Double-loaded sweet potato protein-based core-shell structure nanofiber for delivering water-insoluble nutrients and preparation method of double-loaded sweet potato protein-based core-shell structure nanofiber

Abstract

The invention discloses a double-loaded sweet potato protein-based core-shell structure nanofiber for delivering water-insoluble nutrients and a preparation method thereof. The preparation method comprises the following steps: (1) extracting sweet potato protein; (2) modifying the sweet potato protein by gamma-ray irradiation assisted acetylation; (3) taking a water-soluble nutrient / sweet potato protein / pullulan as a continuous phase, and taking a water-insoluble nutrient / plant essential oil as a dispersed phase; (4) mixing the continuous phase and the dispersed phase, homogenizing and emulsifying to prepare an oil-in-water emulsion capable of being electrospun; and (5) preparing the sweet potato protein-based nanofiber instant membrane with the core-shell structure through emulsion electrostatic spinning. The sweet potato protein-based core-shell structure nanofiber delivery carrier which delivers water-insoluble nutrients and is instant in water is efficiently prepared by taking a continuous phase as a shell layer fluid and a dispersed phase as a core layer fluid, so that the problem that the existing nutrients are difficult to deliver due to water insolubility is solved; the application value of the sweet potato protein in the fields of food, health care products, medicines, cosmetics and the like is greatly expanded.

Description

Technical Field

[0001] The present invention belongs to the technical field of encapsulation and delivery of hydrophobic nutrients, and particularly relates to a dual-loaded sweet potato protein-based core-shell structured nanofiber for delivering water-insoluble nutrients and a preparation method thereof. Background Art

[0002] Functional foods are foods that can regulate the body functions by adding specific nutrients, are suitable for specific populations to consume, and are beneficial to the human body or can prevent diseases. With the rapid increase in the number of sub-healthy people, consumers are more inclined to choose functional foods with health promotion effects. This trend not only reflects the growing public attention to health but also promotes the booming development of the functional food market. Such foods are often rich in specific nutritional components that can be delivered, such as natural edible nutrients, including catechins, vitamin C, and astaxanthin, etc. Thanks to their strong ability to quench singlet oxygen and capture free radicals, these nutrients have shown excellent curative effects in clinical trials for anti-aging, anti-inflammatory, anti-obesity, anti-tumor, and immune enhancement, and are considered an important part of disease prevention and restorative health care. However, including edible nutrients such as astaxanthin, β-carotene, lycopene, vitamin D, vitamin E, and curcumin, etc., all have poor water solubility, physicochemical stability (being very sensitive to environmental factors such as light, oxygen, and heat), and bioaccessibility. Disadvantages such as low encapsulation efficiency and difficult delivery greatly limit their applications in the food industry. Therefore, developing an edible instant carrier for delivering water-insoluble nutrients is of great significance for improving the bioavailability of water-insoluble nutrients, formulating specific nutrient delivery systems for special populations (such as the elderly, young children, and other people with swallowing difficulties), expanding the functional food market, and promoting the health of the national society.

[0003] The core-shell structured nanofibers prepared by the electrospinning process are a good nutrient delivery carrier. With the deepening of the exploration in the field of nanotechnology, the electrospinning technology has shown great potential in the field of active substance encapsulation. This method is an electrohydrodynamic non-thermal processing technique, which avoids the thermal oxidation of nutrients caused by extreme temperatures in existing encapsulation processes (such as solution casting, extrusion, and blow molding processes). Due to its sustainability, low energy consumption, and low cost, it has become an ideal choice for preparing nutrient delivery carriers. Generally, the preparation of core-shell structured electrospun nanofibers includes two approaches: coaxial electrospinning and emulsion electrospinning. However, the disadvantages of low efficiency, complex operation, and high requirements for process variables limit the wide application of coaxial electrospinning in industrial spinning. Compared with coaxial electrospinning, the emulsion electrospinning process can complete the preparation of core-shell structured nanofibers only through a single-axis syringe (i.e., one kind of spinning solution), and can encapsulate water-insoluble nutrients without any organic solvents. Its simple operation and high encapsulation efficiency make it a promising strategy for encapsulating active substances. The water-insoluble nutrients / plant essential oils are loaded as the dispersed phase in the core layer of the fiber, which is beneficial to maintaining their physicochemical stability; the continuous phase of water-soluble nutrients / sweet potato protein / pullulan forms the fiber shell layer. While protecting the internal core layer, the hydrophilic shell layer material also promotes the rapid dissolution and release process of nutrients in the water in the dual-loading system. On the other hand, the special structure with a high specific surface area and high porosity also enables the nanofibers with a dual-loading system to improve the water solubility and bioavailability of water-insoluble nutrients, thereby rapidly releasing and delivering the natural edible nutrients in the shell layer and core layer to achieve a therapeutic effect.

[0004] Sweet potato is a natural polymer widely used in the food industry, mainly for the production of sweet potato starch, which has the advantages of low cost, biodegradability, non-toxicity and odorlessness. However, a large amount of protein-containing wastewater is generated during the starch extraction process. If not properly treated, it will pollute the water source and produce a pungent odor, causing inestimable harm to the environment. Therefore, extracting sweet potato protein with emulsifying properties from the wastewater as an emulsifier for the double-loaded system emulsion of water-insoluble nutrients is undoubtedly an ideal strategy for sustainable development, environmental protection and improving the utilization rate of sweet potatoes. However, its limited emulsifying performance makes it difficult to meet the processing requirements of the modern food industry, and adverse changes such as protein denaturation caused by extreme conditions during the extraction process also reduce its functional properties and nutritional value. Therefore, there is an urgent need to modify sweet potato protein to improve its emulsifying properties. Compared with a single modification method, the composite modification method assisted by γ-ray irradiation and acetylation can significantly improve the emulsifying properties of sweet potato protein, expand the application scope of sweet potato protein in the food industry, and is an ideal precursor for preparing double-loaded core-shell structure nanofibers for delivering water-insoluble nutrients. However, there is no report on the double-loaded sweet potato protein-based core-shell structure nanofiber delivery carrier for delivering water-insoluble nutrients prepared by emulsion electrospinning technology using modified sweet potato protein as an emulsifier. Summary of the Invention

