Whey protein isolate-phospholipid complex embedded fucoxanthine emulsion
By encapsulating fucoxanthin with whey protein isolate-phospholipid complex, the problems of unstable fucoxanthin structure and low bioavailability were solved, achieving efficient and stable delivery and antioxidant effects, and improving bioavailability.
Patent Information
- Application Number
- CN202511405571.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-12-16
AI Technical Summary
Fucoxanthin has an unstable structure and is easily affected by external conditions. It is also intolerant to low pH environments in human gastric juice and has low bioavailability. Existing emulsifiers have poor stability under unsuitable conditions, resulting in poor delivery performance.
Fucoxanthin was encapsulated using a whey protein isolate-phospholipid complex. A whey protein isolate-phospholipid complex O/W emulsion was prepared, and a fucoxanthin solution was added dropwise to it to form stable nanoparticles. Phosphatidylglycerol was then mixed with buffer and homogenized. The solvent was removed by rotary evaporation and sonication to form a stable fucoxanthin emulsion.
It improves the encapsulation efficiency and bioavailability of fucoxanthin, enhances the stability and antioxidant capacity of the emulsion, has a particle size of less than 200 nm, a zeta potential of less than -30 mV, and a bioavailability of more than 70%.
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Figure CN121129764A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a fucoxanthin emulsion encapsulated with a whey protein isolate-phospholipid complex, belonging to the technical field of emulsion encapsulation systems. Background Technology
[0002] Fucoxanthin, also known as fucoxanthin, is a natural pigment belonging to the lutein class of carotenoids, and is abundant in edible brown algae. Fucoxanthin possesses unique propenyl and oxygen-containing functional groups, which endow it with strong biological activity, exhibiting anti-cancer, anti-diabetic, anti-obesity, antioxidant, and anti-inflammatory effects, as well as excellent biocompatibility, making it a promising candidate for applications in the pharmaceutical and food industries.
[0003] However, fucoxanthin is structurally unstable and easily affected by external conditions such as thermal processing, air exposure, light, and chemical oxidation. Furthermore, fucoxanthin cannot tolerate the low pH environment of human gastric juice, and due to its lipophilic nature, it is poorly absorbed in the water-based environment of the intestine, resulting in low bioavailability. Therefore, to achieve efficient delivery and utilization of fucoxanthin, it is necessary to select suitable materials for its encapsulation.
[0004] O / W emulsions are oil-in-water emulsion systems formed by dispersing an oil phase in an aqueous phase, and they have wide applications in delivering hydrophobic active substances. However, emulsions are thermodynamically unstable systems, with relatively high Gibbs free energy (interfacial tension) at the contact between the oil and aqueous phases. This leads to a tendency for these systems to decompose over time, through processes such as gravity separation, flocculation, aggregation, or Ostwald ripening. Therefore, emulsifiers are often needed to maintain the long-term stability of emulsions.
[0005] Whey protein isolate (WPI) is extracted from whey through processes such as centrifugation, filtration, and concentration. It has a strong hydrophobic surface and is a commonly used emulsifier. However, in practical applications, using a single type of emulsifier has certain drawbacks. WPI is highly sensitive to environmental factors; under unsuitable pH, temperature, and salt concentration conditions, its solubility and stability decrease, and it exhibits gelation and coagulation. Therefore, using WPI as the sole delivery carrier results in significant structural changes during production, storage, and utilization, leading to poor actual delivery performance. Combining WPI with other substances to form binary complexes to improve its delivery capabilities is gradually becoming a new research direction.
[0006] Phospholipids (PL) are molecules composed of glycerol, two fatty acids, a phosphate group, and a polar head group. PL has been widely used in the preparation of liposome nanoformulations for local, oral, and parental drug delivery systems, offering advantages such as improved bioavailability, reduced toxicity, and enhanced membrane permeability. Summary of the Invention
[0007] In view of the above-mentioned prior art, the present invention provides a fucoxanthin emulsion encapsulated with whey protein isolate-phospholipid complex.
