CN116831287A

CN116831287A - Pea protein-curcumin nanoparticle and preparation method thereof

Info

Publication number: CN116831287A
Application number: CN202310746065.XA
Authority: CN
Inventors: 谷瑞增; 刘文颖; 李国明; 卢知浩; 任杰; 张新雪; 吴晗硕; 付少委; 陈亮; 王憬; 毕园
Original assignee: China National Research Institute of Food and Fermentation Industries
Current assignee: China National Research Institute of Food and Fermentation Industries
Priority date: 2023-06-21
Filing date: 2023-06-21
Publication date: 2023-10-03

Abstract

The application discloses pea protein-curcumin nano-particles and a preparation method thereof, comprising the following steps: adding pea protein into distilled water, magnetically stirring, and standing to obtain pea protein suspension; adjusting the pH value of the pea protein suspension to 12-13 by using a NaOH solution, after magnetic stirring and balancing, adding a curcumin solution into the pea protein suspension, and stirring in a dark place at room temperature to obtain a mixed solution; adjusting the pH value of the mixed solution to 7-8 by using an HCl solution, stirring in a dark place, centrifuging the sample solution, and dialyzing the supernatant to obtain pea protein-curcumin nano-particles; compared with the pea protein with reduced particle size, the prepared pea protein-curcumin nanoparticle has the advantages of changed secondary structure, higher stability, higher bioactivity and higher bioavailability compared with free curcumin.

Description

Pea protein-curcumin nanoparticle and preparation method thereof

Technical Field

The application relates to the field of nano biological products, in particular to pea protein-curcumin nano particles and a preparation method thereof.

Background

Curcumin has very pleasant aroma and good coloring effect, and is widely used as a colorant and a spice to be added into foods. Curcumin is currently one of the most commercially available natural food colors and is approved for use by health organizations, such as the world health organization and the U.S. food and drug administration, both nationally and worldwide. Curcumin is produced in large quantities in asian countries such as japan, india, china, etc., and has a very long history and a very wide range of uses in asian countries. According to Chinese food additive use Standard, curcumin can be added to carbonated beverages and snacks according to production. Curcumin has been shown to have antioxidant, anti-aging, anti-inflammatory and anti-cancer effects. In particular, curcumin has the anticancer capability, is nontoxic and harmless to human bodies, is efficient and low in cost because of the natural advantages of plant extraction, and is regarded as one of potential cancer treatment means. Up to now, more than 100 curcumin-related clinical trials have been completed, demonstrating their safety.

The active methylene and β -diketone molecules subject to curcumin render them poorly soluble in water, thus making their bioavailability, stability poor. Curcumin has extremely low bioavailability in the human body of about 1%, i.e. most of curcumin taken orally is present in feces or urine, and only a very small part can be detected in blood. Curcumin has low water solubility, especially in acidic and neutral environments, and is susceptible to inactivation by gastric acid and bile acid in the stomach, and low permeability in the small intestine, several factors together leading to very low bioavailability of curcumin in the human body. Meanwhile, curcumin is rapidly metabolized in the intestinal tract after oral administration into several reduction products (di-, tetra-, hexahydrocurcumin and hexahydrocurcumol) and its glucuronic acid or sulfuric acid conjugates.

In order to improve the water solubility and chemical stability of curcumin, researchers have proposed a number of strategies such as nano-delivery systems of emulsions, nanoparticles, liposomes, microcapsules and micelles to encapsulate curcumin. These delivery systems are mainly prepared from natural polymers, for example, curcumin is complexed with nanoparticles of inorganic particles, polysaccharides, proteins, etc. to improve the water dispersibility and chemical stability of curcumin, especially protein-based nanoparticles, which have been widely used as nanocarriers of curcumin due to their non-toxicity and biocompatibility, and are simple to prepare without complex equipment and toxic chemicals, and at the same time, protein-based nanoparticles can effectively improve the solubility and bioavailability of curcumin in fat-free environments, making them suitable for application in functional food and beverage systems.

