Dual-protein nanocapsule based on fibroin / zein as well as preparation method and application of dual-protein nanocapsule

Abstract

The invention discloses a fibroin / zein-based dual-protein nanocapsule and a preparation method and application thereof.The dual-protein nanocapsule is prepared through a microfluidic-assisted nano-coprecipitation technology and comprises the following steps that silk fibroin is dissolved in a citric acid aqueous solution, and a mixed solution is obtained; the preparation method comprises the following steps: dissolving zein, curcumin and linseed oil in ethanol, rapidly mixing through a microchannel, and carrying out nano coprecipitation to obtain the dual protein nanocapsule. Through electrostatic interaction and amphiphilic regulation, a stable double protein shell structure is formed, curcumin is effectively protected from being damaged by gastric acid and digestive juice, targeted release of intestinal tracts is achieved, and the bioavailability of curcumin is remarkably improved. The obtained nanocapsule is uniform in particle size and good in dispersity, is suitable for a functional beverage system, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of protein nanocapsule preparation, and in particular relates to a silk fibroin / zein-based double protein nanocapsule and a preparation method and application thereof. Background Art

[0002] As people's health awareness increases, special-purpose beverages with specific health functions are gaining more and more attention. Among them, curcumin, as a natural polyphenol compound, is favored for its significant liver protection effect. Studies have shown that curcumin protects the liver through multiple mechanisms such as scavenging free radicals, inhibiting the expression of inflammatory factors, and regulating the activation of hepatic stellate cells. However, curcumin has extremely poor water solubility (<1 μg / mL) and is easily degraded under light and high temperature conditions, which severely limits its application in beverage systems.

[0003] In the prior art, nano-encapsulation technology is usually used to improve the stability and bioavailability of curcumin. For example, patent CN202211179994.9 discloses a nano-delivery system based on globulin 1S, a preparation method and an application thereof. Although this method increases the stability of curcumin in the human body and prolongs its release time, the single protein encapsulation layer is more easily destroyed during the simulated gastrointestinal digestion process, resulting in premature release of curcumin and failure to effectively target and deliver it to the intestine, thereby reducing its bioavailability.

[0004] Curcumin is mainly absorbed in the intestine and exerts its physiological functions. Therefore, the ideal delivery system should have the following characteristics: (1) maintain structural integrity in the acidic environment of the stomach; (2) achieve targeted release in the alkaline environment of the intestine; (3) have good photothermal stability and long-term storage stability; (4) be suitable for the dispersibility and transparency of beverage systems. However, it is difficult for existing technologies to meet the above requirements at the same time. For example, single-layer protein encapsulation systems (such as zein or silk fibroin) are prone to disintegration during digestion; when preparing nanoparticles by traditional co-precipitation method, it is difficult to accurately control the interface assembly process, resulting in uneven particle size distribution and poor stability of nanoparticles, which cannot meet the storage and digestion stability requirements of functional beverages.

[0005] Therefore, developing a dual protein nanocapsule technology that can effectively control the interface assembly process, balance processing efficiency and structural stability, and is suitable for large-scale production has become a technical problem that needs to be urgently solved in this field. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a double protein nanocapsule based on silk fibroin / zein and a preparation method thereof, in view of the poor water solubility, low stability and insufficient bioavailability of fat-soluble functional ingredients such as curcumin in the prior art in beverage systems, especially the technical defects that the single-layer protein encapsulation system is easy to disintegrate during digestion and the traditional co-precipitation method is difficult to accurately control the interface assembly process.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing a double protein nanocapsule based on silk fibroin / zein comprises the following steps: (1) dissolving silk fibroin in a citric acid aqueous solution to obtain a silk fibroin solution; (2) dissolving zein, curcumin, and linseed oil in an ethanol aqueous solution to obtain a zein mixed solution; (3) The silk fibroin solution obtained in step (1) and the zein mixed solution obtained in step (2) are quickly mixed by a microchannel mixer for nano-coprecipitation, and the mixture is allowed to stand to evaporate the ethanol.

[0008] Specifically, in step (1), the concentration of the citric acid aqueous solution is 0.19-0.96% w / v, and the pH value is 4-5.

[0009] Preferably, in step (1), the concentration of the silk fibroin in the citric acid aqueous solution is 1 mg / mL-10 mg / mL.

[0010] Specifically, in step (2), the volume concentration of the ethanol aqueous solution is 75-85%; the concentration of the zein in the ethanol aqueous solution is 10-60 mg / mL; the concentration of the curcumin in the ethanol aqueous solution is 1-8 mg / mL; and the concentration of the linseed oil in the ethanol aqueous solution is 5-100 μL / mL.

