Preparation method of pomegranate protein fiber-diglyceride nano emulsion gel microcapsule, obtained product and application

Abstract

The invention belongs to the field of food biology, and particularly relates to a preparation method of pomegranate protein fiber-diglyceride nano emulsion gel microcapsules, an obtained product and application. The pomegranate seed protein fiber and diglyceride are compounded to prepare the protein fiber-diglyceride composite nano-emulsion, liquid drops of the composite emulsion are more uniform than those of single diglyceride emulsion, and meanwhile, the stability of the emulsion is improved; the nano-emulsion gel microcapsule is prepared from a sodium alginate solution and a composite nano-emulsion through ionic crosslinking, the release kinetics of diglyceride in the nano-emulsion gel microcapsule is researched under the condition of the optimal proportion, and the result shows that the prepared emulsion gel microcapsule shows the pH response characteristic; the slow release of the diglyceride in the colon can be realized. In a word, the preparation method provided by the invention provides a technical support for efficient utilization of the diglyceride, and the application value of the diglyceride in the field of functional food development is improved.

Description

Technical Field

[0001] The invention belongs to the field of food biology, and specifically relates to a preparation method of pomegranate protein fiber-diacetylglycerol nanoemulsion gel microcapsule and the obtained product and application. Background Art

[0002] Pomegranate seeds account for 22% of pomegranate fruit, with an average content of 37-143g / kg. Pomegranate seeds are rich in nutrients such as protein, lipids, sugars, crude fiber, etc. In addition, they also contain a large number of flavonoids, anthocyanins, steroid compounds, stigmasterol, etc., and are raw materials for manufacturing medicines, cosmetics and some functional foods. At present, after pomegranates are processed and produced into pomegranate juice, pomegranate wine, jam, etc., a large amount of pomegranate waste residue will be generated, which contains a large number of pomegranate seeds. Most of these pomegranate seeds are discarded, which is easy to cause a waste of resources.

[0003] Pomegranate seeds contain 12.3% protein and a total of 17 amino acids, of which 8 essential amino acids account for 34.83% of the total amino acids, glutamic acid accounts for 23.13% of the total amino acids, the highest content, followed by arginine at 13.44%, which is higher than general protein. In the prior art, there is no record of preparing pomegranate seed protein into microcapsules. Summary of the invention

[0004] In view of the research and development gap in the prior art, the present invention provides a method for preparing pomegranate protein fiber-diacylglycerol nanoemulsion microcapsules.

[0005] The present invention also provides a pomegranate protein fiber-diacylglycerol nanoemulsion microcapsule prepared by the above preparation method.

[0006] Another object of the present invention is to provide the use of the pomegranate protein fiber-diacylglycerol nanoemulsion microcapsules in the preparation of functional foods.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a method for preparing a pomegranate protein fiber-diacylglycerol nanoemulsion, comprising the following steps: (1) dispersing pomegranate seed protein in deionized water to obtain a pomegranate seed protein solution; adjusting the pH and stirring for a period of time; then hydrating the pomegranate seed protein solution at low temperature, centrifuging after hydration, adjusting the pH of the supernatant, and performing heat treatment; after the heat treatment, cooling the solution in an ice water bath, and then freeze-drying to obtain protein fiber PNF; (2)Preparation of nanoemulsion: Tween 80 and protein fiber were added to deionized water and heated to dissolve to form an aqueous phase; diglyceride as the oil phase was added to the aqueous phase and continuously stirred to obtain a mixed solution, and then the mixed solution was homogenized to obtain an initial emulsion; the initial emulsion was ultrasonically broken to obtain nanoemulsion PNF-DG-EM.

[0008] Preferably, in step (1), the concentration of the pomegranate seed protein solution is 50 mg / mL; the pH is adjusted to 2.0 with 6 mol / L HCl; after adjusting the pH, it is stirred at 25 °C for 2 h; the supernatant is adjusted to pH 2.0 with 6 mol / L HCl; the heat treatment is continuous heating and stirring in a 90 °C water bath for 10 h.