Chinese patent document 201810668281.6 discloses a pea protein-curcumin nano-composite, a preparation method and application thereof, wherein the weight ratio of pea protein to curcumin in the composite is 25:1-60:1. The preparation method comprises the following steps: (1) Weighing a certain amount of pea protein, dispersing in distilled water or deionized water, homogenizing under stirring, performing microfluidization treatment, centrifuging, collecting suspension, and lyophilizing to obtain pea protein powder; (2) Optimizing the condition of the microfluidization method by using the pea protein powder and curcumin, and selecting the optimal microfluidization condition; (3) Preparing a pea protein-curcumin nanocomposite emulsion using a microfluidization method according to the optimal microfluidization conditions in the step (2), and then freeze-drying the emulsion to prepare a pea protein-curcumin nanocomposite powder; the process steps are complex and are not suitable for large-scale industrial production.

Disclosure of Invention

In order to solve the defects in the prior art, the application aims to provide the pea protein-curcumin nanoparticle and the preparation method thereof, which have the advantages of simple process operation, no introduction of organic reagent, safety, no toxic or side effect and capability of effectively improving the stability, the bioactivity and the bioavailability of curcumin.

In order to achieve the above purpose, the present application adopts the following technical scheme:

a pea protein-curcumin nanoparticle and a preparation method thereof comprise the following steps:

(1) Adjusting the pH value of the pea protein suspension by using a NaOH solution, after magnetic stirring and balancing, adding a curcumin solution into the pea protein suspension, and stirring in a dark place at room temperature to obtain a mixed solution;

(2) And regulating the pH of the mixed solution by using an HCl solution, stirring in a dark place, centrifuging the sample solution, and dialyzing the supernatant to obtain the pea protein-curcumin nano-particles.

Preferably, the pea protein suspension is prepared as follows: adding pea protein into distilled water, magnetically stirring, and standing to obtain pea protein suspension.

Preferably, the magnetic stirring condition is room temperature and 500-600 r/min for stirring for 1-2 h, and the standing condition is 4 ℃ for standing for 14-18 h.

Preferably, in step (1), the concentration of the pea protein suspension is 40-60 mg/mL.

Preferably, in the step (1), the concentration of the NaOH solution is 1-2 mol/L, the pH of the pea protein suspension is adjusted to be 12-13, and the magnetic stirring condition is 500-600 r/min and the stirring is carried out for 1-2 h.

Preferably, in the step (1), the concentration of the curcumin solution is 0.4-0.8 mg/mL, and the curcumin solution is stirred for 30-60 min under the light-shielding stirring condition of 500-600 r/min.

Preferably, in the step (2), the concentration of the HCl solution is 1-2 mol/L, the pH of the mixed solution is regulated to 7-8, and the mixture is magnetically stirred for 1-2 hours in a dark place under the condition of 500-600 r/min.

Preferably, in the step (2), the centrifugation condition is 20-25 ℃ and the centrifugation is carried out for 15-30 min at 8000 r/min.

Preferably, in step (2), the dialysis conditions are dialysis in a 8000u dialysis bag for 18-22 hours.

Preferably, the pea protein-curcumin nanoparticles are stored at 4 ℃.

The application also discloses pea protein-curcumin nano-particles prepared by the preparation method.

Compared with the prior art, the application has the following beneficial effects:

the pea protein-curcumin nanoparticle prepared by the method can effectively protect curcumin molecules wrapped by the pea protein, slow down the thermal decomposition rate of the pea protein, improve the ultraviolet light stability and gastrointestinal digestion retention rate of the pea protein, and change the chemical stability of reactive groups in curcumin by forming a compound with the curcumin; compared with the pea protein with reduced particle size, the prepared pea protein-curcumin nanoparticle has the advantages of changed secondary structure, higher stability, higher bioactivity and higher bioavailability compared with free curcumin.