[0008] This invention is achieved through the following technical solution: A method for preparing a fucoxanthin emulsion encapsulated with a whey protein isolate-phospholipid complex, comprising the following steps: (1) Preparation of whey protein isolate-phospholipid complex O / W emulsion Phosphatidylglycerol is mixed with a buffer solution and homogenized to obtain a phospholipid emulsion, wherein the phosphatidylglycerol is selected from any one of DHA phosphatidyl monocaprylate (DHAPL-GMC), DHA phosphatidyl monodecanoate (DHAPL-GMD), DHA phosphatidyl monolaurate (DHAPL-GML), DHA phosphatidyl monopalmitate (DHAPL-MP), and DHA phosphatidyl monostearate (DHAPL-MG). The phospholipid emulsion is mixed with the whey protein isolate solution and homogenized to obtain the whey protein isolate-phospholipid complex O / W emulsion. (2) Add the fucoxanthin solution dropwise to the whey protein isolate-phospholipid complex O / W emulsion and stir for 2 to 4 hours to obtain the product.
[0009] Further, in step (1), the concentration of phosphatidylglycerol in the phospholipid emulsion is 5-15 mg / mL, preferably 10 mg / mL; the concentration of whey protein isolate solution is 15-25 mg / mL, preferably 20 mg / mL; and the weight ratio of phosphatidylglycerol to whey protein isolate is 1:(1.5-2.5), preferably 1:2.
[0010] Furthermore, in step (1), the buffer solution is a phosphate buffer, preferably a pH 7.0 10 mM phosphate buffer.
[0011] Further, in step (1), the specific method of "mixing and homogenizing phosphatidylglycerol with buffer solution" is as follows: take phosphatidylglycerol, dissolve it into a fluid state with ethyl butyrate under ultrasonic conditions, add buffer solution, homogenize it for 2 min at 23000 r / min using a homogenizer, and then remove organic solvent by rotary evaporation at 40℃ and 100 MPa for 10 min.
[0012] Further, in step (1), the whey protein isolate solution is pretreated as follows: the whey protein isolate solution is stirred at 500-1000 r / min for 2-4 h, and then placed at 4℃ for hydration for 8-16 hours.
[0013] Further, in step (1), the specific method of "mixing and homogenizing the phospholipid emulsion with the whey protein isolate solution" is as follows: mix the phospholipid emulsion with the whey protein isolate solution and homogenize it for 2 minutes at a speed of 23000 r / min using a homogenizer.
[0014] Further, in step (2), the concentration of the fucoxanthin solution is 0.5 to 1.5 mg / mL, preferably 1 mg / mL, and the solvent is anhydrous ethanol; the volume ratio of the fucoxanthin solution to the whey protein isolate-phospholipid complex O / W emulsion is 1:(3 to 5), preferably 1:4.
[0015] Furthermore, in step (2), the fucoxanthin solution is added at a rate of 1 mL / min while stirring.
[0016] Furthermore, in step (2), the stirring speed is 800-1200 r / min.
[0017] Further, in step (2), after stirring, the ethanol is removed by rotary evaporation at 40°C and 100 MPa for 10 min; after rotary evaporation, the ethanol is rapidly ultrasonically treated for 10 min under ice bath conditions.
[0018] Fucoxanthin emulsion encapsulated in whey protein-phospholipid complex was prepared using the above method.
[0019] A whey protein isolate-phospholipid complex O / W emulsion, prepared by the same method as above.
[0020] The whey protein isolate-phospholipid complex O / W emulsion is used in the encapsulation of hydrophobic active substances and in the preparation of hydrophobic active substance formulations.
[0021] Furthermore, the hydrophobic active substance is selected from fucoxanthin.
[0022] The fucoxanthin emulsion encapsulated with whey protein isolate-phospholipid complex of the present invention exhibits good stability (uniform spherical vesicles), high encapsulation efficiency (above 94%), particle size below 200 nm, PDI less than 0.4, and zeta potential less than -30 mV; it also demonstrates excellent antioxidant capacity and high bioavailability of fucoxanthin (greater than 70%). This research expands the application of novel phospholipids in the construction of stable emulsion systems, providing new ideas and directions for the encapsulation and delivery of active substances.
[0023] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description
[0024] Figure 1The results of particle size determination of fucoxanthin emulsions are given, where the significance of differences is indicated by letters, and the same applies below.
[0025] Figure 2 Results of PDI value determination for fucoxanthin emulsion.
[0026] Figure 3 Results of zeta potential determination of fucoxanthin emulsion.