[0005] In order to overcome the bottleneck of the above-mentioned existing technologies in the encapsulation and delivery applications of water-insoluble nutrients, the primary object of the present invention is to provide a double-loaded sweet potato protein-based core-shell structure nanofiber for delivering water-insoluble nutrients and its preparation method, so as to improve the bioavailability of water-insoluble nutrients, expand the market of functional foods, and promote the healthy development of society.

[0006] Another object of the present invention is to provide an emulsion electrospinning process for preparing core-shell structure nanofibers of a double-loaded system to replace the coaxial electrospinning process with low efficiency, complex operation and high requirements for process variables.

[0007] Another object of the present invention is to provide a composite modification method to improve the emulsifying properties of sweet potato protein to better meet the development needs of the modern food industry.

[0008] Another object of the present invention is to provide a strategy for improving the utilization rate of sweet potato protein, effectively avoiding environmental pollution and resource waste.

[0009] To achieve the above objects, the present invention relates to the following technical solutions: A method for preparing double-loaded sweet potato protein-based core-shell structured nanofibers for delivering water-insoluble nutrients, wherein the sweet potato protein-based nanofiber membrane is composed of a shell layer and a core layer covered inside the shell layer. Among them, the shell layer is formed by electrospinning a hydrophilic material sweet potato protein / pullulan polysaccharide solution with rapid dissolution characteristics, and the core layer is formed by electrospinning water-insoluble nutrients / plant essential oils; the method includes the following steps: (1) Extract sweet potato protein from fresh sweet potatoes by the acid precipitation and alkali dissolution method.

[0010] (2) Modify sweet potato protein by γ-ray irradiation-assisted acetylation.

[0011] (3) Dissolve sweet potato protein and pullulan polysaccharide at high temperature. After cooling and mixing evenly, add water-soluble nutrients and stir evenly at room temperature to obtain a continuous phase.

[0012] (4) Add water-insoluble nutrients to plant essential oils and mix evenly to obtain a dispersed phase.

[0013] (5) Mix the continuous phase and the dispersed phase, homogenize and emulsify, and ultrasonically remove bubbles to obtain an oil-in-water emulsion for electrospinning.

[0014] (6) Perform emulsion electrospinning to prepare double-loaded sweet potato protein-based core-shell structured nanofibers for delivering water-insoluble nutrients.

[0015] Preferably, the extraction of sweet potato protein in step (1) includes the following steps: Place fresh sweet potatoes in a blender to extract sweet potato juice. After filtration, let it stand overnight under refrigeration conditions. Take the supernatant, adjust the pH value to pH = 4.0 with hydrochloric acid, and centrifuge to retain the bottom precipitate. After repeating the whole process twice, dissolve the protein precipitate in deionized water, adjust the pH value to pH = 7.0 with sodium hydroxide solution, and freeze-dry for 48 h to obtain sweet potato protein powder.

[0016] Preferably, the modification of sweet potato protein in step (2) includes the following steps: Place the sweet potato protein powder in a double-layer polyethylene packaging bag, and perform 60 CO-γ-ray irradiation treatment for modification.

[0017] Preferably, the γ-ray irradiation dose in step (2) is 10-20 kGy.

[0018] Preferably, the mass of acetic anhydride in step (2) accounts for 15%-30% of the dry weight of sweet potato protein.

[0019] Preferably, the water-soluble nutrients in the continuous phase in step (3) include at least one of vitamin C, anthocyanin, and catechin.

[0020] Preferably, the addition amount of the water-soluble nutrient in step (3) is 0.01-0.2 g / mL, more preferably 0.02-0.15 g / mL, and most preferably 0.024 g / mL.

[0021] Preferably, in the continuous phase of step (3), the mass fraction of sweet potato protein is 3%-5%, more preferably 5%.

[0022] Preferably, in the continuous phase of step (3), the mass fraction of pullulan polysaccharide is 5%-16%, more preferably 8%-12%, and still more preferably 10%.

[0023] Preferably, in the continuous phase of step (3), the mixed solution of sweet potato protein and pullulan polysaccharide is stirred in a water bath at 30-50 °C for 3-5 h, more preferably stirred in a water bath at 35 °C for 4 h.

[0024] Preferably, the water-insoluble nutrients in step (4) include at least one of astaxanthin, curcumin, β-carotene, vitamin D, vitamin E, lycopene, and lutein; the plant essential oils include at least one of clove essential oil, peppermint essential oil, cinnamon essential oil, thyme essential oil, tea tree essential oil, and rosemary essential oil.

[0025] Preferably, in the dispersion phase of step (4), the addition amount of the water-insoluble nutrient is 0.02-0.2 g / mL, more preferably 0.05-0.1 g / mL, and most preferably 0.06 g / mL.

[0026] Preferably, in the dispersion phase of step (4), the water-insoluble nutrient is added to the plant essential oil and stirred evenly, more preferably stirred at room temperature for 1 h.

[0027] Preferably, the homogenization rate in step (5) is 5000-15000 rpm, more preferably 10000-13000 rpm, most preferably 12000 rpm, and the homogenization time is preferably 1-2 min.

