Lactalbumin cross-linked emulsion loaded with fat-soluble thermosensitive polyphenol and application of whey protein cross-linked emulsion

Abstract

The invention discloses a whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenol and application of the whey protein cross-linked emulsion, and belongs to the technical field of Pickering emulsions. The invention provides a novel method for delivering fat-soluble thermosensitive polyphenol by using whey protein cross-linked emulsion, which comprises the following steps of: preparing the whey protein cross-linked emulsion (PCEs) by using whey protein as a Pickering emulsion raw material and adopting a transglutaminase cross-linking method and an emulsifying solvent evaporation method; the whey protein cross-linked emulsion prepared by the invention shows high stability in temperature changes such as freezing and thawing, high temperature and the like, and has the ability of promoting transdermal permeation.

Description

Technical Field

[0001] The present invention relates to a whey protein cross-linked emulsion loaded with a lipophilic thermosensitive polyphenol and its application, belonging to the technical field of Pickering emulsions. Background Art

[0002] Lipophilic thermosensitive polyphenols are a class of natural compounds with lipophilic characteristics and sensitive to high temperatures, widely present in the secondary metabolites of plants. Due to their unique antioxidant, anti-inflammatory, antibacterial, and anticancer biological activities, these polyphenols have been widely used in the fields of food, cosmetics, medicine, and functional materials.

[0003] Although lipophilic thermosensitive polyphenols have significant application potential in many fields, there are still some problems and deficiencies in their practical applications. Lipophilic thermosensitive polyphenols are very sensitive to temperature due to the presence of groups such as unsaturated double bonds and phenolic hydroxyl groups, and are prone to a decrease in activity due to temperature degradation during processing, storage, and use. In addition, due to their lipophilic properties, lipophilic drugs will precipitate in the gastrointestinal tract, resulting in a slower absorption rate than elimination. And oil-based products cannot be effectively absorbed by the human body, and have a large first-pass effect, which affects their uniform dispersion and bioavailability in medicine, food, and cosmetics. Delivering lipophilic thermosensitive polyphenols in an aqueous medium is beneficial because it will increase their feel, palatability, and usage effects.

[0004] Pickering emulsions have a series of application advantages such as improving drug stability, controlling drug release, and promoting transdermal penetration. Whey protein is a protein of natural origin and has functional properties suitable for stabilizing Pickering emulsion particles. However, since Pickering emulsions are a thermodynamically unstable system, it is difficult to resist environmental changes. Therefore, the delivery system of whey protein-based Pickering emulsions is limited in terms of long-term storage stability, environmental stress tolerance, especially freeze-thaw stability.

[0005] The literature "Novel Pickering emulsion stabilized by glycosylated whey protein isolate: Characterization, stability, and curcumin bioaccessibility" discloses a method for preparing Pickering emulsion and encapsulating curcumin using whey protein and transglutaminase. In this method, modified protein particles are first prepared, and then Pickering emulsion is prepared and encapsulated. The literature "Low temperature extrusion promotes transglutaminase cross-linking of whey protein isolate and enhances its emulsifying properties and water holding capacity" discloses the use of extrusion and transglutaminase treatment to improve the properties of whey protein isolate. However, the Pickering emulsions prepared by the above technical solutions still have problems such as poor stability, inability to be frozen and redissolved.

[0006] Therefore, it has extremely high practical and economic value to develop a Pickering emulsion with stronger stability, easier storage and transportation, and resistance to environmental changes, and further improve the encapsulation effect and percutaneous penetration efficiency of lipophilic thermosensitive polyphenols. Summary of the Invention

[0007] To solve the above problems, the present invention provides a method for preparing a whey protein cross-linked emulsion loaded with lipophilic thermosensitive polyphenols, obtaining a nano-cross-linked emulsion with a high encapsulation rate and drug loading. The present invention solves the problem of poor water solubility of lipophilic thermosensitive polyphenols, such as 8,9-dihydrocannabidiol (H2CBD) and phenethyl resorcinol. At the same time, due to its unique small particle size and safe and green characteristics, it can become a new method for promoting the percutaneous delivery of lipophilic thermosensitive polyphenols.