[0027] Figure 4 Fourier transform infrared spectrum of fucoxanthin emulsion.
[0028] Figure 5 Results of antioxidant capacity determination of fucoxanthin emulsion.
[0029] Figure 6 : TEM image of fucoxanthin emulsion, where a and b represent scale bars of 50 nm and 100 nm, respectively.
[0030] Figure 7 Results of the determination of bioaccessibility of fucoxanthin emulsion. Detailed Implementation
[0031] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0032] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0033] In previous research, the inventors of this application used phospholipase DSp-TLI146 as a catalyst, DHA phosphatidylcholine (DHAPC) extracted from squid eggs as a substrate, and different monoglycerides as co-substrates to efficiently prepare different DHA phosphatidyl monoacylglycerols. The specific preparation methods are as follows: Organic phase: DHAPC was dissolved in cyclopentyl methyl ether at a concentration of 20 mg / mL.
[0034] Aqueous phase: 50 mmol / mL anhydrous CaCl2 dissolved in 20 mmol / mL phosphate buffer (pH 6.0); substrates were glyceryl monocaprylate (GMC), glyceryl monodecanoate (GMD), glyceryl monolaurate (GML), glyceryl monopalmitate (MP), glyceryl monostearate (MG), and glycerol, with a substrate concentration of 30 mg / mL.
[0035] In a brown reaction flask, an organic phase and an aqueous phase with a volume ratio of 1:1 were added and mixed and shaken. The mixture was then reacted in a water bath shaker at 40°C at a speed of 220 r / min for 12 h. After the reaction was completed, the upper reaction solution was collected by centrifugation, and the organic phase was removed to obtain DHA phosphatidyl monocaprylate glycerol (DHAPL-GMC), DHA phosphatidyl monodecanoate glycerol (DHAPL-GMD), DHA phosphatidyl monolaurate glycerol (DHAPL-GML), DHA phosphatidyl monopalmitate glycerol (DHAPL-MP), DHA phosphatidyl monostearate glycerol (DHAPL-MG), and DHA phosphatidylglycerol (DHA-PG).
[0036] Experiment 1: Preparation of whey protein isolate-phospholipid complex O / W emulsion In this experiment, different DHA phosphatidylglycerols were combined with WPI to form O / W emulsions. The preparation method is as follows: The 20 mg / mL WPI solution was stirred at 800 r / min for 3 h on a magnetic stirrer. After stirring, it was placed in a 4°C refrigerator overnight (12 h) for hydration.
[0037] Different phosphatidylglycerols (DHAPL-GMC, DHAPL-GMD, DHAPL-GML, DHAPL-MP, DHAPL-MG, DHA-PG) and DHAPC were first dissolved in 50 μL of ethyl butyrate under sonication to form a fluid state. Then, 950 μL of 10mM phosphate buffer (pH 7.0) was added, and the mixture was homogenized using an IKA homogenizer at 23000 r / min for 2 min to obtain a phospholipid emulsion. The concentrations of phosphatidylglycerol and DHAPC were each 10 mg / mL.
[0038] 1 mL of phospholipid emulsion was rotary evaporated at 100 MPa and 40 °C for 10 min to remove organic solvents. Then, 1 mL of WPI solution was added and homogenized using an IKA homogenizer at 23000 r / min for 2 min to form a whey protein isolate-phospholipid (WPI-PL) complex O / W emulsion.
[0039] Experiment 2 Embedding and Characterization of Fucoxanthin In this experiment, fucoxanthin was encapsulated using a WPI-PL complex O / W emulsion, and its characterization and related studies were conducted.
[0040] (I) Experimental Methods 1. Preparation of Fucoxanthin Emulsion Fucoxanthin was dissolved in anhydrous ethanol to a concentration of 1 mg / mL, ensuring complete dissolution. A freshly prepared WPI-PL complex O / W emulsion (2 mL) (prepared in Experiment 1) was prepared. 500 μL of fucoxanthin solution was added dropwise to the emulsion at a speed of 1 mL / min using a magnetic stirrer, maintaining the stirrer speed at 1000 r / min. The sample was continuously stirred in the dark for 3 h. After stirring, ethanol was removed using a rotary evaporator (40℃, 100 MPa), and the lost volume was replaced with 10 mmol / L phosphate buffer (pH 7.0). After rotary evaporation, the emulsion was rapidly sonicated in an ice bath for 10 min (250 W, 3 s + 3 s) to obtain the WPI-PL emulsion encapsulating fucoxanthin. During preparation, all containers were wrapped with aluminum foil to maximize the protection of fucoxanthin and minimize its loss.