[0028] Preferably, for the emulsion in step (5), the volume ratio of the continuous phase to the dispersion phase is 1:9-5:5, more preferably 2:8-4:6.

[0029] The conductivity of the oil-in-water emulsion in step (5) is 440-800 μs / cm, and the particle size is 90-400 nm.

[0030] Preferably, for the emulsion electrospinning process in step (6), the flow rate of the spinning solution is 0.3-0.6 mL / h, the voltage of the spinning machine is 14-26 kV, the humidity of the spinning environment is 25%-35%, the temperature of the spinning environment is 25-40 °C, the spinning distance is 10-15 cm, the rotation speed of the spinning machine drum is 90-150 rpm, and the spinning time is 5-6 h.

[0031] By the above technical solution, a double-loaded sweet potato protein-based core-shell structure nanofiber for delivering water-insoluble nutrients and a preparation method thereof are obtained. Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention uses an emulsion electrospinning process to prepare an electrospun instant nanofiber loaded with water-insoluble nutrients, which can improve the water solubility and bioavailability of water-insoluble nutrients and promote their delivery process in the water system.

[0032] (2) The present invention uses an emulsion electrospinning process to prepare a core-shell structure nanofiber with a double-loaded system, which improves the loading efficiency of nutrients and improves the physicochemical stability of the water-insoluble nutrients in the core layer.

[0033] (3) The present invention uses an emulsion electrospinning process to prepare a core-shell structure nanofiber, which replaces the coaxial electrospinning process with disadvantages such as low efficiency, complex operation and high requirements for process variables, and successfully loads water-insoluble nutrients without using any organic solvents.

[0034] (4) The present invention uses γ-ray irradiation-assisted acetylation to modify sweet potato protein, which improves the emulsifying properties and functional properties of sweet potato protein and meets the development needs of the modern food industry.

[0035] (5) The present invention extracts sweet potato protein from wastewater, realizes the recycling of resources, avoids environmental pollution, and greatly expands and improves the application scope and value of sweet potato protein in the field of nutrient encapsulation and delivery. Description of the Drawings

[0036] Figure 1 Optical microscope image of a double-loaded sweet potato protein-based emulsion loaded with water-insoluble β-carotene and water-soluble anthocyanin prepared in Example 1; Figure 2 Scanning electron microscope images of double-loaded sweet potato protein-based core-shell structure nanofibers for delivering water-insoluble β-carotene and water-soluble anthocyanin prepared in Example 2 at voltages of 26 kV, 22 kV, 20 kV, 18 kV, 16 kV and 14 kV respectively; Figure 3 Scanning electron microscope images of double-loaded sweet potato protein-based core-shell structure nanofibers with a sweet potato protein mass fraction of 3%, 4% and 5% loaded with water-insoluble β-carotene and water-soluble anthocyanin prepared in Example 3; Figure 4 Transmission electron microscope image of double-loaded sweet potato protein-based core-shell structure nanofibers for delivering water-insoluble astaxanthin and water-soluble vitamin C prepared in Example 4; Figure 5X-ray diffraction patterns of sweet potato protein, pullulan, anthocyanin, astaxanthin, and the nanofiber membrane prepared in Example 4; Figure 6 Release curve of β-carotene in the core-shell structure nanofibers. Detailed implementation mode

[0037] The following is a detailed description of the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0038] Example 1 Place fresh sweet potatoes in a blender to extract sweet potato juice. After filtration, let it stand overnight under refrigeration at 4 °C and take the supernatant. Adjust the pH to pH = 4.0 with 1 mol / L hydrochloric acid, place it in a centrifuge and centrifuge at 4 °C at 12000 rpm, and retain the bottom precipitate. Repeat this process twice. Dissolve the protein precipitate in deionized water, adjust the pH value to pH = 7.0 with 1 mol / L sodium hydroxide solution, and freeze-dry for 48 h to obtain sweet potato protein powder.

[0039] Place the sweet potato protein powder in a double-layer polyethylene packaging bag, and perform 60 CO-γ ray irradiation modification with an irradiation dose of 15 kGy on the sweet potato protein powder. Add the irradiated and modified sweet potato protein powder to deionized water to prepare a 3% sweet potato protein solution by mass fraction. Adjust the pH value to pH = 7.0, and gradually add acetic anhydride accounting for 15% of the dry weight of the sweet potato protein during stirring. After the pH value is stable, dialyze in distilled water for 32 h, and freeze-dry for 48 h to obtain the composite-modified sweet potato protein powder.

[0040] Add sweet potato protein powder with a mass fraction of 5% to 10 mL of deionized water respectively, and then add pullulan with a mass fraction of 10%. Continuously stir at 35 °C for 4 h. After the mixed solution cools down, continue to add 0.24 g of water-soluble vitamin C, and stir for 1 h under normal temperature and light-shielding conditions to obtain the continuous phase. Add 0.15 g of water-insoluble β-carotene to 2.5 mL of clove essential oil, and continuously stir at normal temperature and light-shielding conditions for 1 h to obtain the dispersed phase.

[0041] Mix the continuous phase and the dispersed phase, homogenize and emulsify at 12000 rpm for 1 min, and then ultrasonicate in an ultrasonic machine for 5 min to remove air bubbles to obtain the emulsion for electrospinning.

[0042] Emulsion electrospinning was carried out using an electrospinning device. The voltage of the spinning machine was 16 kV, the flow rate of the spinning solution was 0.3 mL / h, the humidity of the spinning environment was 30%, the temperature of the spinning environment was 30 °C, the spinning distance was 15 cm, the rotation speed of the spinning machine drum was 100 rpm, and the spinning time was 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients were obtained.