[0009] Preferably, in step (2), in the mixed solution, the mass ratio of each raw material is: Tween 80 4%, protein fiber 2%, diglyceride 2%, and the balance is deionized water.

[0010] Preferably, in step (2), the stirring time is 2 h.

[0011] The present invention also provides a method for preparing pomegranate protein fiber-diglyceride nanoemulsion microcapsules using the above-mentioned pomegranate protein fiber-diglyceride nanoemulsion, including the following steps: (a) Sodium alginate was added to deionized water and stirred evenly to obtain a sodium alginate solution; (b) The sodium alginate solution and the pomegranate protein fiber-diglyceride nanoemulsion were mixed and stirred at room temperature to obtain a nanoemulsion-sodium alginate composite sol; the nanoemulsion-sodium alginate composite sol was dropped into a CaCl2 solution to prepare nanoemulsion gel microcapsules.

[0012] Preferably, in step (a), the concentration of the sodium alginate solution is 2%, w / w; the stirring is carried out at room temperature for 24 h.

[0013] Preferably, in step (b), the volume ratio of the sodium alginate solution to the pomegranate protein fiber-diglyceride nanoemulsion is 9 - 1:1 - 9; the stirring is carried out at room temperature for 5 min.

[0014] Preferably, in step (b), the mass concentration of the CaCl2 solution is 2%; the nanoemulsion-sodium alginate composite sol is dropped into the CaCl2 solution using a single-channel microinjection pump.

[0015] Preferably, in step (b), the flow rate of the single-channel microinjection pump is set to 0.6 mL / min, the distance from the syringe needle to the CaCl2 solution is 5 cm; the nanoemulsion gel microcapsules are crosslinked in the CaCl2 solution for 15 min, and after crosslinking, the CaCl2 solution is removed and washed.

[0016] Another object of the present invention is to provide the application of the pomegranate protein fiber-diglyceride nanoemulsion microcapsule prepared by the above method in the preparation of functional foods.

[0017] The pomegranate seed protein used in the present invention is prepared by the following method: The prepared defatted pomegranate seed powder is sieved through a 60-mesh sieve, and then dispersed in deionized water at a ratio of 1 / 8 (g / mL). The pH is adjusted to 11.0 with 1 mol / L NaOH, stirred at 40 °C for 2 h, and the supernatant is collected after centrifugation for 15 min. The pH of the supernatant is adjusted to 4.0 with 1 mol / L HCl, allowed to stand overnight at room temperature, then centrifuged to collect the precipitate, and the precipitate is washed twice with deionized water and freeze-dried to obtain pomegranate seed protein (PSP), which is stored at 4 °C for later use.

[0018] The beneficial effects of the present invention are as follows: (1) In the present invention, a protein fiber-diglyceride composite nanoemulsion is prepared by compounding pomegranate seed protein fiber and diglyceride. The droplets of the composite emulsion are more uniform than those of a single diglyceride emulsion, and at the same time, the stability of the emulsion is improved; (2) In the present invention, nanoemulsion gel microcapsules are prepared by ionic crosslinking of sodium alginate solution and composite nanoemulsion. The effects of different ratios of sodium alginate solution and composite nanoemulsion on the properties of nanoemulsion gel microcapsules are evaluated. By analyzing rheological characteristics, texture properties, swelling properties, and physical and chemical properties, the optimal ratio of sodium alginate solution and composite nanoemulsion is finally determined to be 7 / 3. The release kinetics of diglyceride in the nanoemulsion gel microcapsules is studied under the optimal ratio conditions. The results show that the emulsion gel microcapsules prepared in the present invention exhibit pH-responsive characteristics and can achieve the slow release of diglyceride in the colon. In short, the preparation method provided by the present invention provides a technical support for the efficient utilization of diglyceride and improves the application value of diglyceride in the field of functional food development. Description of the Drawings