[0041] Simultaneously, WPI-embedded fucoxanthin was prepared as a control (preparation method as above, except that the fucoxanthin solution was added dropwise to the WPI solution). DHA phosphatidylglycerol-embedded fucoxanthin was prepared as a control (preparation method as above, except that the fucoxanthin solution was added dropwise to the phospholipid emulsion).
[0042] 2. Determination of encapsulation efficiency Prepare a 1 mg / mL fucoxanthin stock solution and sonicate until homogeneous. Dilute the fucoxanthin stock solution sequentially to concentrations of 1 μg / mL, 2 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL, and 15 μg / mL to prepare standard solutions. Measure the absorbance at 450 nm. Use the concentration of the fucoxanthin standard solution as the x-axis, and OD... 450 Using this as the ordinate, a standard curve for fucoxanthin is plotted.
[0043] One mL of WPI-PL emulsion containing fucoxanthin was filtered through a 0.45 μm polystyrene membrane to remove insoluble or large aggregates. The filtered fucoxanthin emulsion was centrifuged at 12000 r / min for 30 min. The absorbance of the supernatant was measured at 450 nm. A fucoxanthin emulsion containing DHA phosphatidylglycerol was used as a control.
[0044] 3. Particle size and potential measurement The particle size and zeta potential of fucoxanthin emulsions were measured using a ZS90 Malvern laser particle size analyzer. The emulsions were diluted 100-fold with 10 mmol / L phosphate buffer (pH 7.0) before measurement. The refractive index of the particles was set to 1.450, the absorbance to 0.001, and the refractive index of the dispersant to 1.330. A 1 mL sample volume was used for each measurement, and measurements were performed at room temperature. To ensure accuracy, measurements were repeated three times, and the average value was used as the result.
[0045] 4. Fourier transform infrared spectroscopy detection Samples were analyzed using a Fourier transform infrared spectroscopy (FIR) system (Nicolet iS10, Thermo Fisher Scientific). All fucoxanthin emulsions were freeze-dried under vacuum before analysis, mixed with anhydrous potassium bromide (KBr) at a volume ratio of 1:9, and compressed into tablets, with WPI used as a control. All samples and components were kept under infrared lamp irradiation throughout the process and wiped with alcohol swabs before each use. The wavenumber range was 4000 cm⁻¹. -1 ~400 cm -1 The resolution is 4cm. -1 A total of 64 scans were performed.
[0046] 5. Antioxidant activity detection ABTS + Free radical scavenging ability: First, a stock solution of ABTS was prepared by dissolving 7.4 mmol / L 2,2-diazo-bis(3-ethylbenzothiazol-6-sulfonic acid) ammonium salt (ABTS) in deionized water. Then, the 7.4 mmol / L ABTS stock solution was mixed with 2.6 mmol / L potassium persulfate solution at a 1:1 volume ratio and incubated at room temperature in the dark for 14 hours to prepare ABTS. + Solution. Then, dilute ABTS with 10 mmol / L phosphate buffer (pH 7.0). + The solution was prepared until the absorbance at 734 nm reached 0.70 ± 0.02, ensuring its stability at room temperature. 50 μL of fucoxanthin emulsion sample was mixed with 666 μL of ABTS. + The solutions were mixed and reacted at room temperature in the dark for 1 h. After the reaction, the absorbance was measured at 734 nm using a microplate reader. Phosphate buffer solution was used as a blank.
[0047] DPPH free radical scavenging capacity: First, a DPPH stock solution was prepared by dissolving 0.1 mmol / L 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) in anhydrous ethanol. Before use, this solution was diluted to an absorbance of 0.70 ± 0.02 at 515 nm and allowed to stabilize at room temperature. 0.1 mL of fucoxanthin emulsion sample was mixed thoroughly with 3.9 mL of the DPPH free radical solution, and the mixture was allowed to react at room temperature for 30 minutes. After the reaction, the absorbance was measured at 515 nm using a microplate reader. Anhydrous ethanol solution was used as a blank. The free radical scavenging rate is calculated as follows: Scavenging rate % = Where A0 is the absorbance of the blank sample; A1 is the absorbance of the sample with added sample; and A2 is the absorbance of the blank sample without added free radicals.