[0043] Comparative Example 1 Fresh sweet potatoes were placed in a blender to extract sweet potato juice. After filtration, the supernatant was taken after standing overnight under refrigeration conditions at 4 °C. The pH was adjusted to pH = 4.0 using 1 mol / L hydrochloric acid, and it was centrifuged at 12,000 rpm at 4 °C in a centrifuge, and the bottom precipitate was retained. This process was repeated twice. The protein precipitate was dissolved in deionized water, and the pH value was adjusted to pH = 7.0 using 1 mol / L sodium hydroxide solution, and freeze-dried for 48 h to obtain sweet potato protein powder. The sweet potato protein powder was placed in a double-layer polyethylene packaging bag, and the sweet potato protein powder was irradiated with 60 CO-γ rays with irradiation doses of 0 kGy, 10 kGy, 15 kGy, 20 kGy, and 25 kGy to obtain modified sweet potato protein powder. 5% (by mass) of sweet potato protein powder was added to 10 mL of deionized water respectively, and then 10% (by mass) of pullulan was added. It was continuously stirred at 35 °C for 4 h. After the mixed solution was cooled, 0.24 g of water-soluble vitamin C was continuously added, and it was stirred for 1 h under normal temperature and light-shielded conditions to obtain the continuous phase. 0.15 g of water-insoluble β-carotene was added to 2.5 mL of clove essential oil, and it was continuously stirred for 1 h under normal temperature and light-shielded conditions to obtain the dispersed phase. The continuous phase and the dispersed phase were mixed and homogenized and emulsified at 12,000 rpm for 1 min, and then sonicated in an ultrasonic machine for 5 min to remove bubbles to obtain the emulsion for electrospinning. Emulsion electrospinning was carried out using an electrospinning device. The voltage of the spinning machine was 16 kV, the flow rate of the spinning solution was 0.3 mL / h, the humidity of the spinning environment was 30%, the temperature of the spinning environment was 30 °C, the spinning distance was 15 cm, the rotation speed of the spinning machine drum was 100 rpm, and the spinning time was 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients were obtained.

[0044] Determination of protein emulsifying property and emulsifying stability.

[0045] The protein solution (2%) and clove essential oil were homogenized at 13,000 rpm for 2 min in a ratio of 1:1 (v / v) using a homogenizer to obtain an emulsion. First, 50 μL of the emulsion was respectively sucked from the bottom of the emulsions standing for 0 min and 10 min and added to 5 mL of the dilution solution (0.1 g / 100 mL SDS), and quickly mixed evenly using a vortex mixer. Then, the absorbance of the diluted sample at 500 nm was measured using a UV-visible spectrophotometer.

[0046] EAI(m 2 / g)=(2.303×2×A0×N) / (c×Ф×10000) ESI(min)=10×A0 / (A0 - A 10 ) Wherein, N is the dilution coefficient, c is the protein concentration of the solution (g / mL), and Ф is the oil volume fraction. A0 and A 10 are the absorbance values at 0 min and 10 min, respectively.

[0047] After being modified by γ-ray irradiation, the emulsifying property and emulsifying stability of sweet potato protein show a trend of first increasing and then decreasing. When the γ-ray irradiation dose is in the range of 0 - 15 kGy, the emulsifying property and emulsifying stability of sweet potato protein are improved and reach the optimum at 15 kGy. This is because low-dose gamma-ray irradiation can open the tertiary structure of sweet potato protein, and the exposure of internal hydrophobic groups accelerates the adsorption rate of sweet potato protein molecules to the oil-water interface, promotes the rapid formation and stable adsorption of the sweet potato protein-based barrier, and ultimately enhances the emulsifying performance of sweet potato protein. As the irradiation dose increases to 20 - 25 kGy, it will induce the re-aggregation of sweet potato protein molecules, hinder the process of their adsorption to the interfacial protein film, and ultimately lead to a decrease in emulsifying property and emulsifying stability.

[0048] Comparative Example 2 Fresh sweet potatoes were placed in a blender to extract sweet potato juice. After filtration, the juice was left to stand overnight at 4 °C in the refrigerator, and the supernatant was taken. The pH was adjusted to pH = 4.0 using 1 mol / L hydrochloric acid, and the mixture was centrifuged at 4 °C at 12000 rpm in a centrifuge, and the bottom precipitate was retained. This process was repeated twice. The protein precipitate was dissolved in deionized water, and the pH value was adjusted to pH = 7.0 using 1 mol / L sodium hydroxide solution, and then freeze-dried for 48 h to obtain sweet potato protein powder. The sweet potato protein powder was added to deionized water to prepare a 3% (w / w) sweet potato protein solution. The pH was adjusted to pH = 7.0, and acetic anhydride accounting for 5%, 10%, 15%, 20% and 25% of the dry weight of the sweet potato protein was gradually added during stirring. After the pH stabilized, it was dialyzed in distilled water for 32 h and then freeze-dried for 48 h to obtain modified sweet potato protein powder. 5% (w / w) sweet potato protein powder was added to 10 mL of deionized water, and then 10% (w / w) pullulan was added. The mixture was continuously stirred at 35 °C for 4 h. After the mixed solution cooled, 0.24 g of water-soluble vitamin C was added, and the mixture was stirred for 1 h under normal temperature and light-shielded conditions to obtain the continuous phase. 0.15 g of water-insoluble β-carotene was added to 2.5 mL of clove essential oil, and the mixture was continuously stirred at normal temperature and light-shielded conditions for 1 h to obtain the dispersed phase. The continuous phase and the dispersed phase were mixed and homogenized and emulsified at 12000 rpm for 1 min, and then sonicated in an ultrasonic machine for 5 min to remove air bubbles, obtaining the emulsion for electrospinning. Electrospinning of the emulsion was carried out using an electrospinning device. The voltage of the spinning machine was 16 kV, the flow rate of the spinning solution was 0.3 mL / h, the humidity of the spinning environment was 30%, the temperature of the spinning environment was 30 °C, the spinning distance was 15 cm, the rotational speed of the spinning machine drum was 100 rpm, and the spinning time was 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients were obtained.