[0019] Figure 1 For the preparation and characterization of pomegranate seed protein in Example 1; wherein: (A) Flow chart of the pomegranate seed protein preparation process; (B-C) Solubility of pomegranate seed protein at pH = 2-11; (D) Zeta potential of pomegranate seed protein at pH = 2-11; Figure 2 For the preparation of pomegranate seed protein fiber in Example 2; (A) Flow chart of the pomegranate seed protein fiber preparation process; (B) TEM observation of the microstructures of PSP, PNF, and PNF-7; Figure 3 For the ultraviolet absorption spectrum (A) and ThT fluorescence spectrum (B) of pomegranate seed protein fiber; Figure 4 For the circular dichroism spectrum of pomegranate seed protein fiber; Figure 5 For the detection of the physical and chemical properties of pomegranate seed protein fiber. (A) Infrared spectrum; (B) XRD pattern; (C) DSC pattern; Figure 6 For the morphology of DG-EM (A) and PNF-DG-EM (B) and the microscopic morphology of droplets; Figure 7 . Detection of the particle size (A), zeta potential (B) and stability (C) of DG-EM and PNF-DG-EM; Figure 8 . Morphology of nanoemulsion gel microcapsules in different groups; Figure 9 . Rheological properties of nanoemulsion gel microcapsules in different groups. (A) Changes of G′ and G′′ with shear rate; (B) Changes of Tan δ with shear rate; Figure 10 . Hardness (A) and elasticity (B) of nanoemulsion gel microcapsules in different groups; Figure 11 . Thermogravimetric (A) and DSC (B) curves of nanoemulsion gel microcapsules in different groups; Figure 12 . Infrared (A) and XRD (B) patterns of nanoemulsion gel microcapsules in different groups; Figure 13 Swelling ratio of nanoemulsion gel microcapsules in solutions with different pH values; Figure 14 In vitro drug release of nanoemulsion gel microcapsules in simulated gastric fluid, simulated intestinal fluid and simulated colon fluid. Detailed implementation manners

[0020] The technical solutions of the present invention will be further explained and illustrated by specific examples below.

[0021] Example 1 (1) Preparation of defatted pomegranate seed powder First, clean, dry and crush pomegranate seeds to obtain pomegranate seed powder. Use n-hexane to defat the pomegranate seed powder. Treat the pomegranate seed powder and n-hexane according to a solid-liquid ratio of 1 / 10 (g / mL), an ultrasonic treatment time of 1 h, an ultrasonic temperature of 45 °C, and an ultrasonic power of 240 W. After the extraction, the pomegranate seed powder naturally settles and separates from n-hexane, and then is filtered by suction. Subsequently, place the pomegranate seed meal in a fume hood and dry it at room temperature for 12 h. Finally, store the treated defatted pomegranate seed powder in a self-sealing bag at 4 °C for later use.

[0022] (2) Preparation of pomegranate seed protein by alkali extraction and acid precipitation The prepared defatted pomegranate seed powder was passed through a 60-mesh sieve and then dispersed in deionized water at a ratio of 1 / 8 (g / mL). The pH was adjusted to 11.0 with 1 mol / L NaOH, and the mixture was stirred at 40 °C for 2 h. After centrifugation for 15 min, the supernatant was collected. The pH of the supernatant was adjusted to 4.0 with 1 mol / L HCl, and it was left standing overnight at room temperature. Then, the precipitate was collected by centrifugation, washed twice with deionized water, and freeze-dried to obtain pomegranate seed protein (PSP), which was stored at 4 °C for later use.

[0023] Pomegranate seed protein was prepared by the alkali extraction and acid precipitation method ( Figure 1 A). 38.4 g of protein was obtained from 100 g of pomegranate seeds, and the protein purity was determined to be 91.4% by the Kjeldahl method. The whiteness index of the prepared pomegranate seed protein was 58.07, which was similar to that of commercially available whey protein isolate (64.26) and much higher than that of commercially available soy protein isolate (42.23). In summary, pomegranate seed protein has a high yield, high purity, and suitable whiteness, greatly increasing its application value.