[0008] The first object of the present invention is to provide a method for preparing a whey protein cross-linked emulsion loaded with lipophilic thermosensitive polyphenols, comprising the steps of:

[0009] (1) Dissolving lipophilic thermosensitive polyphenols in an oil-phase solution to obtain a bioactive substance oil-phase solution; mixing the bioactive substance oil-phase solution with a whey protein suspension and homogenizing to obtain a primary Pickering emulsion;

[0010] (2) Adding transglutaminase to the primary Pickering emulsion for cross-linking; after cross-linking, cooling, concentrating, and inactivating the enzyme to obtain a whey protein cross-linked emulsion loaded with lipophilic thermosensitive polyphenols.

[0011] In one embodiment, the mass ratio of the fat-soluble thermosensitive polyphenol to the oil-phase solution in step (1) is 0.1-1:1.

[0012] Optionally, the oil-phase solution is a mixed solution of triglyceride caprylate / caprate and a volatile organic solvent; wherein, the volatile organic solvent includes ethyl acetate, petroleum ether, dichloromethane or chloroform.

[0013] In one embodiment, the mass ratio of triglyceride caprylate / caprate to the volatile organic solvent is 1-9:1-9.

[0014] In one embodiment, the concentration of whey protein in the whey protein suspension in step (1) is 0.25-2 wt.%.

[0015] Optionally, the concentration of whey protein is 0.5-2 wt.%.

[0016] In one embodiment, the volume ratio of the bioactive substance oil-phase solution to the whey protein suspension in step (1) is 1-2:1-2.

[0017] Optionally, the volume ratio of the bioactive substance oil-phase solution to the whey protein suspension in step (1) is 1:1.

[0018] In one embodiment, the addition amount of transglutaminase in step (2) is 10-40 U / g of whey protein; the crosslinking is carried out at 30-60 °C for 2-8 h.

[0019] Optionally, the addition amount of transglutaminase is 10-30 U / g of whey protein;

[0020] Optionally, the crosslinking is carried out at 30-50 °C for 3-6 h.

[0021] In one embodiment, the concentration in step (2) is for the volatile oil phase, at 30 °C, and the rotation speed is 10-50 rpm.

[0022] In one embodiment, the fat-soluble thermosensitive polyphenols include 8,9-dihydrocannabidiol, phenethyl resorcinol, tocopherol, bakuchiol, resveratrol, etc.; such fat-soluble substances have poor solubility in the aqueous phase.

[0023] The second object of the present invention is to provide a whey protein crosslinked emulsion loaded with fat-soluble thermosensitive polyphenols prepared by any of the above methods or a powder obtained by drying the whey protein crosslinked emulsion loaded with fat-soluble thermosensitive polyphenols prepared by any of the above methods.

[0024] The third object of the present invention is to provide the application of any of the above-mentioned methods, the above-mentioned whey protein cross-linked emulsion loaded with liposoluble thermosensitive polyphenols or the powder after drying the above-mentioned whey protein cross-linked emulsion loaded with liposoluble thermosensitive polyphenols in the fields of chemical industry, medicine, food, agriculture and environment.

[0025] The fourth object of the present invention is to provide a product, which contains the above-mentioned whey protein cross-linked emulsion loaded with liposoluble thermosensitive polyphenols or the powder after drying the above-mentioned whey protein cross-linked emulsion loaded with liposoluble thermosensitive polyphenols;

[0026] Optionally, the product includes functional materials, cosmetics, foods and drugs.

[0027] In one embodiment, the dosage forms of the cosmetics and drugs are not limited to liniments, but can also be used for transdermal ointments, plasters, cataplasms, patches, aerosols, sprays, foams and microsponges, microneedle patches and other dosage forms.

[0028] In one embodiment, the forms of the foods are not limited to liquid foods, but can also be used for solid foods, semi-solid foods, emulsified foods, suspensions, foam foods, carbonated beverages, frozen foods and fermented foods and other types.

