CN111420064A

CN111420064A - protein-EGCG composite nanoparticle and antioxidant Pickering high internal phase emulsion

Info

Publication number: CN111420064A
Application number: CN202010315392.6A
Authority: CN
Inventors: 唐传核; 彭丽萍
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-04-21
Filing date: 2020-04-21
Publication date: 2020-07-17
Anticipated expiration: 2040-04-21
Also published as: CN111420064B

Abstract

The invention discloses a protein-EGCG composite nanoparticle and an antioxidant Pickering high internal phase emulsion, wherein the preparation method of the protein-EGCG composite nanoparticle comprises the following steps of 1) adding water into soybean β -globulin for dispersing, and hydrating to obtain a protein solution, 2) adding water into EGCG for dispersing to obtain an EGCG solution, 3) adding the protein solution into a polar organic solvent to denature protein, and then adding the EGCG solution to obtain a mixed solution, and 4) dialyzing the mixed solution to remove the polar organic solvent and free EGCG to obtain the protein-EGCG composite nanoparticle.

Description

protein-EGCG composite nanoparticle and antioxidant Pickering high internal phase emulsion

Technical Field

The invention relates to a protein-EGCG composite nano-particle and an antioxidant Pickering high internal phase emulsion.

Background

Epigallocatechin gallate (EGCG) is catechin monomer separated from folium Camelliae sinensis, has antibacterial, antiviral, antioxidant, arteriosclerosis resisting, thrombosis resisting, angiogenesis resisting, antiinflammatory, and antitumor effects, and can be used for preventing diseases such as thrombosis, cancer, diabetes, cardiovascular disease, and neurodegenerative disease. Therefore, it is an intrinsic need to develop health industry to develop some functional foods by utilizing their excellent properties. However, EGCG has poor stability, is susceptible to temperature, pH and oxygen, and has low oral bioavailability, which also makes such polyphenol actives lack significant efficacy in clinical applications.

Researches show that the bioavailability of EGCG can be effectively improved by a nano embedding technology (namely, active substances are embedded in the interior of nanoparticles or adsorbed on the surfaces of the nanoparticles). Food protein-based nanoparticles have the advantages of being non-toxic, degradable, low in price, good in biocompatibility and the like, and are therefore often used as nano-carriers to improve the stability and bioavailability of active substances (including polyphenols). However, the existing protein-based nano-carrier preparation methods such as covalent cross-linking, nano-compounding, emulsification-evaporation and the like generally have the problems of low loading capacity, burst drug release, use of cross-linking agents and the like, for example: CN 110393683A discloses that tea polyphenol is packaged and carried under specific conditions by adopting food protein modified by multiple physical inducements as a carrier, and then a ternary composite stable system is constructed by combining chitosan to improve the stability and the activity of the tea polyphenol, but the preparation process is complex, time-consuming and labor-consuming, high in cost and difficult to popularize and apply. Therefore, how to prepare the carrier with low cost, high drug loading, good stability and small biological toxicity by a simple process is a problem to be solved urgently in the field of food and medicines at present.

High internal phase emulsions (oil phase volume not less than 74%) have been widely used in the fields of cosmetics, foods, pharmaceuticals, and chemical industry. In recent years, the use of protein-based particles with characteristics of low cost, small dosage, environmental friendliness, high safety and the like as a stabilizer for Pickering high internal phase emulsions (Pickering HIPEs) has become a hot direction of research. However, the high internal phase emulsion has limited application in food because of high oil content and easy oxidative deterioration of oil phase, and many nutrients are fat-soluble active substances, which are not easy to be absorbed by human body or lose efficacy in human body too early, and are difficult to be slowly released to play a targeting role. In addition, food products are susceptible to various external factors during processing, storage and transportation, such as: temperature, humidity, etc. Therefore, how to develop a Pickering stabilizer with good stability and strong oxidation resistance to prepare an antioxidant Pickering high internal phase emulsion is a problem to be solved urgently in the field of food and medicines at present.

Disclosure of Invention

The invention aims to provide a protein-EGCG composite nano-particle and an antioxidant Pickering high internal phase emulsion.