[0049] Determination of protein emulsifying property and emulsifying stability.

[0050] The protein solution (2%) and clove essential oil were homogenized at 13000 rpm for 2 min in a 1:1 (v / v) ratio using a homogenizer to obtain an emulsion. First, 50 μL of the emulsion was taken from the bottom of the emulsion standing for 0 min and 10 min respectively and added to 5 mL of the diluent (0.1 g / 100 mL SDS), and quickly mixed using a vortex mixer. Then, the absorbance of the diluted sample at 500 nm was measured using a UV-visible spectrophotometer.

[0051] EAI (m 2 / g)=(2.303×2×A0×N) / (c×Ф×10000) ESI (min)=10×A0 / (A0 - A 10 ) where N is the dilution factor, c is the protein concentration of the solution (g / mL), and Ф is the oil volume fraction. A0 and A 10 are the absorbance values at 0 min and 10 min, respectively.

[0052] After acetylation modification, the emulsifying property of sweet potato protein was slightly improved, while the emulsion stability was significantly enhanced. This may be because acetylated sweet potato protein can form an emulsion network structure similar to hydrophilic colloid, which is beneficial to the maintenance of emulsion stability. However, the overall emulsifying property and emulsion stability are lower than those of sweet potato protein modified by γ-ray irradiation.

[0053] Comparative Example 3 Fresh sweet potatoes were placed in a blender to extract sweet potato juice. After filtration, the supernatant was obtained by standing overnight under refrigeration at 4°C. The pH was adjusted to pH = 4.0 using 1 mol / L hydrochloric acid, and then centrifuged at 4°C at 12,000 rpm in a centrifuge, and the bottom precipitate was retained. The whole process was repeated twice. The protein precipitate was dissolved in deionized water, and the pH value was adjusted to pH = 7.0 using 1 mol / L sodium hydroxide solution, and freeze-dried for 48 h to obtain sweet potato protein powder. The sweet potato protein powder was placed in a double-layer polyethylene packaging bag, and the sweet potato protein powder was irradiated with 60 CO-γ rays at an irradiation dose of 15 kGy to obtain modified sweet potato protein powder. The modified sweet potato protein powder was added to deionized water to prepare a 3% (by mass) sweet potato protein solution, and the pH value was adjusted to pH = 7.0. During stirring, acetic anhydride accounting for 15% of the dry weight of sweet potato protein was gradually added. After the pH value was stabilized, dialysis was carried out in distilled water for 32 h, and then freeze-dried for 48 h to obtain composite-modified sweet potato protein powder. 5% (by mass) sweet potato protein powder was added to 10 mL of deionized water, and then 10% (by mass) pullulan was added. The mixture was continuously stirred at 35°C for 4 h. After the mixed solution was cooled, 0.24 g of water-soluble vitamin C was further added, and the mixture was stirred at room temperature in the dark for 1 h to obtain the continuous phase. 0.15 g of water-insoluble β-carotene was added to 2.5 mL of clove essential oil, and the mixture was continuously stirred at room temperature in the dark for 1 h to obtain the dispersed phase. The continuous phase and the dispersed phase were mixed and homogenized and emulsified at 12,000 rpm for 1 min, and then sonicated in an ultrasonic machine for 5 min to remove air bubbles to obtain an emulsion for electrospinning. Electrospinning of the emulsion was carried out using an electrospinning device. The voltage of the spinning machine was 16 kV, the flow rate of the spinning solution was 0.3 mL / h, the humidity of the spinning environment was 30%, the temperature of the spinning environment was 30°C, the spinning distance was 15 cm, the rotation speed of the spinning machine drum was 100 rpm, and the spinning time was 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients were obtained.

[0054] Determination of protein emulsifying property and emulsifying stability: A protein solution (2%) and clove essential oil were mixed at a ratio of 1:1 (v / v), and homogenized for 2 min at 13000 rpm using a homogenizer to obtain an emulsion. First, 50 μL of the emulsion was taken from the bottom of the emulsions standing for 0 min and 10 min respectively and added to 5 mL of a dilution solution (0.1 g / 100 mL SDS), and quickly mixed using a vortex mixer. Then, the absorbance of the diluted sample at 500 nm was measured using a UV-visible spectrophotometer.

[0055] EAI (m 2 / g) = (2.303 × 2 × A0 × N) / (c × Ф × 10000) ESI (min) = 10 × A0 / (A0 - A 10 ) In the formula, N is the dilution coefficient, c is the protein concentration in the solution (g / mL), and Ф is the oil volume fraction. A0 and A 10 are the absorbance values at 0 min and 10 min respectively.

[0056] Compared with single γ-ray irradiation treatment or acetylation treatment, the composite modification method of γ-ray-assisted acetylation significantly improved the emulsifying property and emulsifying stability of sweet potato protein. This is because after γ-ray irradiation modification, more hydrophobic groups were exposed on the sweet potato protein, and the hydrophilic / lipophilic property on the surface of the sweet potato protein molecule was significantly improved, thus significantly enhancing the emulsifying property; while the subsequent acetylation modification introduced acetyl groups on the free amino groups, making the amino cations closed, weakening the electrostatic attraction between protein molecules and thus reducing the aggregation effect between proteins, thereby significantly improving the emulsifying stability of sweet potato protein. In summary, the composite modification method of γ-ray-assisted acetylation improved the functional properties of sweet potato protein, enabling it to meet the processing requirements of the modern food industry, and laid a foundation for the preparation of sweet potato protein-based double-loaded core-shell structure nanofibers for delivering water-insoluble nutrients.