The application provides a preparation method of pea protein-curcumin nano-particles, which can obtain pea protein-curcumin nano-particles with high curcumin loading rate by adjusting the pH value of pea protein suspension to 12-13 and adjusting the pH value of mixed solution to 7-8.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a scanning electron microscope (A is the morphology of pea protein; B is the microscopic morphology of pea protein-curcumin nanoparticles) of PPI, PPI-Cur;

FIG. 2 is a graph showing pea protein and pea protein-curcumin circular dichroism and secondary structure composition (A is a graph showing pea protein and pea protein-curcumin circular dichroism; B is a graph showing pea protein and pea protein-curcumin secondary structure composition);

FIG. 3 shows the results of heat treatment experiments on curcumin, pea protein-curcumin nanoparticles;

FIG. 4 shows the results of the ultraviolet stability test of curcumin, pea protein-curcumin nanoparticles;

fig. 5 is a graph showing the results of a simulated gastrointestinal stability experiment of curcumin, pea protein-curcumin nanoparticles.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. Of course, the specific embodiments described herein are merely illustrative of the application and are not intended to limit the application.

Unless otherwise specified, both chemical reagents and materials in the present application are purchased through a market route or synthesized from raw materials purchased through a market route.

Pea protein powder (PPI, > 72% protein) was purchased from the tobacco stand Oriental protein technologies Co., ltd;

curcumin (Cur, > 98%) was purchased from Saen chemical technology Co., ltd (Shanghai, china).

The application will be further illustrated by the following examples.

Example 1

(1) Adding pea protein into distilled water, magnetically stirring at room temperature and 500r/min for 1h, and standing at 4deg.C for 16h to obtain pea protein suspension.

(2) Adjusting the pH value of 50mg/mL pea protein suspension to 13 by using 1mol/L NaOH solution, magnetically stirring at 500r/min for 1h, adding 0.6mg/mL curcumin solution into the pea protein suspension, and stirring at room temperature at 500r/min in a dark place for 30min to obtain a mixed solution;

(3) Adjusting the pH of the mixed solution to 8 by using 1mol/L HCl solution, magnetically stirring for 1h at 500r/min in a dark place, centrifuging the sample solution at 25 ℃ for 15min at 8000r/min, and dialyzing the supernatant in a 8000u dialysis bag for 20h to obtain the pea protein-curcumin nano-particles.

Example 2

(3) Adjusting the pH of the mixed solution to 7 by using 1mol/LHCl solution, magnetically stirring for 1h at 500r/min in a dark place, centrifuging the sample solution at 25 ℃ for 15min at 8000r/min, and dialyzing the supernatant in a 8000u dialysis bag for 20h to obtain the pea protein-curcumin nano-particles.

Example 3

(2) Adjusting the pH value of 50mg/mL pea protein suspension to 13 by using 1mol/L NaOH solution, magnetically stirring at 500r/min for 1h, adding 0.4mg/mL curcumin solution into the pea protein suspension, and stirring at room temperature at 500r/min in a dark place for 30min to obtain a mixed solution;

Example 4

Comparative example 1

(3) Adjusting the pH of the mixed solution to 6 by using 1mol/L HCl solution, magnetically stirring for 1h at 500r/min in a dark place, centrifuging the sample solution at 25 ℃ for 15min at 8000r/min, and dialyzing the supernatant in a 8000u dialysis bag for 20h to obtain the pea protein-curcumin nano-particles.

Comparative example 2

(2) Adjusting the pH value of 50mg/mL pea protein suspension to 9 by using 1mol/L NaOH solution, magnetically stirring at 500r/min for 1h, adding 0.4mg/mL curcumin solution into the pea protein suspension, and stirring at room temperature at 500r/min in a dark place for 30min to obtain a mixed solution;

(3) Adjusting the pH of the mixed solution to 6 by using 1mol/LHCl solution, magnetically stirring for 1h at 500r/min in a dark place, centrifuging the sample solution at 25 ℃ for 15min at 8000r/min, and dialyzing the supernatant in a 8000u dialysis bag for 20h to obtain the pea protein-curcumin nano-particles.

For the pea protein-curcumin nanoparticles prepared in examples 1-4, comparative examples 1-2, 0.2mL of the curcumin loaded pea protein-curcumin nanoparticles were mixed with 1.8mL of ethanol and vigorously shaken and vortexed. The mixture was centrifuged at 25℃at 10000r/min for 15min, and the obtained supernatant was then diluted appropriately with 95% ethanol and measured at 426nm using an ultraviolet-visible spectrophotometer. The standard curve of free curcumin (0-10 μg/mL) dissolved in 95% ethanol was used to calculate the measured curcumin loading concentration.

Curcumin loading was calculated using formula (1):

the specific results are shown in Table 1.