[0024] (3)Detection of solubility and zeta potential of pomegranate seed protein at different pH values Accurately weigh the prepared pomegranate seed protein and dissolve it in solutions with different pH values (2 - 11) to detect the solubility and zeta potential of pomegranate seed protein.

[0025] Solubility = (total initial protein - undissolved protein) / total initial protein * 100% In Figure 1 B - C, it was observed that the prepared pomegranate seed protein had a typical U-shaped solubility curve, and the solubility was the lowest at pH = 5.0 (indicating the isoelectric point), which was 20.2%. The solubility of pomegranate seed protein at neutral pH was 92.5%, which was much higher than that of other oilseed crop proteins. Its ability to maintain high solubility in a wide pH range (≥7) makes it have broad prospects in food applications.

[0026] Zeta potential is a method to describe the surface charge of proteins. It affects the interaction between proteins and solvent molecules, thus affecting the solubility of proteins. As Figure 1 shown in D, when the pH value was between 5 and 6, the zeta potential of the pomegranate seed protein solution decreased sharply and passed through the point with a net charge of 0 (isoelectric point), which was consistent with the solubility distribution. When the pH value changed from 11 to 3, due to the deprotonation of amino and carboxyl groups, the net surface charge changed from the negative maximum value (-32.6 mV) to the positive maximum value (24.7 mV). Under strong acidic conditions at pH = 2.0, the zeta potential decreased to 20.4 mV, which may be due to the hydrolysis of glutamine and asparagine into glutamic acid and aspartic acid, respectively. Figure 1

[0027] ​Preparation of Protein Fibers in Example 2 (1)Disperse the prepared pomegranate seed protein in deionized water (50 mg / mL), adjust the pH to 2.0 with 6 mol / L HCl, and stir the solution at 25 °C for 2 h. Then keep the solution overnight at 4 °C for complete hydration, centrifuge for 20 min, adjust the pH of the supernatant to 2.0 with 6 mol / L HCl, and continuously heat and stir in a water bath at 90 °C for 10 h. After heat treatment, cool the solution in an ice-water bath and then lyophilize to obtain protein fibers (PNF), which are stored at 4 °C for later use. At the same time, adjust the pH of the heat-treated solution to 7.0 and lyophilize to obtain sample PNF-7.

[0028] Observe the microstructure of the protein fibers by transmission electron microscopy (TEM).

[0029] Protein fibers are prepared from pomegranate seed protein by acid hydrolysis method ( Figure 2 A). To analyze the morphology of the formed protein fibers at the microscale, TEM images of the samples were taken. As Figure 2 shown in B, linear and curly fibers can be observed at pH values of 2.0 (PNF) and 7.0 (PNF-7), and untreated PSP has no fiber structure. When the pH value is 2.0, PSP can form a stable fiber structure, while when the pH value is adjusted to 7.0, the protein fibers maintain a short linear and curly appearance.

[0030] (2)Characterize the obtained protein fibers by UV absorption, fluorescence spectroscopy, infrared, circular dichroism, DSC, and XRD.

[0031] (a)UV Absorption and ThT Fluorescence Detection of Pomegranate Seed Protein Fibers First, the UV absorption spectra of PSP and PNF were detected. As Figure 3 shown in A, the maximum UV absorption of both PSP and PNF is at 237 nm, indicating that fibrillation has no effect on the UV absorption of the protein. Figure 3 Figure B shows the ThT detection results of PSP and PNF. The fluorescence intensity of PNF increases, indicating that this sample has a higher content of β-turn secondary structure, and further indicating that the protein has undergone fibrillation.

[0032] (b)Circular Dichroism Detection of Pomegranate Seed Protein Fibers Analyze the changes in secondary structure during the fibrillation of pomegranate seed protein by circular dichroism. As Figure 4As shown, the spectrum of PSP has an obvious minimum near 208 nm and a slight minimum near 212 nm, indicating that the secondary structure of PSP mainly consists of α-helix and irregular coils. In the range of 200 - 240 nm, PSP and PNF show obvious negative mean residue ellipticity, which indicates that it is mainly β-turn structure, and the β-turn secondary structure of PNF is more obvious than that of PSP, which is consistent with the results of ThT fluorescence detection.