[0029] The fifth object of the present invention is to provide a method for simultaneously improving the performance of the whey protein cross-linked emulsion, and the performance includes one or more of redissolution, freeze-thaw stability, thermal stability and transdermal effect; the method includes:

[0030] (1) Dissolve liposoluble thermosensitive polyphenols in an ethyl acetate-caprylic / capric triglyceride mixed oil phase solution to obtain a bioactive substance oil phase solution; mix the bioactive substance oil phase solution with a whey protein suspension and homogenize to obtain a primary Pickering emulsion;

[0031] (2) Add transglutaminase to the primary Pickering emulsion for cross-linking; after cross-linking, cool, concentrate and inactivate the enzyme to obtain a whey protein cross-linked emulsion loaded with liposoluble thermosensitive polyphenols;

[0032] Optionally, in step (1), the oil phase solution is a mixed solution of caprylic / capric triglyceride and a volatile organic solvent; wherein, the volatile organic solvent includes ethyl acetate, petroleum ether, dichloromethane or chloroform; the mass ratio of the liposoluble thermosensitive polyphenols to the oil phase solution is 1-9:1-9;

[0033] Optionally, the concentration of whey protein in the whey protein suspension in step (1) is 0.25-2 wt.%; optionally, the concentration of whey protein is 0.5-2 wt.%;

[0034] Optionally, the volume ratio of the bioactive substance oil phase solution to the whey protein suspension in step (1) is 1-2:1-2;

[0035] Optionally, the addition amount of transglutaminase in step (2) is 10-40 U / g of whey protein; the crosslinking is carried out at 30-60 °C for 2-8 h;

[0036] Optionally, the addition amount of transglutaminase is 10-30 U / g of whey protein;

[0037] Optionally, the crosslinking is carried out at 30-50 °C for 3-6 h;

[0038] Optionally, the lipophilic thermosensitive polyphenols include 8,9-dihydrocannabidiol, cannabidiol, phenethyl resorcinol, tocopherol, bakuchiol, resveratrol, etc.

[0039] Advantages of the present invention

[0040] (1) The present invention provides a new method for delivering lipophilic thermosensitive polyphenols by whey protein crosslinked emulsion. Using the Pickering emulsion stabilized by whey protein as the raw material, the whey protein crosslinked emulsion (PCEs) is prepared by the transglutaminase crosslinking method and the emulsification solvent evaporation method; the whey protein crosslinked emulsion prepared by the present invention shows high stability in temperature changes such as freeze-thaw and high temperature, and has the ability to promote transdermal penetration.

[0041] (2) The whey protein crosslinked emulsion obtained by the present invention has a relatively high encapsulation ability for nutrients with poor oil and water solubility. The system according to the present invention is not limited to loading H2CBD as a single emulsion carrier, and some other lipophilic bioactive substances (conventional lipophilic active substances such as phenethyl resorcinol and tocopherol) can also be used under slight adjustment or without change of the system.

[0042] (3) The whey protein crosslinked emulsion loaded with H2CBD of the present invention has good safety, no cytotoxicity, and good skin permeability. Description of the drawings

[0043] Figure 1 It is a schematic diagram of the preparation process of PCEs;

[0044] Figure 2 It is a transmission electron microscope (TEM) image of PCEs;

[0045] Figure 3 It is a microscopic image of PUEs prepared at different whey protein concentrations;

[0046] Figure 4 It is the influence of different crosslinking temperatures on the crosslinking degree, particle size and homogeneity (PDI) of PCEs;

[0047] Figure 5 Effect of different TGase concentrations on the crosslinking degree, particle size and homogeneity of PCEs;

[0048] Figure 6 Effect of different crosslinking times on the crosslinking degree, particle size and homogeneity of PCEs;

[0049] Figure 7 Laser confocal image of PCEs;

[0050] Figure 8 Results of solubility detection of PUEs and PCEs;

[0051] Figure 9 Results of freeze-thaw stability detection of PCEs;

[0052] Figure 10 Results of freeze-thaw stability detection of PUEs;

[0053] Figure 11 Results of thermal stability detection of PCEs;

[0054] Figure 12 Results of thermal stability detection of PUEs;

[0055] Figure 13 Unit area cumulative permeation amount and cumulative permeation amount of PCEs. Detailed implementation manners

[0056] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0057] Tetrahydrocannabinol (THC) and cannabidiol (CBD) are the components with relatively high contents in cannabinoids. Different from tetrahydrocannabinol, cannabidiol has no mental addiction and has anticonvulsant, sedative hypnotic, anti-anxiety, antipsychotic, anti-inflammatory and neuroprotective effects, becoming a natural active ingredient with great application prospects.