TABLE 1 curcumin loading Rate

In comparative examples 1-2, the loading rate of curcumin is less than 5%, which indicates that the hydrophobic groups in the pea protein cannot be fully developed in the weak alkaline environment with pH values of 9, 10 and 11, so that the pea protein is not fully contacted with curcumin, the contact area with curcumin molecules is reduced, the amount of adhered curcumin is reduced in a fixed time, and the curcumin load is affected; in a weakly acidic environment at pH 6, the absence of a "ball-melt" structure also affects the loading of pea proteins with curcumin.

The pea protein-curcumin nanoparticle prepared in example 1 was subjected to a structure characterization study as follows, and its stability and bioavailability under heat treatment, uv treatment, simulated gastrointestinal environment were evaluated by experiments.

(1) Particle size

In the evaluation of nano-delivery vehicles, particles having an average particle diameter of 1 to 1000nm are strictly referred to as nanoparticles. The size of the nanoparticles is closely related to the delivery characteristics of the bioactive substance and to its synergy with respect to bioavailability. When the particle size of the carrier is less than 500nm, the bioavailability of the delivered bioactive substance can be significantly improved. After diluting the pea protein, pea protein-curcumin nanoparticles to the appropriate concentration, their average particle size and zeta potential were measured using a laser particle sizer, and specific data are presented in table 2. The average particle size of the pea protein is 1619+/-34.22 nm, and the average particle size of the pea protein-curcumin nano particle prepared by the method provided by the application is 254.5 +/-9.0 nm, so that the particle size is reduced by about 85% after the treatment. The Zeta potential is an indicator of the surface charge of the nanoparticle, in general, the smaller the molecule or dispersion particle, the higher the absolute value of Zeta potential, the more stable the system, the Zeta potential of pea protein is-28.7 + -0.71 mV, after loading with curcumin, the Zeta potential of pea protein-curcumin nanoparticle becomes significantly (p < 0.05) to-39.7 + -0.14 mV, which demonstrates the binding of curcumin to pea protein molecules and the pea protein-curcumin system is stable.

TABLE 2 average particle size, zeta potential of pea protein, pea protein-curcumin

	Z-Average/d.nm	Zeta/mV
			Pea protein	1619±34.22 ^a	-28.7±0.71 ^b
Pea protein-curcumin	254.5±9.0 ^b	-39.7±0.14 ^a

(2) Scanning electron microscope

The morphological characteristics of pea protein and pea protein-curcumin nano-particles are observed by using a scanning electron microscope, a metal spraying sample is adopted, the sample powder is subjected to a metal spraying operation, so that the sample powder is covered by a layer of metal, and then the microstructure of the sample is observed at 3 kv.

Fig. 1A is an SEM photograph of a pea protein sample using a secondary electron imaging mode with an acceleration voltage of 3kV and a magnification of 1000 x. The sample is mainly composed of irregularly shaped particles of varying sizes, the size distribution of which ranges from about 10 to 50 μm. At the same time the surface of these particles is smooth, with varying degrees of surface relief, which should be the protein particles in the sample. Some relatively small sized particles are tightly packed with other particles due to interaction forces and even adsorb to the surface of other larger particles.

Fig. 1B is an SEM photograph of a pea protein-curcumin sample using a secondary electron imaging mode with an acceleration voltage of 3kV and a magnification of 20000 x. It is clearly observed that the surface of the protein particles is distributed with a plurality of granular nano-structures with small size, and the size of the nano-particles is about 200 nm. Because the surface of the protein particles is smooth and the surface area is large, the tiny nano particles have better dispersibility, and no obvious agglomeration phenomenon occurs between the nano particles.

(3) Two-stage structure

Pea protein-curcumin nanoparticles, pea protein were diluted to equal concentration and scanned using circular dichroism. The step length is 0.2nm, the bandwidth is 2.0nm, and the spectrum range is 180-260 nm.

The secondary structure of pea protein-curcumin nanoparticles was studied by means of circular dichroism. In fig. 2A, pea protein and pea protein-curcumin nanoparticles produced negative peaks at 210-220 nm, indicating that β -sheet is the primary secondary structure of pea protein. And compared with pea protein, the peak of the pea protein-curcumin nanoparticle has red shift, which proves that the preparation method changes the secondary structure of the pea protein.