[0057] Example 2 The composite modified sweet potato protein was prepared using the preparation method of Example 1.

[0058] Add 5% sweet potato protein powder by mass fraction to 10 mL of deionized water respectively, and then add 10% pullulan by mass fraction. Continuously stir at 35 °C for 4 h. After the mixed solution cools down, continue to add 0.24 g of water-soluble vitamin C and stir for 1 h under normal temperature and light-shielded conditions to obtain the continuous phase. Add 0.15 g of water-insoluble β-carotene to 2.5 mL of clove essential oil and continuously stir for 1 h under normal temperature and light-shielded conditions to obtain the dispersed phase. Mix the continuous phase and the dispersed phase, homogenize and emulsify at 12000 rpm for 1 min, and then ultrasonicate for 5 min in an ultrasonic machine to remove air bubbles to obtain the emulsion for electrospinning. Use an electrospinning device to perform emulsion electrospinning. The voltage of the spinning machine is 16 kV, the flow rate of the spinning solution is 0.3 mL / h, the humidity of the spinning environment is 30%, the temperature of the spinning environment is 30 °C, the spinning distance is 15 cm, the rotational speed of the spinning machine drum is 100 rpm, and the spinning time is 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients are obtained.

[0059] Optical microscope image of the emulsion: Before testing, dilute the emulsion 1000 times with deionized water to eliminate the multiple light scattering effect, and observe the microscopic morphology of the sweet potato protein-based emulsion under a 40-fold optical microscope.

[0060] As Figure 1 shown, the double-loaded system emulsion delivering water-insoluble β-carotene and water-soluble anthocyanins has a relatively small particle size range (nanoscale), indicating that the emulsion has good stability; in addition, the emulsion is relatively uniformly dispersed without droplet coalescence, and has a good microscopic morphology of the emulsion. This is because sweet potato protein has good emulsifying properties and can quickly adsorb to the oil / water interface and form a barrier, preventing the aggregation between droplets.

[0061] Comparative Example 4 Prepare the composite modified sweet potato protein using the preparation method of Example 1.

[0062] Add 5% sweet potato protein by mass fraction to 10 mL of deionized water, and then add 10% pullulan by mass fraction. Continuously stir at 35 °C for 4 h. After the mixed solution cools down, continue to add 0.24 g of water-soluble vitamin C and stir for 1 h under normal temperature and light-shielded conditions to obtain the continuous phase. Add 0.15 g of water-insoluble β-carotene to 2.5 mL of clove essential oil and continuously stir for 1 h under normal temperature and light-shielded conditions to obtain the dispersed phase. Mix the continuous phase and the dispersed phase, homogenize and emulsify at 12,000 rpm for 1 min, and then ultrasonicate for 5 min in an ultrasonic machine to remove bubbles to obtain the emulsion for electrospinning. Use an electrospinning device to perform emulsion electrospinning. The voltages of the spinning machine are set to 26 kV, 22 kV, 20 kV, 18 kV, 16 kV, and 14 kV respectively. The flow rate of the spinning solution is 0.3 mL / h, the humidity of the spinning environment is 30%, the temperature of the spinning environment is 30 °C, the spinning distance is 15 cm, the rotational speed of the spinning machine drum is 100 rpm, and the spinning time is 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients are obtained.

[0063] Scanning electron microscope images of nanofibers: Under vacuum conditions, the microscopic surface morphology of electrospun nanofibers at different voltages was observed using a SU3500 scanning electron microscope.

[0064] As Figure 2 shown, when the electrospinning voltage of the emulsion electrospinning process is between 16 - 18 kV, the double-loaded sweet potato protein-based core-shell structure nanofibers delivering water-insoluble β-carotene and water-soluble anthocyanins have good fiber morphology. When it is greater than 18 kV and less than 16 kV, the fibers are difficult to form. This is because a higher voltage will cause the emulsion to demulsify during the electrospinning process, resulting in the dispersed phase in the core layer overflowing from the inside to the outside; while a lower voltage will make it difficult to break the surface tension of the emulsion, resulting in the phenomenon of dripping from the needle tip.

[0065] Comparative Example 5 Add sweet potato protein with mass fractions of 3%, 4% and 5% respectively to 10 mL of deionized water. Subsequently, add pullulan with a mass fraction of 10%, and continuously stir at 35 °C for 4 h. After the mixed solution cools, continue to add 0.24 g of water-soluble vitamin C, and stir for 1 h under normal temperature and light-shielded conditions to obtain the continuous phase. Add 0.15 g of water-insoluble β-carotene to 2.5 mL of clove essential oil, and continuously stir for 1 h under normal temperature and light-shielded conditions to obtain the dispersed phase. Mix the continuous phase and the dispersed phase, homogenize and emulsify at 12,000 rpm for 1 min, and then ultrasonically treat for 5 min in an ultrasonic machine to remove bubbles to obtain the emulsion for electrospinning. Use an electrospinning device to perform emulsion electrospinning. The voltage of the spinning machine is 18 kV, the flow rate of the spinning solution is 0.3 mL / h, the humidity of the spinning environment is 30%, the temperature of the spinning environment is 30 °C, the spinning distance is 15 cm, the rotation speed of the spinning machine drum is 100 rpm, and the spinning time is 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients are obtained.

[0066] Scanning electron microscope images of nanofibers: Under vacuum conditions, the microscopic surface morphology of electrospun nanofibers with different sweet potato protein contents was observed using a SU3500 scanning electron microscope.