The technical scheme adopted by the invention is as follows:

a preparation method of protein-EGCG composite nanoparticles comprises the following steps:

1) dispersing soybean β -conglobulin in water, and hydrating to obtain protein solution;

2) adding water into EGCG for dispersing to obtain EGCG solution;

3) adding the protein solution into a polar organic solvent to denature the protein, and adding an EGCG solution to obtain a mixed solution;

4) and dialyzing the mixed solution to remove the polar organic solvent and the free EGCG so as to obtain the protein-EGCG composite nano-particles.

Preferably, the mass ratio of the soybean β -conglobulin to the EGCG is 1 (0.01-0.3).

Preferably, the concentration of the protein solution in the step 1) is 0.5 wt% to 10 wt%.

Preferably, the following operations are also performed before the hydration in step 1): adding a sodium azide solution with the mass fraction of 0.08%, adjusting the pH of the solution to 7, stirring uniformly, and centrifuging to remove insoluble impurities.

Preferably, the concentration of the EGCG solution in the step 2) is 0.2-2 wt%.

Preferably, the concentration of the soybean β -conglobulin in the mixed solution in the step 3) is 2-20 mg/m L.

Preferably, the volume percentage of the polar organic solvent in the mixed solution in the step 3) is 30-40%.

Preferably, the polar organic solvent in step 3) is one of ethanol, methanol and acetone.

Preferably, the dialysis temperature in the step 4) is 2-6 ℃, and the dialysis time is 64-72 hours.

Preferably, the dialysate used for dialysis in step 4) is deionized water, and is replaced every 8 h.

A protein-EGCG composite nanoparticle is prepared by the method.

An antioxidant Pickering high internal phase emulsion comprises the protein-EGCG composite nano-particles as a stabilizer.

Preferably, the volume ratio of the dispersed phase to the continuous phase in the antioxidant Pickering high internal phase emulsion is 4: 1.

The principle of the invention is as follows: under the action of a polar organic reagent, the conformation of a natural protein structure is changed, the protein structure is unfolded and denatured, so that hydrophobic sites buried in the protein are exposed on the surface, and then in the process of removing the polar organic reagent and free EGCG through dialysis, the exposed hydrophobic sites of the protein pull the EGCG to be enriched in the inner core of the protein self-assembly carrier, so that the stability of an active substance is improved, and new functional characteristics are endowed to particles.

The invention has the beneficial effects that: the protein-EGCG composite nano-particle has good biocompatibility, and the Pickering high internal phase emulsion prepared by using the protein-EGCG composite nano-particle as a stabilizer has excellent antioxidant property, thermal stability and storage stability.

Specifically, the method comprises the following steps:

1) the protein-EGCG composite nano-particle has good biocompatibility, and both the soybean β -conglobulin (β -CG) and the EGCG are rich in nutrition and wide in source, and are beneficial to human health;

2) the protein-EGCG composite nano-particles can efficiently stabilize Pickering high internal phase emulsion, can endow the Pickering high internal phase emulsion with excellent antioxidant property, can inhibit the oxidation of grease, and can be used as a potential protective container and a delivery system of fat-soluble active substances (such as β -carotene), and meanwhile, the Pickering high internal phase emulsion has good thermal stability and storage stability;

3) the preparation method of the protein-EGCG composite nano-particles is simple, safe, low in cost, low in energy consumption, controllable in operation, suitable for large-scale industrial production and processing, and has wide application prospects in the industries of foods, health products, daily chemical products and medicines.

Drawings

FIG. 1 is an appearance diagram of a nanoparticle solution obtained in example 1, wherein the concentration of soybean β -conglobulin is 1 wt%, the volume fraction of ethanol is 40%, and the concentration of EGCG is 0-0.3 wt% in sequence before and after dialysis.

FIG. 2 is a graph showing the hydrated particle size of nanoparticles obtained in example 1, in which the concentration of soybean β -conglobulin is 1 wt%, the volume fraction of ethanol is 40%, and the concentration of EGCG is 0 to 0.3 wt% in this order.

FIG. 3 is a graph of the load amount of β -CG to EGCG in the protein-EGCG composite nanoparticle obtained when the concentration of soybean β -conglobulin is 1 wt%, the volume fraction of ethanol is 40%, and the concentration of EGCG is 0.05 wt% -0.3 wt% in sequence in example 1.

FIG. 4 is a diagram illustrating the mechanism of nanoparticle formation when the concentration of soybean β -conglobulin is 1 wt%, the volume fraction of ethanol is 40%, and the concentration of EGCG is 0-0.3 wt% in sequence in example 1.