It was found from fig. 2B that after the pea protein and curcumin were prepared into pea protein-curcumin nanoparticles, the β -sheet structure was significantly reduced from 33.6% to 25%. In contrast, random coil increases from 23% by about 8% to 31%. Beta-sheet structure is an important spatial structure in proteins, and its reduction may mean that the protein becomes more loose and soft. While an increase in random coil structure may result in an increase in protein solubility. The pea protein which is treated by the preparation method and loaded with curcumin has higher solubility.

(4) Thermal stability

Respectively taking 2mL pea protein-curcumin nano particles and a curcumin sample, placing the pea protein-curcumin nano particles and the curcumin sample into a test tube, placing the test tube into an enzymolysis device at 75 ℃,85 ℃ and 95 ℃ for heating for 2 hours in a water bath, taking 200 microliter of the sample every 30 minutes, diluting and extracting the sample with ethanol, measuring an absorbance value at 426nm wavelength to obtain the concentration of the curcumin, and calculating the retention rate of the curcumin in a thermal environment through a formula (2) to judge the thermal stability of the curcumin.

As shown in fig. 3, approximately 40% of curcumin in water is decomposed by heat during the first 30min of heat treatment at 75 ℃. After 30min of heat treatment, the rate of thermal decomposition of curcumin slowed down. After 2h of heat treatment, the retention of curcumin was about 40%. In contrast, protein-protected pea protein-curcumin nanoparticles retained more than 85% of curcumin in the first 30min heat treatment, and maintained a stable decomposition rate during heat treatment, and still retained more than 65% of curcumin after 2h heat treatment.

Heat treatment at 85 ℃ resulted in more than 50% of the curcumin being thermally decomposed, resulting in more curcumin being thermally decomposed than heat treatment at 75 ℃ and at a faster rate than heat treatment at 75 ℃. After 2h of heat treatment, only about 13% of curcumin was retained. After the heat treatment of the pea protein-curcumin nano-particles is finished in the first 30min, the retention rate of curcumin is about 68%, and compared with the heat treatment at 75 ℃, the retention rate of curcumin is reduced by about 22%. After the final 2h heat treatment, the retention rate of curcumin is 35%, and compared with 75 ℃, the retention rate of curcumin is greatly reduced, but the retention rate is still obviously higher than that of curcumin nano-particles which are not protected by protein. The result of the heat treatment at 95 ℃ is basically consistent with that of the heat treatment at 85 ℃. The rate of thermal decomposition of curcumin increases due to the increase in temperature, resulting in a slight decrease in the curcumin retention rate of each group.

Therefore, under the heat treatment condition, the pea protein can effectively protect the curcumin molecules wrapped by the pea protein and slow down the thermal decomposition rate of the pea protein. And its ability to protect decreases with increasing heat treatment temperature, but still significantly higher than curcumin not protected by protein.

(5) Ultraviolet stability

10mL of each of the pea protein-curcumin nanoparticle samples prepared in examples 1 and 2 and the free curcumin sample were placed in a petri dish, exposed to ultraviolet light with a wavelength of 340nm for 3 hours, 200 microliter of the sample was taken every 30 minutes, diluted and extracted with ethanol, absorbance was measured at a wavelength of 426nm to obtain curcumin concentration, and the retention rate of curcumin in a thermal environment was calculated by the formula (3) to determine the ultraviolet light stability of curcumin.

The retention of pea protein-curcumin nanoparticles and free curcumin was obtained by fitting the retention calculated at each time point using origin 2019 software to obtain a trend line.

As shown in fig. 4, the retention of free curcumin was reduced to about 55% by uv treatment for about 3 hours. The retention of pea protein-curcumin nanoparticles was reduced to about 90% by uv treatment for about 3 hours. It can be seen that the rate of decomposition of curcumin under uv light can be effectively slowed down by making pea protein-curcumin nanoparticles. Indicating that proteins have the ability to retard photodegradation of bioactive components, proteins may also alter the chemical stability of reactive groups in curcumin by forming complexes with curcumin.