[0058] 8,9-Dihydrocannabidiol (H2CBD) is a fully synthetic CBD analogue. Its molecular structure replaces the C=C bond of cannabidiol (CBD) with a saturated C-C bond outside the limonene unit ring, so that H2CBD cannot be converted into neurotoxic Δ9-tetrahydrocannabinol (Δ9-THC). H2CBD also has antibacterial and antioxidant properties similar to those of CBD and better safety. However, since H2CBD is a lipophilic thermosensitive polyphenol and insoluble in water, it has the disadvantages of poor water solubility and poor transdermal delivery effect, and there are problems such as low bioavailability and poor stability under different environmental conditions in the actual application process.

[0059] Therefore, in the present invention, 8,9-dihydrocannabidiol is used as an example of a lipophilic thermosensitive polyphenol to prepare a Pickering solution loaded with 8,9-dihydrocannabidiol.

[0060] Raw materials used in the examples:

[0061] Whey protein was purchased from Agropur Ingredients;

[0062] Ethyl acetate was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0063] Caprylic capric triglyceride was purchased from Shandong Yousuo Chemical Technology Co., Ltd.;

[0064] TGase was purchased from Taixing Dongsheng Biotechnology Co., Ltd.

[0065] H2CBD was synthesized by the research group (according to the method in patent CN113372196B), and the specific steps are as follows:

[0066] Dissolve oleanol (18 g, 0.1 mol) and α-phellandrene (17 g, 0.125 mol) in toluene (10 mL), add ferric chloride (0.162 g, 1 mmol), stir the mixture at room temperature (25 °C) for reaction, monitor with TLC and HPLC until the yield of 8,9-dihydrocannabidiol no longer increases, filter, wash the obtained filtrate with saturated brine (3 × 50 mL), distill off the solvent under reduced pressure, separate the crude product by silica gel column chromatography, and elute with a mixture of petroleum ether and ethyl acetate (petroleum ether:ethyl acetate = 40:1), and track and detect the elution by TLC. Collect and combine the silica gel column eluate containing the target product, and concentrate to obtain 8,9-dihydrocannabidiol (28.44 g, yield 90%).

[0067] Testing methods:

[0068] 1. Determination of crosslinking degree:

[0069] The crosslinking degree is determined by calculating the change in the content of free amino groups. Since TGase catalyzes the crosslinking reaction between the γ-carboxamide group of the glutamine residue of whey protein and the ε-amino group of the lysine residue to form an ε-(γ-glutamyl)lysine bond, and the free amino groups of whey protein are involved in the formation of the ε-(γ-glutamyl)lysine bond, the decrease in the content of free amino groups can reflect the increase in the ε-(γ-glutamyl)lysine bond in the whey protein sample, that is, the increase in the degree of protein crosslinking. The free amino group of lysine in whey protein serves as an acyl acceptor, and the change in its content to a certain extent indicates the occurrence of the TGase crosslinking reaction and can also indirectly reflect the crosslinking degree of whey protein.

[0070] The o-phthalaldehyde method (OPA) was used to determine free amino groups. Using 0.62 M boric acid as a diluent (0.1% SDS), 10 mg of o-phthalaldehyde was dissolved in 4 mL of ethanol and mixed with this solution. Then, 176 mg of 1,4-dithiothreitol was added and stirred until the solution returned to room temperature. Finally, the volume was adjusted to 200 mL with distilled water to obtain the OPA reagent (the OPA reagent should be prepared immediately before use).