Fig. 5 is an appearance view and an optical microscope image of a Pickering high internal phase emulsion in example 2.

FIG. 6 is a graph of the elastic modulus (G', solid) and viscous modulus (G ", hollow) as a function of frequency for the Pickering high internal phase emulsion of example 2.

Fig. 7 is an appearance view and an optical microscope image of the Pickering high internal phase emulsion in example 2 before and after being stored at room temperature for 50 days.

Fig. 8 is an appearance view and an optical microscopic view of the Pickering high internal phase emulsion before and after heat treatment and an appearance view after one freeze-thaw treatment in example 2.

FIG. 9 is a graph of the content of primary oxidation product hydroperoxide and secondary oxidation product malondialdehyde produced in the Pickering high internal phase emulsion of example 2 over time during storage in a 50 ℃ incubator for 21 days.

FIG. 10 is an appearance of the Pickering high internal phase emulsion of example 3 after heat treatment.

FIG. 11 is a graph of the retention of β -carotene after heat treatment of the Pickering high internal phase emulsion of example 3.

Detailed Description

The invention will be further explained and illustrated with reference to specific examples.

Example 1:

a nanoparticle, the method of making comprising the steps of:

1) dispersing 5g of soybean β -conglobulin powder in 100g of distilled water, stirring at room temperature for 2h, adding 1 drop of 0.08 mass percent sodium azide solution (to prevent the growth of microorganisms), adjusting the pH value of the solution to 7 by using 0.2 mol/L HCl solution and 0.2 mol/L NaOH solution, stirring for 2h, putting the solution in a refrigerator at 4 ℃ overnight, taking the solution out of the refrigerator, returning the solution to room temperature, and centrifuging (8000rpm, 15min) to remove insoluble impurities to obtain a protein solution with the concentration of 5 wt%;

2) dispersing 0.4g of EGCG powder in 20g of distilled water, stirring for 2h at room temperature, and placing in a refrigerator at 4 ℃ overnight to obtain EGCG solution with the concentration of 2 wt%;

3) taking 5 goblet samples No. 1, 2, 3, 4 and 5 with the capacity of 50m L, adding 20m L of absolute ethyl alcohol into all the beakers, sequentially adding 20m L, 18.75m L, 17.5m L, 15m L and 12.5m L of deionized water into the samples No. 1, 2, 3, 4 and 5, uniformly stirring, then respectively adding 10m L of protein solution, uniformly stirring, then dropwise adding EGCG solution while stirring, sequentially adding 0m L, 1.25m L, 2.5m L, 5m L and 7.5m L of EGCG solution into the 5 samples, enabling the protein concentration in the final system to be 10mg/m L, the volume fraction of the ethyl alcohol to be 40%, the concentration of the EGCG to be 0mg/m L, 0.5mg/m L, 0.1mg/m L, 0.2mg/m L mg/m 632 and 6863 h, and continuously stirring to obtain a mixed solution;

4) and (3) dialyzing the mixed solution in a refrigerator at 4 ℃ for three days (the dialyzate is distilled water, the cut-off molecular weight of a dialysis bag is 3500Da, and the dialyzate is replaced every 8 hours), and adjusting the pH value to 7 to obtain a nanoparticle solution (the sample No. 1 is protein particles, and the samples No. 2-5 are protein-EGCG composite nanoparticles).

And (3) performance testing:

the appearance figures of the nano-particle solution obtained when the concentration of the soybean β -conglobulin is 1 wt%, the volume fraction of the ethanol is 40% and the concentration of the EGCG is 0-0.3 wt% in sequence before and after dialysis are shown in figure 1.

As can be seen from fig. 1: the appearance and turbidity of the protein particle solution of the sample No. 1 and the protein-EGCG composite nanoparticle solution of the sample No. 2-5 before and after dialysis are not obviously changed, and the samples are transparent, so that nanoscale particles are formed.

The hydrated particle size of the nanoparticles obtained when the concentration of the soybean β -conglobulin is 1 wt%, the volume fraction of the ethanol is 40%, and the concentration of the EGCG is 0-0.3 wt% in sequence is shown in FIG. 2 (a-e in the figure are marked by significant differences).