(6) Simulating gastrointestinal stability

2.5g of pepsin was taken and added to 1mol/L of diluted hydrochloric acid, and the volume was fixed to 250mL using diluted hydrochloric acid, which was regarded as a gastric simulated digestive juice. 2.5g trypsin was added to PBS buffer at pH 7.4 to a volume of 250mL to prepare an intestinal simulated digest. The pea protein-curcumin nanoparticle and curcumin samples of example 1 were added to a conical flask with 10mL and 10mL gastric simulated digest, placed in a stirrer, heated in a 37 ℃ water bath for 1h, removed, adjusted to pH 7.4, extracted with ethanol and tested for absorbance at 426 nm. And (3) calculating the retention rate of curcumin in simulated gastric fluid through a formula (4), and judging the stabilizing capacity of the curcumin.

And (3) entering an intestinal digestion simulation experiment, adding 40mL of intestinal digestion simulation liquid into each conical flask, heating and stirring in a water bath at 37 ℃ for 1h, taking out, taking part of samples in the conical flasks, adding into a centrifuge tube, and centrifuging for 15min at the rotating speed of 10000r/min to obtain supernatant. The supernatant was extracted with ethanol and the absorbance of the sample was measured at 426 nm. And (5) calculating the retention rate of curcumin in the simulated intestinal juice through a formula (5), and judging the bioavailability of the curcumin.

As shown in fig. 5, after 1h of simulated gastric fluid digestion, the retention of unprotected curcumin by protein was only about 63%. In contrast, the protection effect of the pea protein-curcumin nanoparticles on curcumin is quite obvious, and only about 3% of curcumin is lost in the simulated stomach environment. After the pH value is regulated to about 7.4, the retention rate of curcumin and pea protein-curcumin nano-particles dispersed in water is not changed obviously in the weak alkaline environment simulating human intestinal tracts. At the same time, about 96% of curcumin was released from pea protein-curcumin nanoparticles after intestinal trypsin digestion.

The pea protein-curcumin nano-particles have obvious protective effect in stomach environment compared with curcumin, and can effectively prevent curcumin from being decomposed in gastric acid and gastric enzyme environment. And in the simulated intestinal environment, the composition can be effectively released, and has the potential of being absorbed by human bodies.

The foregoing is only a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any person skilled in the art, who is within the scope of the present application, should make equivalent substitutions or modifications according to the technical scheme of the present application and the inventive concept thereof, and should be covered by the scope of the present application.

Claims

1. A pea protein-curcumin nanoparticle and a preparation method thereof, which is characterized by comprising the following steps:

2. The method of preparation of claim 1, wherein the method of preparation of the pea protein suspension is as follows: adding pea protein into distilled water, magnetically stirring, and standing to obtain pea protein suspension.

3. The preparation method according to claim 2, wherein the magnetic stirring condition is room temperature, stirring is carried out for 1-2 hours at 500-600 r/min, and the standing condition is that standing is carried out for 14-18 hours at 4 ℃.

4. The method of claim 1, wherein in step (1), the concentration of the pea protein suspension is 40-60 mg/mL.

5. The preparation method of claim 1, wherein in the step (1), the concentration of NaOH solution is 1-2 mol/L, the pH of pea protein suspension is adjusted to 12-13, and the magnetic stirring condition is 500-600 r/min for 1-2 h.

6. The preparation method of claim 1, wherein in the step (1), the concentration of the curcumin solution is 0.4-0.8 mg/mL, and the curcumin solution is stirred for 30-60 min under a dark stirring condition of 500-600 r/min.

7. The preparation method of claim 1, wherein in the step (2), the concentration of HCl solution is 1-2 mol/L, the pH of the mixed solution is adjusted to 7-8, and the mixture is magnetically stirred in a dark place for 1-2 hours under a dark stirring condition of 500-600 r/min.

8. The method according to claim 1, wherein in the step (2), the centrifugation is performed at 8000r/min at 20 to 25℃for 15 to 30 minutes.

9. The method according to claim 1, wherein in the step (2), the dialysis condition is dialysis in a 8000u dialysis bag for 18-22 hours.

10. Pea protein-curcumin nanoparticle prepared by the preparation method according to any one of claims 1 to 9.