[0011] Specifically, in step (3), the microchannel mixer is any one of a serpentine channel, a Tesla channel, a herringbone channel, and a butterfly channel; the width of the microchannel mixer is 0.1 mm-1.5 mm, the height is 0.1 mm-1.5 mm, the liquid holding capacity is 5-20 mL, and one end of the microchannel mixer has two injection ports for simultaneously injecting two solutions, and the other end has a liquid outlet for collecting the reaction product.

[0012] Specifically, in step (3), the volume flow rate of the silk fibroin solution when injected into the microchannel mixer is 2 mL / h-50 mL / h, and the volume flow rate of the zein mixed solution when injected into the microchannel mixer is 0.1 mL / h-1 mL / h.

[0013] Preferably, in step (3), the mixing volume ratio of the silk fibroin solution to the zein mixed solution is 20:1 to 500:1.

[0014] Furthermore, the double protein nanocapsules prepared by the above preparation method are also within the protection scope of the present invention.

[0015] Furthermore, the present invention also claims to protect the use of the above-mentioned double protein nanocapsules in the preparation of functional beverages.

[0016] Furthermore, the present invention also protects a functional beverage, comprising the following components by mass: 10-50 parts of a dispersion of the double protein nanocapsules prepared by the above method in a citric acid solution; 3-10 parts sweetener; 5-20 parts of citric acid; 100-500 parts of water; In the dispersion of the double protein nanocapsules in the citric acid solution, the concentration of the double protein nanocapsules is 1 mg / mL-12 mg / mL; The sweetener is selected from any one of neotame, xylitol, aspartame, acesulfame potassium, glycyrrhizinate, maltitol, sorbitol, steviol glycosides, cyclamate, and erythritol, or a mixture of two or more of the above.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts microfluidics-assisted nano-coprecipitation technology to achieve efficient mixing of silk fibroin solution and zein mixed solution, which is suitable for industrial production. The average particle size of the obtained nanocapsules is 287.4 nm, which has good dispersibility and transparency in the beverage system, and solves the precipitation and turbidity problems common in traditional encapsulation technology. The dual protein nanocapsule structure effectively protects fat-soluble functional ingredients such as curcumin from photothermal degradation, extends the shelf life of the product, and ensures its stability during storage and transportation. Through the precise structural control of the dual protein nanocapsules, fat-soluble functional ingredients such as curcumin are effectively protected during the digestion process, avoiding the destruction of gastric acid and digestive juices, thereby improving their bioavailability, enabling them to better play their liver protection, antioxidant and anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0019] Figure 1Microscope image of the dispersion of silk fibroin / zein dual protein nanocapsules in a citric acid aqueous solution at pH = 4.5.

[0020] Figure 2 From left to right are the dispersion of zein particles in deionized water, the dispersion of zein particles in silk fibroin aqueous solution, and the dispersion of dual protein nanocapsules in citric acid solution at pH = 4.5. DETAILED DESCRIPTION

[0021] The present invention can be better understood with reference to the following examples.

[0022] Example 1

[0023] (1) Dissolve silk fibroin in a citric acid aqueous solution at pH 4.5 at a concentration of 4 mg / mL, stir evenly and allow to stand for degassing to obtain a silk fibroin solution.

[0024] (2) Zein (25 mg / mL), curcumin (3 mg / mL), and linseed oil (25 μL / mL) were dissolved in 85% ethanol solution and ultrasonically dispersed for 20 min to obtain a zein mixed solution.

[0025] (3) A serpentine microchannel (diameter 0.5 mm, height 0.5 mm, liquid holding capacity 5 mL) was used to inject the silk fibroin solution into the microchannel at a flow rate of 20 mL / h, and the zein mixed solution was injected into the microchannel at a flow rate of 0.5 mL / h. The volume ratio of the two phases was 40:1.

[0026] (4) Collect the mixed solution at the outlet of the microchannel, and let the mixture stand to evaporate the ethanol, thereby obtaining a dispersion of the silk fibroin / zein dual protein nanocapsules in the citric acid solution, such as Figure 1 As shown, the obtained solution has a core-shell structure and is in the form of uniform spheres, which proves that uniform nanocapsules were successfully prepared after rapid nano-coprecipitation.

[0027] Example 2

[0028] The difference between Example 2 and Example 1 is that in step (3), the volume ratio of the silk fibroin solution to the zein mixed solution is 20:1.

[0029] Example 3

[0030] The difference between Example 3 and Example 1 is that the ethanol solution used in step (2) is an 80% ethanol solution.

[0031] Example 4

[0032] (1) The dispersion of the dual protein nanocapsules prepared in Example 1 in citric acid solution was added into water at a ratio of 10 wt.%.

[0033] (2) Add 5 wt.% of xylitol and 4 wt.% of citric acid to the solution prepared in step (1) and stir well.

[0034] (3) The mixed solution obtained in step (2) was homogenized at a pressure of 15 MPa and a temperature of 50°C.