[0071] The sample was dispersed in phosphate buffer (10 mM, pH 7.4) at a concentration of 10 mg / mL. The sample suspension (600 μL) was mixed with 4.5 mL of the OPA reagent and incubated at 25 °C in the dark for 5 min. The absorbance of the reaction mixture was measured at 340 nm using a UV / visible spectrophotometer, and the content of free amino groups was calculated. The crosslinking degree of the sample was calculated based on the content of free amino groups in Equation (1).

[0072]

[0073] In the formula, M1 is the content of free amino groups in the initial whey protein, and M2 is the content of free amino groups in the whey protein after crosslinking with TGase.

[0074] 2. Transdermal test method:

[0075] The Franz-type vertical diffusion method was used to perform the permeation test on porcine skin. A piece of intact and undamaged porcine skin was taken, the fine hairs on its surface were removed with tweezers, and the subcutaneous fat was removed with a scalpel. Then it was washed clean with phosphate buffer (PBS). Phosphate buffer containing 5% ethanol and 1% polysorbate 80 was used as the receiving medium. The receiving cell was filled with the release medium, and the porcine skin with the epidermal side facing the dosing chamber was mounted on the receiving cell. It was kept at a constant temperature of 37 °C, and a magnetic stirrer was placed in the receiving cell with a rotation speed of 300 rpm / min to prevent the boundary layer effect. Free H2CBD was dissolved in the same concentration in triglyceride caprylate as a control. HPLC sampling analysis was performed on the H2CBD concentration in the receiving cell at certain time intervals (1, 2, 4, 7, 10, 24 h), and an equal volume of fresh release medium was supplemented after each sampling. The cumulative penetration amount Q was calculated by the following formula (2):

[0076]

[0077] In the formula, C n and C i are the concentrations (mg / mL) of H2CBD in the release medium measured at the nth sampling point and the concentration (mg / mL) of H2CBD in the release medium at the ith sampling; the effective permeation area A is 2.2 cm 2 ; the volume V of the receiving chamber is 7 mL; the volume V i of the receiving medium taken at each interval is 0.5 mL.

[0078] Freeze-drying method:

[0079] The sample was frozen for 24 h to ensure the formation of a uniform ice crystal structure, and then placed in a freeze dryer under a vacuum of 10 Pa for 48 h, with the cold trap temperature maintained at about -50 °C.

[0080] Example 1: Preparation of whey protein cross-linked emulsion (PCEs) encapsulating dihydrocannabidiol

[0081] A method for preparing whey protein cross-linked emulsion (PCEs) encapsulating dihydrocannabidiol, the process schematic diagram of which is as Figure 1 shown, specifically including the following steps:

[0082] (1) Dissolve 5 g of H2CBD in 50 mL of a mixed oil phase solution of ethyl acetate - glyceryl caprylocaprate (mass ratio of ethyl acetate to glyceryl caprylocaprate is 9:1) to obtain an H2CBD oil phase solution; mix the H2CBD oil phase solution and a whey protein suspension (whey protein concentration is 1 wt.%, stirred at room temperature for 2 h, and placed in the refrigerator overnight to ensure complete hydration) according to a volume ratio of 1:1, homogenize and emulsify at 10000 rpm for 2 min, repeat three times to obtain a primary Pickering emulsion, and after removing ethyl acetate by rotary evaporation at 30 °C for the primary emulsion, a whey protein uncross-linked emulsion (PUEs) loaded with dihydrocannabidiol is obtained;

[0083] (2) Add 20 U / g of TGase (transglutaminase) to the primary Pickering emulsion, cross-link at 50 °C for 4 h; immediately cool to 30 °C in an ice bath after cross-linking, then remove ethyl acetate by rotary evaporation at 30 °C, inactivate TGase by heating at 85 °C for 15 min, and cool to room temperature to obtain a whey protein cross-linked emulsion (PCEs) loaded with dihydrocannabidiol.

[0084] After the whey protein cross-linked emulsion (PCEs) was treated, it was observed using a transmission electron microscope (TEM), and the results are as Figure 2 shown. The results show that PCEs are spherical whey protein cross-linked nanocapsules, presenting a uniform and smooth spherical surface structure, forming a polydisperse system, with an average particle size of 400 nm, PDI less than 0.16, and Zeta potential at 37 mV.

