A method for improving stability and bioavailability of wheat peptides

Abstract

The application discloses a method for improving the stability and bioavailability of wheat peptide, and constructs a wheat peptide delivery system through a nano-liposome encapsulation technology. The wheat peptide has various biological activities, but has the problems of poor stability, easy degradation in a physiological environment and low bioavailability. The application uses L-alpha-phosphatidyl ethanolamine and cholesteryl chloroformate as raw materials, and the optimized conditions are that the mass ratio of PE to Chol is 1:5, and the peptide-encapsulating amount is 30%. The peptide-encapsulating liposome is prepared through the steps of dissolving, injecting, ultrasonicating and rotary evaporating. The liposome has a particle size of 235.4+ / -1.18 nm, a PDI of 0.20+ / -0.02, a stable system, uniform particle size, an encapsulation rate of 95.16% and a loading rate of 67.55%. Structural characterization proves that the wheat peptide is successfully encapsulated, and the two form a stable supramolecular structure through non-covalent interaction. Compared with free wheat peptide, the stability of the wheat peptide in the pH, temperature, salt ion fluctuation and simulated gastrointestinal fluid is significantly improved, the in-vitro antioxidant activity is greatly enhanced, an innovative scheme is provided for oral delivery of the wheat peptide, and the application has important scientific value and industrial potential.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide delivery technology, and more specifically to a method for improving the stability and bioavailability of wheat peptides. Background Technology

[0002] Wheat, one of the world's three major grains, contains proteins that can be processed into wheat peptides (WP). Wheat peptides possess various biological activities, including antioxidant activity, blood sugar regulation, immune enhancement, gastrointestinal mucosal protection, and anti-inflammatory effects, showing broad application prospects in functional foods, nutritional supplements, and drug development. However, wheat peptides themselves have poor stability and are easily degraded prematurely in the complex physiological environment of the gastrointestinal tract (acidic pH, digestive enzymes, metabolic processes, etc.), leading to low absorption efficiency and significantly reduced bioavailability, severely limiting the effective exertion of their physiological activities.

[0003] To address the aforementioned challenges, nanodelivery systems have become a research hotspot, with liposomes being widely used due to their unique advantages. Liposomes possess a cell membrane-like phospholipid bilayer structure, exhibiting excellent biocompatibility and preventing rapid clearance by the immune system. Their amphiphilic structure allows for the simultaneous encapsulation of both hydrophilic and hydrophobic substances, enabling synergistic delivery of multiple components. Surface modification with functional groups enables targeted delivery or long-term circulation, and their transmembrane efficiency can be enhanced through transport via intestinal epithelial cells. While liposome encapsulation has been proven to improve the stability of bioactive peptides, the encapsulation conditions for wheat peptides have not yet been optimized, resulting in low encapsulation efficiency, insignificant stability improvement, and complex preparation processes, making it difficult to meet the demands of industrial applications.

[0004] Based on this, the present invention constructs an efficient wheat peptide liposome delivery system by optimizing the ratio of lipid raw materials, the amount of peptides encapsulated, and the preparation process, which significantly improves the stability and bioavailability of wheat peptides, solves the bottlenecks of existing technologies, and provides technical support for the large-scale application of wheat peptides. Summary of the Invention

[0005] In view of this, the present invention aims to overcome the shortcomings of poor stability and low bioavailability of existing wheat peptides, and provide a simple, reproducible, and highly efficient nanoliposome encapsulation method. The peptide-encapsulated liposomes prepared by this method can significantly improve the stability and in vitro antioxidant activity of wheat peptides in complex environments, and expand their applications in the food, pharmaceutical and health care fields.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for improving the stability and bioavailability of wheat peptides includes the following steps:

[0008] (1) Weigh the raw materials according to the mass ratio of L-α-phosphatidylethanolamine to cholesterol chloroformate 1:5, add anhydrous ethanol, and stir magnetically at 45°C until completely dissolved to form a homogeneous lipid ethanol solution.

[0009] (2) Use a sterile syringe to inject the lipid ethanol solution prepared in step (1) into a wheat peptide aqueous solution preheated at 45°C, control the amount of peptide to be 30% of the total mass of lipid raw materials, and stir at 45°C for 40-60 min to form colostrum.