[0067] As Figure 3 shown, when the mass fraction of sweet potato protein is 5%, spindle-shaped structures are randomly and evenly distributed on the fibers, with good fiber morphology. When the mass fraction is less than 5%, the fibers show entanglement, aggregation, and uneven distribution. This is because at low sweet potato protein contents, the emulsifying performance decreases, and it is difficult for the emulsifier to cover the surface of the emulsion droplets, resulting in larger-sized emulsion droplets, leading to emulsion demulsification and fiber adhesion during the fiber forming process. Therefore, within a certain range, a higher level of sweet potato protein is beneficial for generating fibers with good morphology.

[0068] Example 3 Add 5% sweet potato protein by mass to 10 mL of deionized water, and then add 10% pullulan polysaccharide by mass. Continuously stir the mixture at 35 °C for 4 h. After the mixed solution cools down, continue to add 0.24 g of water-soluble anthocyanin, and stir for 1 h under normal temperature and light-shielded conditions to obtain a continuous phase. Add 0.15 g of water-insoluble astaxanthin to 2.5 mL of peppermint essential oil, and continuously stir for 1 h under normal temperature and light-shielded conditions to obtain a dispersed phase. Mix the continuous phase and the dispersed phase, and homogenize and emulsify them at 12000 rpm for 1 min. Then, ultrasonicate for 5 min in an ultrasonic machine to remove air bubbles, obtaining an emulsion for electrospinning. Use an electrospinning device to perform emulsion electrospinning. The voltage of the spinning machine is 18 kV, the flow rate of the spinning solution is 0.3 mL / h, the humidity of the spinning environment is 30%, the temperature of the spinning environment is 30 °C, the spinning distance is 15 cm, the rotational speed of the spinning machine drum is 100 rpm, and the spinning time is 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients are obtained.

[0069] Transmission electron microscopy image of nanofibers: The core-shell structure of the nanofibers was observed using a JEM-1400 transmission electron microscope at a magnification of 10000 times.

[0070] As Figure 4 shown, there are obvious internal dark regions and external bright regions in the double-loaded sweet potato protein-based core-shell structure nanofibers delivering water-insoluble astaxanthin and water-soluble vitamin C. This is due to the different transmittance of the electron beam when passing through the dispersed phase inside the fiber and the continuous phase outside. Therefore, it is proved that there is a core-shell structure in the double-loaded sweet potato protein-based core-shell structure nanofibers delivering water-insoluble astaxanthin and water-soluble vitamin C, and two nutrients, astaxanthin and vitamin C, are successfully loaded.

[0071] X-ray diffraction pattern: Diffraction patterns of sweet potato protein, pullulan polysaccharide, water-soluble anthocyanin, and water-insoluble astaxanthin were obtained using an Ultima Ⅳ-185 X-ray diffractometer at a scanning rate of 0.02° / s in the range of 4 - 40° (2θ). The working voltage and current were set to 40 kV and 40 mA, respectively.

[0072] As Figure 5 , clear characteristic diffraction peaks of astaxanthin exist at around 2θ of 11.1°, 13.3°, 15.8°, 18.2°, 20.7°, 23.4°, 25.2°, 27.5°, and 29.2°. The relatively high crystallinity of astaxanthin is the main reason why astaxanthin is difficult to be delivered as a dietary supplement in the water system. After the emulsion electrospinning process, the sharp diffraction peaks of astaxanthin do not appear in the nanofibers, indicating that the highly crystalline astaxanthin has been encapsulated into the core layer of the fiber after the emulsion electrospinning process, completing the transformation from the crystalline state to the amorphous state, and the bioavailability has been significantly improved.

[0073] Example 4 Add sweet potato protein with a mass fraction of 5% to 10 mL of deionized water, and then add pullulan with a mass fraction of 10%. Continuously stir at 35 °C for 4 h. After the mixed solution cools, continue to add 0.24 g of water-soluble anthocyanin, and stir for 1 h under normal temperature and light-shielded conditions to obtain the continuous phase. Add 0.15 g of water-insoluble β-carotene to 2.5 mL of peppermint essential oil, and continuously stir for 1 h under normal temperature and light-shielded conditions to obtain the dispersed phase. Mix the continuous phase and the dispersed phase, homogenize and emulsify at 12,000 rpm for 1 min, and then ultrasonically treat for 5 min in an ultrasonic machine to remove air bubbles to obtain the emulsion for electrospinning. Use an electrospinning device to perform emulsion electrospinning. The voltage of the spinning machine is 18 kV, the flow rate of the spinning solution is 0.3 mL / h, the humidity of the spinning environment is 30%, the temperature of the spinning environment is 30 °C, the spinning distance is 15 cm, the rotational speed of the spinning machine drum is 100 rpm, and the spinning time is 6 h. After completion, double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients are obtained.

[0074] Release curve of β-carotene in double-loaded sweet potato protein-based core-shell structure nanofibers capable of delivering water-insoluble nutrients.

[0075] Dissolve Na2HPO4 (2.38 g), KH2PO4 (0.19 g), NaCl (8.00 g), and α-amylase (0.06 g) in 1000 mL of deionized water, and adjust the pH to pH = 6.8 with phosphoric acid to prepare artificial saliva. Then, immerse the nanofibers (5 mg) in 25 mL of artificial saliva at 37 °C. Remove the nanofiber solution every once in a while (20 s), and according to the standard curve y = 0.082x + 0.05 (R 2 = 0.966), analyze the content of β-carotene in the dissolved solution sample with a TU-1900 ultraviolet spectrophotometer.