[0033] (c) Detection of the physicochemical properties of pomegranate seed protein fiber The physicochemical properties of pomegranate seed protein fiber were detected, such as Figure 5 As shown in Figures A and 5B, the infrared spectra and XRD patterns of PSP and PNF are basically the same, indicating that fibrillation did not change the physical structure of the protein. However, by comparing the DSC spectra of PSP and PNF ( Figure 5 C), it can be seen that the melting temperature of PSP is about 101 °C, and the melting temperature of PNF is about 120 °C, indicating that fibrillation improves the thermal stability of the protein.

[0034] Example 3 Preparation of protein fiber - diglyceride nanoemulsion (1) Preparation of nanoemulsion The nanoemulsion consists of an aqueous phase, an oil phase, and a surfactant. The oil phase is selected as diglyceride, and the surfactant is selected as Tween 80. Tween 80 and protein fiber were added to deionized water and heated to dissolve to form the aqueous phase. Diglyceride was added to the aqueous phase and continuously stirred for 2 h to obtain a mixed solution (the mass percentages of each raw material are: Tween 80: 4%, protein nanofiber: 2%, diglyceride: 2%, and the balance is deionized water), and then the mixed solution was homogenized to obtain an initial emulsion. The initial emulsion was ultrasonically broken to obtain a nanoemulsion (PNF-DG-EM). At the same time, a nanoemulsion without protein fiber was prepared as a control (DG-EM).

[0035] As Figure 6 shown, both the diglyceride nanoemulsion (DG-EM) and the protein fiber - diglyceride nanoemulsion (PNF-DG-EM) are homogeneous emulsions, showing isotropy. It can be seen under a microscope that the droplets are all regular spheres. In comparison, the particle size of PNF-DG-EM is more uniform, and the results show that protein fiber helps the emulsion droplets to be more regular.

[0036] (2) Characterization of nanoemulsion The particle size and potential of the nanoemulsion were detected, and the stability was evaluated. Take 10 mL of the nanoemulsion and measure the particle size and PDI of the sample at room temperature using a particle size analyzer, and observe at intervals of 2 days for a total of 14 days.

[0037] The average particle size of DG-EM was 109.87 ± 1.29 nm, and that of PNF-DG-EM increased to 114.64 ± 2.72 nm, indicating that the addition of protein fibers had little effect on the particle size of emulsion droplets ( Figure 7 A). The charge interaction between particles and the medium was measured as the Zeta potential, which helped to maintain the separation of droplets and thus contributed to the improvement of emulsion stability. The Zeta potential value of DG-EM was -23.2 ± 0.4 mV, and that of PNF-DG-EM was -27.7 ± 0.3 mV. The decrease in the Zeta potential of PNF-DG-EM indicated that the PNF-DG-EM system was more stable ( Figure 7 B). At the same time, the particle size of the nanoemulsion did not change significantly within 14 days, indicating that the prepared emulsion had good stability over time ( Figure 7 C).

[0038] Preparation of nanoemulsion gel microcapsules in Example 4 (1) 2 g of sodium alginate was added to 98 g of deionized water to obtain a sodium alginate solution (2%, w / w). The mixture was stirred at room temperature for 24 h to obtain a sodium alginate solution (SA). The sodium alginate solution and the prepared protein fiber-diglyceride nanoemulsion were mixed in different ratios (SA / PNF-DG-EM = 10 / 0, 9 / 1, 7 / 3, 5 / 5, 3 / 7, 1 / 9), and stirred at room temperature for 5 min to obtain a nanoemulsion-sodium alginate composite sol. The obtained composite sol was dropped into a 2% CaCl2 solution using a single-channel microinjection pump to prepare nanoemulsion gel microcapsules. The flow rate of the injection pump was set at 0.6 mL / min, and the distance from the syringe needle to the CaCl2 solution was 5 cm. The nanoemulsion gel microcapsules were crosslinked in the CaCl2 solution for 15 min. After crosslinking, the CaCl2 solution was removed, and the microcapsules were washed twice with deionized water to obtain stable nanoemulsion gel microcapsules.