[0010] (3) After ultrasonic treatment of colostrum, anhydrous ethanol is removed to obtain peptide liposome dispersion, thus completing the liposome encapsulation of wheat peptides.

[0011] Preferably, the purity of the L-α-phosphatidylethanolamine is ≥98%, and the purity of the cholesterol chloroformate is ≥98%.

[0012] Preferably, in step (1), distearylphosphatidylethanolamine-polyethylene glycol 2000 is added as a stabilizer, wherein the amount of distearylphosphatidylethanolamine-polyethylene glycol 2000 added is 5% of the mass of the lipid ethanol solution.

[0013] Preferably, the concentration of the wheat peptide aqueous solution in step (2) is 0.1 mg / mL, and the volume ratio of the lipid ethanol solution to the wheat peptide aqueous solution is 1:9.

[0014] Preferably, the injection rate of the lipid ethanol solution in step (2) is 1 mL / min.

[0015] Preferably, in step (3), the ultrasonic power is 150w, the working time is 2s and the stopping time is 2s, and the processing time is 60min.

[0016] Preferably, in step (3), ethanol is removed by rotary evaporation at 45°C for 30-60 minutes.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for improving the stability and bioavailability of wheat peptides, which has the following beneficial effects:

[0018] (1) Process optimization: The liposome preparation process is simplified, and key parameters (raw material ratio, peptide amount, ultrasound and de-alcoholization conditions) are optimized. It has strong repeatability and is suitable for industrial-scale production.

[0019] (2) Excellent encapsulation efficiency: the encapsulation rate reaches 95.16% and the loading rate reaches 67.55%, which is significantly higher than the existing wheat peptide liposome encapsulation process, and can retain the active ingredients of wheat peptides to the maximum extent.

[0020] (3) Significantly improved stability: The liposome bilayer structure forms a physical barrier and combines with supramolecular interactions, so that wheat peptides have a retention rate of ≥80% in pH 2-8, 20-80℃, 0.01-0.2M NaCl environment and simulated gastrointestinal fluid, effectively resisting degradation;

[0021] (4) Enhanced bioactivity: The DPPH and ABTS free radical scavenging rates of wheat peptides were significantly increased after encapsulation, and the biocompatibility of liposomes could promote the transmembrane absorption of wheat peptides and improve oral bioavailability.

[0022] (5) Wide range of applications: It can be used for food fortification, pharmaceutical functional preparations and health care product development, and provides a general technical solution for the delivery of easily degradable bioactive peptides. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The diagram shows the optimized preparation conditions for Lips@WP, including: (A) Transmission electron microscopy (TEM) images of Blank lips; (B) Transmission electron microscopy (TEM) images of Lips@WP; (C) Potential distribution of Blank lips and Lips@WP with a PE:Chol ratio of 1:5; (D) Particle size and PDI changes with different ratios of PE and Chol; (E) Particle size and PDI changes of Lips@WP liposomes with different concentrations of WP at a PE:Chol ratio of 1:5; (F) Zeta potential of Blank lips and Lips@WP liposomes with a PE:Chol ratio of 1:5.

[0025] Figure 2 Particle size distribution of Lips@WP liposomes with different phospholipid and cholesterol ratios;

[0026] Figure 3 The structural characterization analysis of Lips@WP includes: (A) XRD spectra of Lips@WP, WP+Lips, Blank lips and WP; (B) UV spectra of Lips@WP, WP+Lips, Blank lips and WP; and (C) IR spectra of Lips@WP, WP+Lips, Blank lips and WP.

[0027] Figure 4The graph shows the stability analysis of different phospholipid and cholesterol ratios, where: (A) temperature stability of different phospholipid and cholesterol ratios; (B) ionic stability of different phospholipid and cholesterol ratios; (C) pH stability of different phospholipid and cholesterol ratios; different letters a–c indicate that at the same PE:Chol ratio, but with different temperatures, sodium chloride concentrations, and pH values, there are significant differences.

[0028] Figure 5 The diagram shows the stability analysis of peptide-containing liposomes, where: (A) pH stability of liposomes with a PE:