass of the burdock powder, hydrolyzing at the temperature of 60 ℃ and the speed of 440rpm, and then heating in a boiling water bath to inactivate the enzyme; adjusting the pH of the mixed solution to 4.5, adding 1% saccharifying enzyme by mass of burdock powder, hydrolyzing at 60 deg.C and 440rpm, and inactivating enzyme by boiling water bath treatment; centrifuging to separate supernatant, concentrating at 40 deg.C under reduced pressure to 1/4, adding mixed solvent (4: 1 of chloroform and n-butanol) 1/3 volume of supernatant, stirring, centrifuging to remove organic solvent layer and denatured protein layer, and repeating for 5 times; adding anhydrous ethanol with 4 times volume of the aqueous phase, standing at 4 deg.C, centrifuging to obtain precipitate, and lyophilizing to obtain burdock glycoprotein.
3. The method of preparing a burdock glycoprotein based nutrient delivery system according to claim 1, wherein: dissolving the burdock glycoprotein in the step 2 in water to reach the concentration of 5mg/mL, carrying out ultrasonic crushing treatment under the condition of an external ice bath, and adjusting the power of ultrasonic crushing to be 200-800W and the time to be 2-10 min.
4. The method of preparing a burdock glycoprotein based nutrient delivery system according to claim 1, wherein: and (3) adjusting the power of ultrasonic crushing to 200W, 400W or 600W during ultrasonic crushing treatment in the step 2.
5. The method of preparing a burdock glycoprotein based nutrient delivery system according to claim 1, wherein: in the step 2, the power and time of ultrasonic crushing are adjusted to regulate the molecular mass and chain length of the glycoprotein.
6. The method of preparing a burdock glycoprotein based nutrient delivery system according to claim 1, wherein: in the step 4, under the condition of magnetic stirring, the volume fraction of the anhydrous ethanol continuously dropwise added into the mixed solution in the step 3 reaches 25%, then the mixed solution is adjusted to pH 4.5 by using 1mol/L HCl, stirred for 12-15 h at 4 ℃, sealed in a polyethylene self-sealing bag, kept for 10-15 min under the condition of 400-600 MPa under the condition of ultrahigh pressure to form core-shell structured nanoparticles, subjected to ultrahigh pressure treatment, transferred into a dialysis bag with the molecular weight cutoff of 8,000-14000 kDa, and dialyzed for 5-10h to remove solvents such as ethanol and ethyl acetate and unencapsulated nutrients.
7. A burdock glycoprotein based nutrient delivery system, which is characterized in that: made by the method of any one of claims 1-6.
8. Use of a burdock glycoprotein based nutrient delivery system, characterized in that: loading nutrient or medicine into the burdock glycoprotein-based nutrient delivery system of claims 1-6, assembling, making into liquid or lyophilized powder, and adding into food or beverage.
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