[0048] 6. Transmission electron microscopy (TEM) detection The morphology of fucoxanthin emulsions was observed using a JEOL JEM-2100 TEM. Fucoxanthin emulsion samples were dropped onto a copper grid supported by a carbon film, and excess sample was removed with filter paper after 5 min. The samples were then negatively stained with 1% (w / v) phosphotungstic acid and dried at room temperature for 12 h before being observed under TEM.
[0049] 7. In vitro simulated digestion We used an in vitro digestion model to analyze the bioavailability of fucoxanthin during digestion.
[0050] Simulated gastric digestion (SGF): Equal volumes of fucoxanthin emulsion and simulated gastric juice (3.20 mg / mL pepsin, 0.15 mol / L sodium chloride) were mixed. The pH was adjusted to 2.0 with 6 mol / L hydrochloric acid, and the mixture was incubated in a water bath with stirring for 2 h (37℃, 100 r / min).
[0051] Simulated intestinal digestion (SIF): After gastric digestion, the SGF result mixture was immediately adjusted to pH 7.0 using 1 mol / L sodium hydroxide solution. An equal volume of the SGF sample solution was mixed with simulated small intestinal fluid (trypsin 1.0 mg / mL, bile extract 20.0 mg / mL, pH 7.0) and incubated in a water bath shaker for 2 h (37°C, 100 r / min).
[0052] After simulated intestinal digestion, the sample was centrifuged at 12000 r / min for 30 min at 4°C, and the supernatant was collected. The supernatant consisted of a micelle layer containing fucoxanthin, and the absorbance was measured at 450 nm using a microplate reader. The bioavailability of fucoxanthin was calculated using the following formula: Bioavailability % = , where C micelle The concentration of fucoxanthin in the micelle layer (g / mL), Coriginal The concentration of fucoxanthin in the original digestive fluid (g / mL).
[0053] 8. Data Analysis Methods All experiments were repeated three times, and data analysis was performed using SPSS Statistic 27 software. Differences between groups were assessed using one-way ANOVA, with a significance level set at [value missing]. p <0.05. Based on the results of the homogeneity of variance test, the LSD (Least Significant Difference) or Waller-Duncan method is selected for further analysis of variance.
[0054] (II) Results and Discussion 1. Encapsulation ratio The results showed that the encapsulation efficiency of fucoxanthin encapsulated by O / W emulsions formed from single substances such as DHA phosphatidylglycerol or DHAPC was below 80%, indicating a less than ideal encapsulation effect. In contrast, the encapsulation efficiency of fucoxanthin encapsulated by WPI-PL was above 94%. This suggests that the O / W emulsion structure formed by the combination of DHAPL and WPI is more compact, making core material less prone to leakage and thus improving the encapsulation efficiency of fucoxanthin.
[0055] 2. Particle size and potential analysis The particle size, PDI, and potential of the fucoxanthin emulsion are as follows: Figure 1 , Figure 2 , Figure 3 As shown, all O / W emulsions formed after WPI-PL encapsulation of fucoxanthin are in the nanoscale range, with particle sizes below 200 nm. Further analysis revealed that the particle sizes of emulsions formed after encapsulation with DHAPG, DHAPC, and WPI are less than 150 nm, with DHAPC encapsulation achieving particle sizes below 100 nm. This may be due to the smaller side chain groups of PL and the smaller structure of WPI itself. Analysis of the polydispersity index (PDI) of the encapsulated fucoxanthin emulsions... Figure 2The O / W emulsions formed by six synthesized DHAPLs (DHAPL-GMC, DHAPL-GMD, DHAPL-GML, DHAPL-MP, DHAPL-MG, and DHA-PG) and WPI showed relatively uniform dispersion (PDI < 0.4) in the system after encapsulation of fucoxanthin. This indicates that these emulsions have a narrower particle size distribution and are more stable than DHAPC and WPI. DHAPG had the lowest PDI value, reaching 0.241. The PDI values of DHAPC and WPI were 0.499 and 0.737, respectively, indicating that they easily formed aggregates after encapsulation of fucoxanthin, resulting in poor emulsion stability. The PDI values of each fucoxanthin emulsion were consistent with the analysis results of zeta potential, which is one of the indicators reflecting the stability of the dispersion system. Figure 3 It can be seen that the absolute values of the zeta potentials of DHAPC and WPI emulsions are relatively small, at -16.83 mV and -11.57 mV respectively, which is consistent with the PDI values. The zeta potentials of other synthesized DHAPL and WPI emulsions are all less than -30 mV, indicating that these emulsions possess a certain degree of electrostatic stability. Furthermore, a high negative charge increases the repulsive force between droplets, preventing emulsion aggregation and resulting in a more stable structure. Based on the analysis results of particle size, PDI, and zeta potential, the emulsions formed by embedding fucoxanthin with DHAPL-GMC, DHAPL-GMD, DHAPL-GML, DHAPL-MP, and DHAPL-MG exhibit more uniform droplet size and a more stable structure.