As can be seen from fig. 2: the average particle diameters of the protein particles of the sample No. 1 and the protein-EGCG composite nanoparticles of the sample No. 2-5 are 25.7nm, 34.43nm, 41.16nm, 47.46nm and 62.12nm in sequence.

The load quantity graph of β -CG to EGCG in the protein-EGCG composite nano-particles obtained when the concentration of the soybean β -conglobulin is 1 wt%, the volume fraction of the ethanol is 40% and the concentration of the EGCG is 0.05 wt% -0.3 wt% in sequence is shown in figure 3.

As can be seen from FIG. 3, the loading amounts of β -CG to EGCG in the protein-EGCG composite nanoparticles of samples No. 2-5 are 3.32%, 4.92%, 7.02% and 8.99% in sequence.

The forming mechanism diagram of the nano-particles is shown in figure 4 when the concentration of the soybean β -conglobulin is 1 wt%, the volume fraction of the ethanol is 40%, and the concentration of the EGCG is 0-0.3 wt% in sequence.

Example 2:

a Pickering high internal phase emulsion is prepared by the following steps:

the protein particle solution of the sample No. 1 and the protein-EGCG composite nano particle solution of the sample No. 2-5 in the example 1 are respectively mixed with linseed oil according to the volume ratio of 1:4, added into a homogenizer, and homogenized at 5000rpm for 1min to obtain Pickering high internal phase emulsion (the Pickering high internal phase emulsion corresponding to the sample No. 1 does not have an antioxidant function, and the Pickering high internal phase emulsion corresponding to the sample No. 2-5 has an antioxidant function).

And (3) performance testing:

the appearance and optical microscopy of Pickering high internal phase emulsions is shown in FIG. 5.

The elastic modulus (G', solid) and viscous modulus (G ", hollow) of Pickering high internal phase emulsions as a function of frequency are shown in figure 6.

As can be seen from fig. 5 and 6: the Pickering high internal phase emulsion is stable, the size of the emulsion drops is uniform, the emulsion drops do not flow when being inverted, the gel emulsion mainly comprising elastic modulus is formed, and the optical microscope observes that most of the emulsion drops are closely packed and two adjacent emulsion drops can be seen to share the same interface layer.

Appearance and optical microscopy of Pickering high internal phase emulsions before and after 50 days storage at room temperature are shown in figure 7.

As can be seen from fig. 7: the appearance and microstructure of the Pickering high internal phase emulsion are not obviously changed after storage, and the storage stability is excellent.

The appearance and optical microscopy before and after Pickering high internal phase emulsion heat treatment (100 ℃ C. for 15min → water bath for 15min) and the appearance after one freeze-thaw treatment (-20 ℃ C. for 24h → 25 ℃ C. for 2h) are shown in FIG. 8.

As can be seen from fig. 8: after heat treatment, the Pickering high internal phase emulsion can still be inverted and does not flow, the phenomena of emulsion breaking and oil leakage are avoided, the appearance and the microstructure are not greatly changed, and the thermal stability is better; the Pickering high internal phase emulsion has higher freeze-thaw stability after heat treatment, can still keep the original state after one freeze-thaw, and only has a few cases of emulsion breaking and oil leakage, while the Pickering high internal phase emulsion which is not subjected to heat treatment is subjected to one freeze-thaw, namely emulsion breaking and oil leakage, which shows that the heat treatment can improve the freeze-thaw stability of the Pickering high internal phase emulsion, and fully shows that the Pickering high internal phase emulsion has excellent storage stability and thermal stability.

The time-dependent changes in the contents of the primary oxidation product hydroperoxide and the secondary oxidation product malondialdehyde generated in the Pickering high internal phase emulsion of example 2 during 21 days of storage in a 50 ℃ incubator are shown in FIG. 9 (blank control is flaxseed oil in bulk, sample control is Tween-20 stable high internal phase emulsion with a concentration of 2 wt%).