[0039] Sodium alginate solution (SA) and protein fiber-diglyceride nanoemulsion (PNF-DG-EM, hereinafter directly referred to as PNF) were used to prepare nanoemulsion gel microcapsules by ionic crosslinking in different ratios. As Figure 8 shown, the prepared gel microcapsules had uniform particles with a particle size of about 1 mm. When SA / PNF = 10 / 0, 9 / 1, 7 / 3, 5 / 5, the particle morphology showed regular spherical shapes; when SA / PNF = 3 / 7, the particles began to show a trailing situation; when SA / PNF = 1 / 9, gel particles could not be formed, which might be due to the too low concentration of sodium alginate resulting in low ionic crosslinking degree and inability to form a stable spatial network structure.

[0040] (2) Detection of rheological properties of nanoemulsion gel microcapsules The rheological properties of nanoemulsion gel microcapsules in different groups were determined by a rheometer. The shear storage modulus (G′), loss modulus (G′′), and loss tangent (Tanδ) values of the samples were measured using a flat plate with a diameter of 40 mm in the angular frequency range of 0.1 - 100 rad / s.

[0041] The linear viscoelastic region of the samples was determined by rheological frequency sweep method, which can indirectly reflect the stability of the samples. G′ is the storage modulus, reflecting the elasticity of the system; G′′ is the loss modulus, reflecting the viscosity of the system. As Figure 9 shown in A, with the increase of shear frequency, both G′ and G′′ of all samples increased, indicating that the cross - links in the network structure were relatively dense. In the whole frequency range (0.1 - 100 rad / s), G′′ of all samples was higher than G′, indicating that a gel structure mainly composed of viscous gel was formed. When SA / PNF = 7 / 3, the G′ and G′′ of the sample were relatively high, which may be due to the increase in cross - linking degree leading to the enhancement of the network structure, thus forming a composite gel with better viscoelasticity. The larger the Tanδ value, the greater the internal friction of the system. Tanδ < 1 indicates that the elasticity of the sample is dominant, while Tanδ > 1 indicates that the viscosity of the sample is dominant. As Figure 9 shown in B, the Tanδ values of all samples were less than 1, indicating that solid - state gel microcapsules mainly composed of elasticity were formed.

[0042] (3) Texture analysis of nanoemulsion gel microcapsules At room temperature, a texture analyzer was used to measure the hardness and elasticity of nanoemulsion gel microcapsules in each group. The probe was P50, the pre - test speed was 2.0 mm / s, the measurement speed was 1.0 mm / s, and the post - test speed was 5.0 mm / s. The nanoemulsion gel microcapsules were compressed to 40% of the original height, and the hardness and elasticity values were recorded.

[0043] Hardness and elasticity are two basic properties of gel strength, reflecting the internal spatial structure of the gel. Higher hardness and elasticity of the gel indicate a high degree of cross - linking in the system, strong molecular interactions, a compact structure, and good overall stability. As Figure 10 shown, when the ratio of SA and PNF is different, the hardness and elasticity of the gel microcapsules are also different. When SA / PNF = 7 / 3, the sample has the best hardness and elasticity. When the proportion of PNF increases or decreases, the hardness and elasticity of the material both decrease significantly. The differences in sample hardness and elasticity reflect the differences in the internal network structure.

[0044] (4) Physicochemical property analysis of nanoemulsion gel microcapsules The physicochemical properties of nanoemulsion gel microcapsules were analyzed by physical and chemical detection techniques, including thermogravimetry, DSC, infrared, and XRD.

[0045] The thermodynamic properties of nanoemulsion gel microcapsules were analyzed by the thermogravimetric and DSC curves of CG and ME-CG. As Figure 11 shown, the thermal stabilities of nanoemulsion gel microcapsules obtained with different SA / PNF ratios were also different. Among them, the sample with SA / PNF = 7 / 3 had the highest thermal stability. The results showed that protein fibers helped to improve the thermal stability of gel microcapsules.