[0056] 3. Fourier transform infrared spectroscopy analysis FTIR can characterize the encapsulation of fucoxanthin in the O / W emulsion formed by WPI-PL. The Fourier transform infrared spectrum of the fucoxanthin emulsion is shown below. Figure 4 As shown, the characteristic infrared absorption peak of free fucoxanthin is at 1927 cm⁻¹. -1 This corresponds to the typical functional group of fucoxanthin—the propylene bond (C=C=C). However, this characteristic peak disappeared in the infrared spectra of several emulsions containing fucoxanthin, indicating that fucoxanthin has been effectively encapsulated in the O / W emulsion formed by WPI-PL.
[0057] 4. Antioxidant activity analysis ABTS + The DPPH free radical scavenging assay and other methods are two main approaches to assess antioxidant activity. ABTS reacts with a suitable oxidant to generate blue-green ABTS cationic free radicals (ABTS). +DPPH, a stable nitrogen-centered chromogenic free radical, produces a characteristic absorption peak at 734 nm and a characteristic absorption peak at 517 nm. ABTS exhibits this characteristic absorption peak in the presence of antioxidants. + The production of both DPPH and DPPH will be inhibited, resulting in a decrease in absorbance.
[0058] The results of the antioxidant capacity test of fucoxanthin emulsion are as follows: Figure 5 As shown. By measuring ABTS + The study revealed that free fucoxanthin exhibited low antioxidant activity, which may be due to the presence of numerous olefinic bonds and acetyl groups in fucoxanthin, leading to its reduced ABTS scavenging ability. + The scavenging ability was relatively low. O / W emulsions formed from several complexes exhibited higher antioxidant properties after encapsulation of fucoxanthin than free fucoxanthin, and WPI-encapsulated fucoxanthin also showed higher ABTS free radical scavenging ability. Among them, the emulsion formed from DHAPL-GMC showed the best ABTS free radical scavenging ability. + The ABTS removal ability was the best, increasing from 47.52% for free fucoxanthin to 63.86%. Furthermore, emulsions containing fucoxanthin encapsulated with DHAPL-GMC, DHAPL-GMD, DHAPL-GML, DHAPL-MP, and DHAPL-MG effectively removed ABTS. + Their abilities are all greater than 50%, and the first four are even higher than 60%.
[0059] The DPPH scavenging ability also showed the same trend. Free fucoxanthin had a significantly weaker DPPH scavenging ability, only 32.04%, while DHAPL-GMC still had the highest scavenging activity (51.47%). Several other complexes and WPI showed strong DPPH scavenging abilities after encapsulating fucoxanthin, all greater than 40%.
[0060] Analysis of the antioxidant activity of the two substances showed that the antioxidant capacity was improved after encapsulating fucoxanthin, indicating that O / W emulsion encapsulation of fucoxanthin can better exert its antioxidant effect.
[0061] 5. Microstructure of emulsions The morphology and structure of fucoxanthin encapsulated in O / W emulsions were observed using TEM, such as... Figure 6 As shown, the O / W emulsion formed by the WPI-PL complex, after encapsulating fucoxanthin, produces relatively uniform spherical vesicles with no significant deformation or leakage; the addition of unsaturated PL does not alter the spherical morphology. However, the vesicles formed by WPI encapsulating fucoxanthin are larger and have less regular edges, and some spherical objects with rough surfaces exist in the system, indicating that the emulsion is less stable and that fucoxanthin may leak or ooze out.