As can be seen from fig. 9: after being stored for 21 days at 50 ℃, the high internal phase emulsion stabilized by No. 1 particles, the high internal phase emulsion stabilized by No. 2, No. 3, No. 4 and No. 5 particles (protein-EGCG composite nano-particles), the high internal phase emulsion stabilized by Tween 20(2.0wt percent), and the grease generated by the loose linseed oil after being stored for 21 days at 50 ℃, wherein the hydroperoxide content is 273.49mmol/kg, 170.83mmol/kg, 130.96mmol/kg, 101.86mmol/kg, 71.20mmol/kg, 1057.24mmol/kg and 763.07mmol/kg, the secondary oxidation products, namely the malonaldehyde content is 1.51mmol/kg, 1.21mmol/kg, 0.78mmol/kg, 0.65mmol/kg, 0.47mmol/kg, 4.64mmol/kg and 3.79mmol/kg in turn, can be found that the grease oxidation inhibition capacity is obviously enhanced after the EGCG load is increased and the EGCG concentration is increased, is beneficial to delaying the oxidation of the grease in the storage and transportation process and improving the stability of the grease.

Example 3:

referring to the procedure of example 2, a Pickering high internal phase emulsion was prepared by replacing the dispersed phase linseed oil with n-dodecane with 0.003 wt% β -carotene.

And (3) performance testing:

the appearance of the Pickering high internal phase emulsion after heat treatment (storage in an electric thermostat at 70 ℃ for 18h) is shown in FIG. 10, and the retention of β -carotene is shown in FIG. 11 (in the figure, a-f are marked by significant differences).

As shown in the graphs of FIG. 10 and FIG. 11, after 18h of storage at 70 ℃, the high internal phase emulsion stabilized by No. 1 (without EGCG), the high internal phase emulsion stabilized by No. 2, No. 3, No. 4 and No. 5 particles (protein-EGCG composite nanoparticles), the high internal phase emulsion stabilized by Tween 20(2.0 wt%), and the high internal phase emulsion stabilized by Tween 20(2.0 wt%), have the retention rates of β -carotene in n-dodecane which is the same volume of dispersion liquid of 88.8%, 91.3%, 93.9%, 95.1%, 96.8%, 9.53% and 67.2%, respectively, so that the soybean β -conglobulin-EGCG assembly nanoparticles can effectively protect fat-soluble active substances in grease and prevent the fat-soluble active substances from thermal degradation, and the protection effect is in direct proportion to the concentration of EGCG, namely the protection effect is more obvious when the concentration is higher.

In conclusion: the particles prepared by using ethanol to induce protein self-assembly loading EGCG are in a nanometer grade, when the concentration of the EGCG is 0.3 wt%, the protein loading amount reaches 8.99 wt%, the particle size of a sample is increased along with the increase of the concentration of the EGCG, and the EGCG-loaded particles are usedThe constructed nanoparticles are stable in high internal phase emulsion (c)_β-CG1 wt% and phi 0.8), and endows the fat-soluble active substance with excellent antioxidant function on the basis of original good thermal stability and storage stability, can effectively protect the oxidative degradation of β -carotene serving as a fat-soluble active substance and effectively inhibit primary oxidation and secondary oxidation of grease, and has wide application prospect and potential application value.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A preparation method of protein-EGCG composite nano-particles is characterized by comprising the following steps: the method comprises the following steps:

2) adding water into EGCG for dispersing to obtain EGCG solution;

2. The preparation method according to claim 1, wherein the mass ratio of the soybean β -conglobulin to the EGCG is 1 (0.01-0.3).

3. The production method according to claim 1 or 2, characterized in that: step 1) the concentration of the protein solution is 0.5 wt% -10 wt%; the concentration of the EGCG solution in the step 2) is 0.2-2 wt%.

4. The method according to claim 1 or 2, wherein the concentration of the soybean β -conglobulin in the mixture of step 3) is 2-20 mg/m L.

5. The production method according to claim 1 or 2, characterized in that: the volume percentage of the polar organic solvent in the mixed solution in the step 3) is 30-40%.

6. The production method according to claim 1 or 2, characterized in that: and 3) the polar organic solvent is one of ethanol, methanol and acetone.

7. The production method according to claim 1 or 2, characterized in that: and 4) dialyzing at the temperature of 2-6 ℃ for 64-72 h.

8. A protein-EGCG composite nanoparticle, which is characterized in that: prepared by the method of any one of claims 1 to 7.

9. An antioxidant Pickering high internal phase emulsion is characterized in that: the stabilizer is the protein-EGCG composite nanoparticle of claim 8.

10. The antioxidant Pickering high internal phase emulsion of claim 9, wherein: the volume ratio of the dispersed phase to the continuous phase was 4: 1.