[0035] (4) Sterilize at 135°C for 3 seconds.

[0036] (5) After cooling to room temperature, aseptic filling is performed.

[0037] Example 5

[0038] Compared with Example 4, Example 5 differs in that step (1) is changed to: adding the dispersion of the double protein nanocapsules prepared in Example 2 in citric acid solution into water at a ratio of 10 wt.%.

[0039] Example 6

[0040] Compared with Example 4, Example 6 differs in that step (1) is changed to: adding the dispersion of the double protein nanocapsules prepared in Example 3 in citric acid solution into water at a ratio of 10 wt.%.

[0041] Comparative Example 1

[0042] (1) Zein (25 mg / mL), curcumin (3 mg / mL), and linseed oil (25 μL / mL) were dissolved in 85% ethanol solution and ultrasonically dispersed for 20 min to obtain a zein mixed solution.

[0043] (2) A serpentine microchannel (diameter 0.5 mm, height 0.5 mm, liquid holding capacity 5 mL) was used. Deionized water was injected into the microchannel at a flow rate of 20 mL / h, and the zein mixed solution was injected into the microchannel at a flow rate of 0.5 mL / h. The volume ratio of the two phases was 40:1.

[0044] (3) After mixing, a dispersion of zein nanocapsules in water is obtained.

[0045] Comparative Example 2

[0046] (1) Dissolve silk fibroin in deionized water at a concentration of 4 mg / mL, stir well, and let stand to obtain a silk fibroin aqueous solution.

[0047] (2) Zein (25 mg / mL), curcumin (3 mg / mL), and linseed oil (25 μL / mL) were dissolved in 85% ethanol solution and ultrasonically dispersed for 20 min to obtain a zein mixed solution.

[0048] (3) Using a serpentine microchannel (diameter 0.5 mm, height 0.5 mm, liquid holding capacity 5 mL), the silk fibroin aqueous solution was injected into the microchannel at a flow rate of 20 mL / h, and the zein mixed solution was injected into the microchannel at a flow rate of 0.5 mL / h. The volume ratio of the two phases was 40:1.

[0049] (4) After mixing, a dispersion of silk fibroin / zein nanoparticles in water is obtained.

[0050] Comparative Example 3

[0051] (1) Dissolve silk fibroin in a citric acid aqueous solution at pH = 4.5 at a concentration of 4 mg / mL, stir evenly and allow to stand for degassing to obtain a silk fibroin solution.

[0052] (2) Zein (25 mg / mL), curcumin (3 mg / mL), and linseed oil (25 μL / mL) were dissolved in 85% ethanol solution and ultrasonically dispersed for 20 min to obtain a zein mixed solution.

[0053] (3) The zein mixed solution was added dropwise to the silk fibroin solution at a flow rate of 0.5 mL / h, with a stirring speed of 500 rpm and continuous stirring for 30 min.

[0054] (4) The mixture is allowed to stand to evaporate the ethanol, thereby obtaining a dispersion of the silk fibroin / zein dual protein nanocapsules in a citric acid solution.

[0055] Comparative Example 4

[0056] The dispersion of zein nanocapsules prepared in Comparative Example 1 in water was added into water at a ratio of 10 wt.%. The remaining steps were the same as those in Example 4.

[0057] Comparative Example 5

[0058] The difference between Comparative Example 5 and Example 4 is that the dispersion liquid is a dispersion liquid of the silk fibroin / zein nanoparticles prepared in Comparative Example 2 in water.

[0059] Comparative Example 6

[0060] The difference between Comparative Example 6 and Example 4 is that the dispersion liquid is a dispersion liquid of the silk fibroin / zein dual protein nanocapsules prepared in Comparative Example 3 in a citric acid solution.

[0061] Effect Example 1

[0062] The dispersions of the double protein nanocapsules prepared in Example 1 and the zein particles prepared in Comparative Examples 1 and 2 in deionized water and silk fibroin aqueous solution were as follows: Figure 2 As shown, from left to right are the dispersion of zein particles in deionized water, the dispersion of zein particles in silk fibroin aqueous solution, and the dispersion of double protein nanocapsules in citric acid solution at pH = 4.5, indicating that the double protein nanocapsules have higher stability, are clearer and more transparent, and are suitable for special-purpose beverage systems.

[0063] The potential of the mixed solution prepared in Example 1 and Comparative Example 1 was measured, and the results are shown in Table 1, indicating that the silk fibroin was successfully adsorbed on the surface of the zein particles to form a double protein shell, which not only protected the curcumin but also facilitated its adsorption in the intestine.