[0046] (5) Infrared and XRD analyses of nanoemulsion gel microcapsules The physicochemical properties of nanoemulsion gel microcapsules were further analyzed by infrared and XRD. As Figure 12 shown, the infrared and XRD spectra of each group of samples were basically consistent, indicating that no new chemical bonds were formed during the formation of gel microcapsules, and the samples maintained their original crystal forms.

[0047] (6) Swelling performance detection of nanoemulsion gel microcapsules The nanoemulsion gel microcapsules were freeze-dried, and the initial weight of the freeze-dried sample was recorded as W0. Then the samples were respectively immersed in HCl solution (pH = 2.0), HCl solution (pH = 5.0), PBS solution (pH = 7.0) and NaOH solution (pH = 9.0), and replaced every 10 h. In the swelling study, the samples were weighed after 0.5, 1, 2, 4, 6, 8, 10, 15, 20 h, and the weight was recorded as Wt. The swelling ratio (SR%) of the sample was calculated as follows: SR (%) = (Wt - W0) / W0 × 100% The swelling property is a phenomenon in which the volume of the gel significantly increases after absorbing liquid, reflecting the internal spatial structure of the gel. As Figure 13 shown, the samples showed different swelling properties in HCl solution (pH = 2.0), HCl solution (pH = 5.0), PBS solution (pH = 7.0) and NaOH solution (pH = 9.0) respectively. The swelling ratio of the sample in HCl solution was significantly lower than that in PBS solution and NaOH solution, indicating that the sample was quite sensitive to alkalinity. When SA / PNF = 7 / 3, the swelling index of the sample was the largest. These results also showed that in different solutions, the time for the sample to reach swelling equilibrium was different. In NaOH solution, the shortest time to reach swelling equilibrium was 8 h, while in HCl solution (pH = 2.0), the longest time to reach swelling equilibrium was more than 20 h. The increase in internal cross-linking of the gel made the spatial network structure more tightly connected, thus increasing the resistance of external water molecules to penetrate the hydrogel. All swelling tests showed that nanoemulsion gel microcapsules had good pH sensitivity and could effectively delay drug release.

[0048] Effect Example: Study on the release kinetics of diglyceride The in vitro release amounts of diglycerides in nanoemulsion gel microcapsules were determined in different solutions of simulated gastric fluid (pH = 1.2), intestinal fluid (pH = 6.8), and colonic fluid (pH = 7.4), respectively. The freeze-dried nanoemulsion gel microcapsules were dispersed in different release media and stirred at room temperature. High-performance liquid chromatography was used to determine the release levels of diglycerides in the release media. The in vitro release curves were plotted based on the cumulative release amounts of diglycerides.

[0049] To analyze the release kinetics of PEA in PEA@ME3-CG3, the following different mathematical models were used to fit the in vitro drug release curves: Zero-order model: Q = k × t First-order model: Q = 1 - exp. (k × t) Higuchi model: Q = k × t 1 / 2 Where Q is the cumulative release amount of diglycerides, t is the release time, and k is the kinetic constant.

[0050] To study the release effects of diglycerides in nanoemulsion gel microcapsules in different media, in vitro release tests were conducted in this invention, and the pH sensitivity of nanoemulsion gel microcapsules was determined (the sample with SA / PNF = 7 / 3 was selected as the research object). There were significant differences in the cumulative release amounts (%) of diglycerides in nanoemulsion gel microcapsules in different release media ( Figure 14 ), and the cumulative release amount in simulated colonic fluid was significantly higher than that in gastric fluid and intestinal fluid. In the simulated colonic fluid release experiment, the release equilibrium was reached at about 6 h, and in gastric fluid and intestinal fluid, the release equilibriums were reached at about 8 h and 7 h, respectively. The results showed that the release of nanoemulsion gel microcapsules was sensitive to the alkaline environment, realizing the pH-controlled release of diglycerides.