[0062] 6. Bioavailability of fucoxanthin The bioavailability of fucoxanthin is generally defined as the portion of fucoxanthin transferred from the food matrix to the mixed micelles, thus becoming available for subsequent absorption by the intestinal mucosa. The results of the bioavailability assay for fucoxanthin emulsions are as follows: Figure 7 As shown, fucoxanthin, as a carotenoid, contains unsaturated conjugated double bonds, making it easily oxidized and degraded in the low pH of gastric juice. Therefore, it suffers significant loss in SGF (stomach-encapsulated gastric juice), resulting in lower release levels in simulated small intestinal fluid. The bioavailability of free fucoxanthin in SIF is only 28%. O / W emulsions formed by several WPI-PL complexes protect fucoxanthin, forming a strong barrier that allows for greater absorption of fucoxanthin in the small intestine. Therefore, the bioavailability of fucoxanthin after emulsion encapsulation is higher than that of free fucoxanthin. During in vitro simulated digestion, trypsin, with its powerful decomposition ability, can enzymatically break down complexes adsorbed at the oil-water interface. This process disrupts the interfacial properties of the system, leading to the release of a large amount of active ingredients from the droplets. Simultaneously, bile salts have the property of dissolving lipids in water, which can enhance the absorption and digestion of lipid-soluble active substances in the small intestine. In summary, after encapsulating fucoxanthin, the bioavailability of fucoxanthin in the O / W emulsions formed by several WPI-PL formulations was greater than 70%, which is higher than the bioavailability of fucoxanthin encapsulated by WPI (67%). Furthermore, the bioavailability of emulsions formed by DHAPG and DHAPC was only around 70%, while the bioavailability of fucoxanthin encapsulated by other DHA phosphatidyl monoglycerides was above 75%. Therefore, the fucoxanthin in DHAPL-GMC, DHAPL-GMD, DHAPL-GML, DHAPL-MP, and DHAPL-MG can be better absorbed and utilized in the small intestine.
[0063] (III) Conclusion This experiment successfully encapsulated fucoxanthin with WPI-PL O / W emulsion using a combination of reduced-pressure rotary evaporation and sonication. The fucoxanthin emulsion was then physically characterized, and the specific results are as follows: (1) The encapsulation effect of the synthesized DHAPL (DHAPL-GMC, DHAPL-GMD, DHAPL-GML, DHAPL-MP, DHAPL-MG) complex with WPI is higher than that of DHAPG and DHAPC, and the encapsulation rate can reach more than 94%.
[0064] (2) The O / W emulsions formed by WPI-PL after encapsulating fucoxanthin are all in the nanoscale range, and the particle size range is all below 200 nm. The PDI of the O / W emulsion formed by synthesized DHAPL (DHAPL-GMC, DHAPL-GMD, DHAPL-GML, DHAPL-MP, DHAPL-MG, DHAPG) and WPI is less than 0.4, and the zeta potential of the emulsion formed with WPI is less than -30 mV.
[0065] (3) FTIR analysis revealed that the fucoxanthin was 1927 cm⁻¹ -1 The characteristic infrared absorption peak disappeared after encapsulation, indicating that the O / W emulsion successfully encapsulated fucoxanthin.
[0066] (4) The effect of the emulsion formed by DHAPL-GMC on ABTS + The removal ability was the best, increasing from 47.52% to 63.86% compared to free fucoxanthin. DHAPL-GMC, DHAPL-GMD, DHAPL-GML, DHAPL-MP, and DHAPL-MG remove ABTS. + The ability to [achieve certain benefits] is greater than 50%. Furthermore, as the fatty acid chain of DHAPL decreases, its effect on ABTS [is reduced / decreases]. + The scavenging ability showed an increasing trend. Similarly, the scavenging ability of the complexes against DPPH was greater than 40%.
[0067] (5) TEM observation results showed that the microstructure of WPI-PL embedded emulsion was uniform spherical vesicles, while the edge of WPI-embedded fucoxanthin emulsion was blurred, and fucoxanthin was leaking or seeping out.