[0085] Example 2: Effect of whey protein concentration on PCEs

[0086] On the basis of Example 1, the whey protein concentrations in step (1) were changed to 0.25, 0.5, and 2 wt.%, respectively, and the remaining steps were the same as those in Example 1, and the formation of the primary Pickering emulsion was detected.

[0087] Microscopic images of primary Pickering emulsions at different whey protein concentrations are as follows Figure 3 shown. The results show that stable emulsion droplets cannot be obtained at low whey protein concentrations (below 1 wt.%). Primary Pickering emulsions can be formed when the whey protein concentration is greater than about 1 wt.%. When the concentration increases further, the average droplet size has basically remained unchanged, that is, when the concentration is 1 wt.%, the whey protein is basically completely covered at the oil / water interface.

[0088] Example 3: Effect of crosslinking temperature on PCEs

[0089] On the basis of Example 1, the crosslinking temperature in step (2) was changed to 30, 40, and 60 °C, and the remaining steps were the same as those in Example 1. The crosslinking degree and particle size of PCEs were detected.

[0090] The detection results of the crosslinking degree and particle size of PCEs at different crosslinking temperatures are as follows Figure 4 shown. The results show that within 50 °C, as the crosslinking temperature of TGase increases, the crosslinking degree also increases. The crosslinking degree decreases instead at 60 °C, which is due to the inactivation of some enzymes resulting in a decrease in the crosslinking degree ( Figure 4 A); at low temperatures, whey protein cannot be completely crosslinked, resulting in the aggregation of a large amount of oil phase during the rotary evaporation of ethyl acetate and the formation of large droplets, that is, the particle size is larger ( Figure 4 B; in the figure, the dark broken line is the particle size, and the light broken line is the PDI, the same below).

[0091] Example 4: Effect of TGase concentration on PCEs

[0092] On the basis of Example 1, the addition amount of TGase in step (2) was changed to 10, 30, and 40 U / g of whey protein, and the remaining steps were the same as those in Example 1. The crosslinking degree and particle size of PCEs were detected.

[0093] The detection results of the crosslinking degree and particle size of PCEs at different TGase concentrations are as follows Figure 5 shown. The results show that as the concentration of TGase increases, the crosslinking degree of whey protein increases ( Figure 5 A); when the TGase exceeds 20 U / g, the crosslinking degree of whey protein no longer increases significantly. The particle size of PCEs decreases with the increase of TGase concentration, but when the TGase reaches 40 U / g, its particle size is higher than that of 20 U / g and 30 U / g, which is caused by the crosslinking between emulsion droplets ( Figure 5 B).

[0094] Example 5: Effect of crosslinking time on PCEs

[0095] On the basis of Example 1, the crosslinking time in step (2) was changed to 2, 6, and 8 h respectively, and the remaining steps were the same as those in Example 1. The crosslinking degree and particle size of PCEs were detected.

[0096] The detection results of the crosslinking degree and particle size of PCEs with different crosslinking times are as Figure 6 shown. The results show that when TGase crosslinks for 4 h, the crosslinking degree is higher than that of other samples ( Figure 6 A); at the same time, extending the time does not result in more whey protein crosslinking, and after 4 h, the crosslinked proteins may have undergone hydrolysis reactions, resulting in an increase in the particle size and homogeneity of PCEs ( Figure 6 B).

[0097] Example 6: Detection of PCEs properties

[0098] 1. Characterization of PCEs

[0099] Take the PCEs prepared in Example 1 and observe them using laser confocal microscopy. The results are as Figure 7 shown. The results show that PCEs are in an oil-in-water state (the red in the figure is the H2CBD oil-phase solution, and the green is the crosslinked whey protein).

[0100] 2. Redispersibility

[0101] Take the PUEs and PCEs prepared in Example 1 and detect their redispersibility characteristics. After freeze-drying the PUEs and PCEs, redissolve them in water and detect the emulsion dispersibility of the PUEs and PCEs during redissolution after freeze-drying. The results are as Figure 8 shown.