[0051] Table 1 lists the correlation coefficient (R 2 ) values when different mathematical models were used to fit the release curves of nanoemulsion gel microcapsules. The release curves of nanoemulsion gel microcapsules in different media were most in line with the first-order model, and the R 2 values were all greater than 0.9, indicating the sustained and delayed release of diglycerides in the gastrointestinal tract. The above results showed that nanoemulsion gel microcapsules had good pH responsiveness and could be used as a sustained-release carrier in the colon.

[0052] Table 1 Correlation coefficient (R 2 ) of the release kinetic model of nanoemulsion gel microcapsules

Claims

1. A preparation method of pomegranate protein fiber-diglyceride nanoemulsion, characterized in that, It includes the following steps: (1) Disperse pomegranate seed protein in deionized water to obtain a pomegranate seed protein solution; adjust the pH and then stir for a period of time; Then hydrate the pomegranate seed protein solution at low temperature. After the hydration is completed, centrifuge it, adjust the pH of the supernatant, and perform heat treatment; After the heat treatment, cool the solution in an ice-water bath and then lyophilize it to obtain protein fiber PNF; (2) Preparation of nanoemulsion: Add Tween 80 and protein fiber to deionized water and heat to dissolve it into the aqueous phase; add the oil phase diglyceride to the aqueous phase and continuously stir to obtain a mixed solution, and then homogenize the mixed solution to obtain an initial emulsion; subject the initial emulsion to ultrasonic fragmentation to obtain nanoemulsion PNF-DG-EM.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the pomegranate seed protein solution is 50 mg / mL; adjust the pH to 2.0 with 6 mol / L HCl; stir at 25 °C for 2 h after adjusting the pH; adjust the pH of the supernatant to 2.0 with 6 mol / L HCl; the heat treatment is to continuously heat and stir in a 90 °C water bath for 10 h.

3. The preparation method according to claim 1 or 2, characterized in that, In step (2), in the mixed solution, the mass ratio of each raw material is: Tween 80 4%, protein fiber 2%, diglyceride 2%, and the balance is deionized water.

4. The preparation method according to claim 1 or 3, characterized in that, In step (2), the stirring time is 2 h.

5. A method for preparing pomegranate protein fiber - diglyceride nanoemulsion microcapsules based on the pomegranate protein fiber - diglyceride nanoemulsion described in claims 1 - 4, characterized in that, It includes the following steps: (a) Add sodium alginate to deionized water and stir evenly to obtain a sodium alginate solution; (b) Mix the sodium alginate solution and the protein fiber-diglyceride nanoemulsion and stir at room temperature to obtain a nanoemulsion-sodium alginate composite sol; drop the nanoemulsion-sodium alginate composite sol into a CaCl2 solution to prepare nanoemulsion gel microcapsules.

6. The method according to claim 5, characterized in that, In step (a), the concentration of the sodium alginate solution is 2%, w / w; the stirring is carried out at room temperature for 24 h.

7. The method according to claim 5, characterized in that In step (b), the volume ratio of the sodium alginate solution to the protein fiber-diglyceride nanoemulsion is 9-1:1-9; the stirring is carried out at room temperature for 5 min.

8. The method according to claim 7, characterized in that In step (b), the mass concentration of the CaCl2 solution is 2%; the nanoemulsion-sodium alginate composite sol is dropped into the CaCl2 solution by a single-channel microinjection pump.

9. The method according to claim 5 or 8, characterized in that In step (b), the flow rate of the single-channel microinjection pump is set to 0.6 mL / min, and the distance from the syringe needle to the CaCl2 solution is 5 cm; the nanoemulsion gel microcapsules are crosslinked in the CaCl2 solution for 15 min, and after crosslinking, the CaCl2 solution is removed and washed.

10. Application of the pomegranate protein fiber-diglyceride nanoemulsion microcapsules prepared by the method according to any one of claims 5-9 in the preparation of functional foods.

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