[0064] Table 1 Nanoparticle size and charge of nanocapsules obtained in Example 1 and Comparative Example 1 Nanoparticle size Charge Example 1 287.4 nm -18.4 ± 2.42 mV Comparative Example 1 264.8 nm +10.2 ± 5.53 mV

[0065] In order to evaluate the particle size and dispersibility of the examples, the particle size distribution of the double protein nanocapsules prepared in Example 1 and the double protein nanocapsules prepared in Comparative Example 3 were compared, as shown in Table 2, indicating that the nanocapsules prepared by the microfluidic method have uniform particle size distribution and smaller particle size, which is conducive to the stable storage of the dispersion and is less prone to sedimentation.

[0066] Table 2 Nanoparticle size of nanocapsules obtained in Example 1 and Comparative Example 3 Nanoparticle size Example 1 287.4 nm Comparative Example 3 597.4 nm

[0067] Effect Example 2 In order to evaluate the difference between the examples and the comparative examples in the application of functional beverages, the taste, color and texture of the functional beverages prepared in Examples 4-6 and Comparative Examples 4-6 were evaluated under normal indoor lighting and 40°C for three months. The relevant evaluation results are shown in Table 3.

[0068] Table 3 Evaluation of Example 2 and Comparative Examples 4-6

[0069] The following conclusions can be drawn from Table 3: The functional beverages obtained in Examples 4-6 are uniform liquids without precipitates, indicating that the dual protein nanocapsules in the functional beverages obtained are not easy to agglomerate and are in a stable state. Compared with Comparative Examples 4-6, the functional beverages obtained in Examples 4-6 have obvious advantages, the product has a pure and uniform color, a moderate taste, and the encapsulated curcumin is not easy to decompose.

[0070] The present invention provides a dual protein nanocapsule based on silk fibroin / zein and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a dual protein nanocapsule based on silk fibroin / zein, characterized in that: The steps include: (1) dissolving silk fibroin in a citric acid aqueous solution to obtain a silk fibroin solution; (2) dissolving zein, curcumin, and linseed oil in an ethanol aqueous solution to obtain a zein mixed solution; (3) The silk fibroin solution obtained in step (1) and the zein mixed solution obtained in step (2) are quickly mixed by a microchannel mixer for nano-coprecipitation, and the mixture is allowed to stand to evaporate the ethanol.

2. The method for preparing the dual protein nanocapsules based on silk fibroin / zein according to claim 1, characterized in that: In step (1), the concentration of the citric acid aqueous solution is 0.19-0.96% w / v, and the pH value is 4-5.

3. The method for preparing the dual protein nanocapsules based on silk fibroin / zein according to claim 1, characterized in that: In step (1), the concentration of the silk fibroin in the citric acid aqueous solution is 1 mg / mL-10 mg / mL.

4. The method for preparing the dual protein nanocapsules based on silk fibroin / zein according to claim 1, characterized in that: In step (2), the volume concentration of the ethanol aqueous solution is 75-85%; the concentration of the zein in the ethanol aqueous solution is 10-60 mg / mL; the concentration of the curcumin in the ethanol aqueous solution is 1-8 mg / mL; and the concentration of the linseed oil in the ethanol aqueous solution is 5-100 μL / mL.

5. The method for preparing the dual protein nanocapsules based on silk fibroin / zein according to claim 1, characterized in that: In step (3), the microchannel mixer is any one of a serpentine channel, a Tesla channel, a herringbone channel, and a butterfly channel; the width of the microchannel mixer is 0.1 mm-1.5 mm, the height is 0.1 mm-1.5 mm, the liquid holding capacity is 5-20 mL, and one end of the microchannel mixer has two injection ports for simultaneously injecting two solutions, and the other end has a liquid outlet for collecting the reaction product.

6. The method for preparing the dual protein nanocapsules based on silk fibroin / zein according to claim 1, characterized in that: In step (3), the volume flow rate of the silk fibroin solution when injected into the microchannel mixer is 2 mL / h-50 mL / h, and the volume flow rate of the zein mixed solution when injected into the microchannel mixer is 0.1 mL / h-1 mL / h.

7. The method for preparing the dual protein nanocapsules based on silk fibroin / zein according to claim 1, characterized in that: In step (3), the mixing volume ratio of the silk fibroin solution to the zein mixed solution is 20:1 to 500:

1.

8. The double protein nanocapsule prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the double protein nanocapsule according to claim 8 in preparing functional beverages.

10. A functional beverage, characterized in that: It includes the following components by mass: 10-50 parts of a dispersion of the double protein nanocapsules prepared in claim 1 in a citric acid solution; 3-10 parts sweetener; 5-20 parts of citric acid; 100-500 parts of water; In the dispersion of the double protein nanocapsules in the citric acid solution, the concentration of the double protein nanocapsules is 1 mg / mL-12 mg / mL; The sweetener is selected from any one of neotame, xylitol, aspartame, acesulfame potassium, glycyrrhizinate, maltitol, sorbitol, steviol glycosides, cyclamate, and erythritol, or a mixture of two or more of the above.

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