[0102] The results show that the freeze-dried PUEs are no longer in an emulsion state, while scanning electron microscopy (SEM) shows that the PCEs can still form an emulsion. It can be seen that the interface (crosslinked whey protein) of PCEs has high elasticity, which can avoid the shedding and coalescence of emulsifiers during the freeze-drying dehydration process.

[0103] 3. Freeze-thaw stability

[0104] Take the PCEs and PUEs prepared in Example 1 and conduct freeze-thaw stability tests. Place the PCEs and PUEs in a -20 °C environment and freeze them for 24 h, then melt them completely at room temperature for one cycle. Detect the microscopic, particle size, and appearance changes of the PCEs and PUEs after 5 freeze-thaw cycles.

[0105] The results of PCEs are as Figure 9 shown. The results show that PCEs can maintain a stable emulsion under freeze-thaw conditions, and even after multiple freeze-thaws, PCEs will not break Figure 9A); Meanwhile, the droplet size and PDI of PCEs hardly changed under freeze-thaw conditions, showing excellent freeze-thaw stability( Figure 9 B).

[0106] The results of PUEs are as Figure 10 shown. The results indicate that coalescence occurred in PUEs after two freeze-thaw cycles, and demulsification of PUEs occurred( Figure 10 A); The droplet size of PUEs increased significantly after the third cycle( Figure 10 B), which may be because the oil encapsulated by proteins crystallized during freezing, affecting the mechanical integrity of the crystal network in the emulsion.

[0107] 4. Thermal stability

[0108] Take the PCEs and PUEs prepared in Example 1 for thermal stability detection. Place the PCEs and PUEs in an environment of 50 °C for one month respectively, and detect the changes in the microscope, droplet size and appearance of the PCEs and PUEs.

[0109] The results of PCEs are as Figure 11 shown. The results indicate that PCEs can maintain a stable emulsion at 50 °C( Figure 11 A), and there is basically no change in droplet size and PDI( Figure 11 B). The reason for the enhanced thermal stability may be that covalent cross-linking improves the relative uniformity of the whey protein molecular chain length, increases the spacing between polymer chains, and enhances the flexibility of the surface of PCEs droplets; meanwhile, a stable interfacial film is maintained at the oil-water interface, thus having excellent thermal stability.

[0110] The results of PUEs are as Figure 12 shown. The results indicate that PUEs began to become turbid after being stored in an environment of 50 °C for a period of time, and flocculation occurred under microscopic observation( Figure 12 A); As time goes by, the emulsion droplets gradually aggregate into large aggregates and precipitate. This is because in an environment of 50 °C, non-polar and sulfhydryl-containing amino acids on the surface of whey protein will be more exposed to the aqueous phase, causing the formation of disulfide bonds between the proteins on different droplets, resulting in droplet aggregation.

[0111] 5. Transdermal effect

[0112] Take the PCEs prepared in Example 1 to detect the effect of H2CBD loaded in PCEs on skin absorption and permeation performance. The results are as Figure 13 shown.

[0113] The results indicate that the cumulative transdermal amount of H2CBD in PCEs is significantly higher than that of free H2CBD( Figure 13A). Through in-vivo skin penetration studies, the distribution of H2CBD in different skin layers was detected, further confirming its enhancing effect on the retention of H2CBD in the skin. After 24 hours of penetration, the absorption rate of H2CBD in the dermis layer by PCEs was higher than that of free H2CBD( Figure 13 B). The above results indicate that PCEs have good skin permeability and skin retention ability, which is more conducive to the exertion of their efficacy.

[0114] Example 7: Encapsulation of other lipophilic active substances

[0115] On the basis of Example 1, H2CBD was changed to other lipophilic substances, such as phenethyl resorcinol, and a whey protein cross-linked emulsion loaded with phenethyl resorcinol was prepared.

[0116] After testing, the prepared whey protein cross-linked emulsion loaded with phenethyl resorcinol also has good redissolution, freeze-thaw stability, thermal stability and transdermal effect; it can be seen that the method of Example 1 can be applied to conventional lipophilic active substances.