[0076] As Figure 6 shown, the β-carotene release curve in the simulated oral environment shows a burst release behavior (about 83%) within the first 2 minutes, and the cumulative release is about 91% after 5 minutes. The burst release behavior of the nanofibers indicates that the emulsion electrospinning process overcomes the limitations of poor water solubility and low bioavailability of β-carotene, and completes the efficient delivery of water-insoluble β-carotene in the water system.

[0077] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing double-loaded sweet potato protein-based core-shell structured nanofibers for delivering water-insoluble nutrients, characterized in that: The sweet potato protein-based nanofiber membrane is composed of a shell layer and a core layer covered inside the shell layer, wherein the shell layer is electrospun from a sweet potato protein / pullulan solution, a hydrophilic material with fast dissolution characteristics, and the core layer is electrospun from a water-insoluble nutrient / plant essential oil; the preparation method thereof comprises the following steps: (1) Sweet potato protein was extracted from fresh sweet potato seeds by acid precipitation and alkali dissolution method; (2) Modification of sweet potato protein by γ-ray irradiation-assisted acetylation; (3) preparing a water-soluble nutrient / sweet potato protein / pullulan solution as a continuous phase and preparing a water-insoluble nutrient / plant essential oil as a dispersed phase; (4) mixing the continuous phase and the dispersed phase obtained in step (3), homogenizing and emulsifying, and preparing an oil-in-water emulsion having electrospinnable properties; (5) The oil-in-water emulsion prepared in step (4) is subjected to emulsion electrospinning technology to prepare a sweet potato protein-based nanofiber instant dissolving membrane with a core-shell structure.

2. The method for preparing a double-loaded sweet potato protein-based core-shell structured nanofiber for delivering water-insoluble nutrients according to claim 1, characterized in that: In the step (1), the sweet potato juice is squeezed and refrigerated and left to stand overnight, the supernatant is taken and its pH value is adjusted to pH=4.0 by hydrochloric acid, then centrifuged and the bottom precipitate is retained, and the whole process is repeated twice; finally, the precipitate is dissolved in deionized water and the pH value is adjusted to pH=7.0 by sodium hydroxide, and freeze-dried for 48 hours to obtain the sweet potato protein powder.

3. The method for preparing a double-loaded sweet potato protein-based core-shell structured nanofiber for delivering water-insoluble nutrients according to claim 1, characterized in that: In step (2), by 60 The extracted sweet potato protein powder was irradiated and modified by CO-γ ray.

4. The method for preparing a double-loaded sweet potato protein-based core-shell structured nanofiber for delivering water-insoluble nutrients according to claim 1, characterized in that: In the step (2), the irradiated modified sweet potato protein powder is added to deionized water to prepare a sweet potato protein solution with a mass fraction of 3%, the pH value is adjusted to pH=7.0, acetic anhydride is gradually added during stirring, and after the pH value stabilizes, it is dialyzed in distilled water for 32 hours and freeze-dried for 48 hours to obtain the composite modified sweet potato protein powder.

5. The method for preparing a double-loaded sweet potato protein-based core-shell structured nanofiber for delivering water-insoluble nutrients according to claim 1, characterized in that: In step (3), the water-soluble nutrients include at least one of vitamin C, anthocyanins and catechins; the water-insoluble nutrients include at least one of astaxanthin, curcumin, β-carotene, vitamin D, vitamin E, lycopene and lutein; and the plant essential oil includes at least one of clove essential oil, peppermint essential oil, cinnamon essential oil, thyme essential oil, tea tree essential oil and rosemary essential oil.

6. The method for preparing a double-loaded sweet potato protein-based core-shell structured nanofiber for delivering water-insoluble nutrients according to claim 1, characterized in that: In the step (3), in the water-soluble nutrient / sweet potato protein / pullulan mixed solution, the amount of water-soluble nutrient added is 0.01-0.2 g / mL, the mass fraction of sweet potato protein is 3%-5%, and the mass fraction of pullulan is 5%-16%; Preferably, the sweet potato protein / pululan mixed solution is stirred in a water bath at 30-50° C. for 3-5 hours, and after being stirred evenly and cooled to room temperature, 0.01-0.2 g / mL of water-soluble nutrients are added, stirred evenly at room temperature for 2-3 hours, and ultrasonicated for 5-10 minutes to remove bubbles, to obtain a water-soluble nutrient / sweet potato protein / pululan mixed solution as a continuous phase.

7. The method for preparing a dual-loaded sweet potato protein-based nanofiber membrane for delivering water-insoluble nutrients according to claim 1, characterized in that: In the step (3), the amount of water-insoluble nutrients added is 0.02-0.2 g / mL; and the volume ratio of the dispersed phase to the continuous phase is 1:9-5:

5.

8. The method for preparing a dual-loaded sweet potato protein-based nanofiber membrane for delivering water-insoluble nutrients according to claim 1, characterized in that: In the step (4), the homogenization emulsification includes one or more of rotor-stator homogenization, ultrasonic homogenization and high-pressure homogenization; the conductivity of the water-in-oil emulsion is 440-800 μs / cm, and the particle size is 90-400 nm.

9. The method for preparing a dual-loaded sweet potato protein-based nanofiber membrane for delivering water-insoluble nutrients according to claim 1, characterized in that: In the step (5), the conditions for emulsion electrospinning are: a spinning solution flow rate of 0.3-0.6 mL / h, a spinning machine voltage of 18-20 kV, a spinning environment humidity of 25-35%, a spinning environment temperature of 25-40° C., a spinning distance of 10-15 cm, a spinning machine drum speed of 90-150 rpm, and a spinning time of 5-6 h.

10. Use of sweet potato protein-based nanofibers for delivering water-insoluble nutrients prepared by the preparation method according to any one of claims 1 to 9 in the production of food, health products, medicines and cosmetics.

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