[0068] (6) The bioavailability of fucoxanthin in the O / W emulsion formed by the synthesized DHAPL and WPI is greater than 70%, which is higher than the bioavailability of fucoxanthin after WPI encapsulation (67%).
[0069] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.
Claims
1. A method for preparing a fucoxanthin emulsion encapsulated with a whey protein-phospholipid complex, characterized in that, The steps are as follows: (1) Preparation of whey protein isolate-phospholipid complex O / W emulsion Phosphatidylglycerol is mixed with buffer solution and homogenized to obtain phosphatidyl emulsion. The phosphatidylglycerol is selected from any one of DHA phosphatidyl monocaprylate, DHA phosphatidyl monodecanoate, DHA phosphatidyl monolaurate, DHA phosphatidyl monopalmitate, and DHA phosphatidyl monostearate. The phospholipid emulsion is mixed with the whey protein isolate solution and homogenized to obtain the whey protein isolate-phospholipid complex O / W emulsion. (2) Add the fucoxanthin solution dropwise to the whey protein isolate-phospholipid complex O / W emulsion and stir to obtain the final product.
2. The method for preparing the fucoxanthin emulsion embedded with whey protein-phospholipid complex according to claim 1, characterized in that: In step (1), the concentration of phosphatidylglycerol in the phospholipid emulsion is 5-15 mg / mL; the concentration of whey protein isolate solution is 15-25 mg / mL; and the weight ratio of phosphatidylglycerol to whey protein isolate is 1:(1.5-2.5).
3. The method for preparing the fucoxanthin emulsion embedded with whey protein-phospholipid complex according to claim 1, characterized in that: In step (1), the specific method of "mixing and homogenizing phosphatidylglycerol with buffer solution" is as follows: take phosphatidylglycerol, dissolve it into a fluid state with ethyl butyrate under ultrasonic conditions, add buffer solution, and homogenize it for 2 min at 23000 r / min using a homogenizer; then remove organic solvent by rotary evaporation.
4. The method for preparing the fucoxanthin emulsion embedded with whey protein-phospholipid complex according to claim 1, characterized in that: In step (2), the concentration of the fucoxanthin solution is 0.5–1.5 mg / mL; the volume ratio of the fucoxanthin solution to the whey protein isolate-phospholipid complex O / W emulsion is 1:(3–5).
5. A fucoxanthin emulsion embedded with whey protein isolate-phospholipid complex prepared by the method described in any one of claims 1 to 4.
6. A whey protein isolate-phospholipid complex O / W emulsion, characterized in that, It is prepared by the following method: phosphatidylglycerol is mixed with buffer and homogenized to obtain a phosphatidyl emulsion, wherein the phosphatidylglycerol is selected from any one of DHA phosphatidyl monocaprylic acid glyceride, DHA phosphatidyl monodecanoic acid glyceride, DHA phosphatidyl monolaurate glyceride, DHA phosphatidyl monopalmitoate glyceride, and DHA phosphatidyl monostearate glyceride; the phosphatidyl emulsion is mixed with whey protein isolate solution and homogenized to obtain a whey protein isolate-phosphatidyl complex O / W emulsion.
7. The whey protein isolate-phospholipid complex O / W emulsion according to claim 6, characterized in that: The concentration of phosphatidylglycerol in the phospholipid emulsion is 5–15 mg / mL; the concentration of whey protein isolate solution is 15–25 mg / mL; and the weight ratio of phosphatidylglycerol to whey protein isolate is 1:(1.5–2.5).
8. The whey protein isolate-phospholipid complex O / W emulsion according to claim 6, characterized in that: The specific method of "mixing and homogenizing phosphatidylglycerol with buffer solution" is as follows: take phosphatidylglycerol, dissolve it into a fluid state with ethyl butyrate under ultrasonic conditions, add buffer solution, and homogenize it for 2 min at 23000 r / min using a homogenizer; then remove organic solvent by rotary evaporation.
9. The use of the whey protein isolate-phospholipid complex O / W emulsion according to any one of claims 6 to 8 in encapsulating hydrophobic active substances, or in the preparation of hydrophobic active substance formulations.
10. The application according to claim 9, characterized in that: The hydrophobic active substance is selected from fucoxanthin.