[0117] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing a whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenols, characterized in that: Includes steps: (1) dissolving a fat-soluble thermosensitive polyphenol in an oil phase solution to obtain a bioactive substance oil phase solution; mixing the bioactive substance oil phase solution with a whey protein suspension, and homogenizing to obtain a primary Pickering emulsion; (2) adding transglutaminase to the primary Pickering emulsion for cross-linking; cooling, concentrating, and inactivating the enzyme after cross-linking to obtain a whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenols.

2. The method according to claim 1, characterized in that In step (1), the mass ratio of the fat-soluble thermosensitive polyphenol to the oil phase solution is 0.1 to 1:1; Optionally, the oil phase solution is a mixed solution of caprylic / decanoic acid triglyceride and a volatile organic solvent; wherein the volatile organic solvent includes ethyl acetate, petroleum ether, dichloromethane or chloroform.

3. The method according to claim 1, characterized in that The concentration of whey protein in the whey protein suspension of step (1) is 0.25-2 wt.%; Optionally, the concentration of whey protein is 0.5-2 wt.%.

4. The method according to claim 1, characterized in that: In step (1), the volume ratio of the bioactive substance oil phase solution to the whey protein suspension is 1-2:1-2.

5. The method according to claim 1, characterized in that In step (2), the amount of transglutaminase added to the primary Pickering emulsion is 10 to 40 U / g whey protein; the cross-linking is performed at 30 to 60° C. for 2 to 8 hours; Optionally, the amount of transglutaminase added is 10 to 30 U / g whey protein; Optionally, the crosslinking is carried out at 30-50° C. for 3-6 hours.

6. The method according to claim 1, characterized in that Fat-soluble thermosensitive polyphenols include 8,9-dihydrocannabidiol, phenethylresorcinol, tocopherol, bakuchiol, and resveratrol.

7. The whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenols prepared by the method according to any one of claims 1 to 6 or the powder after drying the whey protein cross-linked emulsion according to claim 2.

8. Use of the method according to any one of claims 1 to 6, the whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenols according to claim 7, or the dried powder of the whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenols according to claim 7 in the chemical, medical and food fields.

9. A product, characterized in that The product contains the whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenols according to claim 7 or the powder obtained by drying the whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenols according to claim 7; Optionally, the products include cosmetics, food, and medicines.

10. A method for simultaneously improving the performance of whey protein cross-linked emulsion, characterized in that: The performance includes one or more of resolubility, freeze-thaw stability, thermal stability, and transdermal effect; the method includes: (1) dissolving a fat-soluble thermosensitive polyphenol in an ethyl acetate-caprylic decanoic acid triglyceride mixed oil phase solution to obtain a bioactive substance oil phase solution; mixing the bioactive substance oil phase solution with a whey protein suspension, and homogenizing to obtain a primary Pickering emulsion; (2) adding transglutaminase to the primary Pickering emulsion for cross-linking; cooling, concentrating, and inactivating the enzyme after cross-linking to obtain a whey protein cross-linked emulsion loaded with fat-soluble thermosensitive polyphenols; Optionally, the oil phase solution in step (1) is a mixed solution of caprylic acid triglyceride and a volatile organic solvent; wherein the volatile organic solvent includes ethyl acetate, petroleum ether, dichloromethane or chloroform; and the mass ratio of the fat-soluble thermosensitive polyphenol to the oil phase solution is 0.1 to 1:1; Optionally, the concentration of whey protein in the whey protein suspension in step (1) is 0.25 to 2 wt.%; Optionally, the concentration of whey protein is 0.5 to 2 wt.%; Optionally, the volume ratio of the bioactive substance oil phase solution to the whey protein suspension in step (1) is 1-2:1-2; Optionally, in step (2), the amount of transglutaminase added is 10 to 40 U / g whey protein; the cross-linking is carried out at 30 to 60° C. for 2 to 8 hours; Optionally, the amount of transglutaminase added is 10 to 30 U / g whey protein; Optionally, the cross-linking is carried out at 30 to 50° C. for 3 to 6 hours; Optionally, the fat-soluble thermosensitive polyphenols include 8,9-dihydrocannabidiol, phenethylresorcinol, tocopherol, bakuchiol, and resveratrol.

Images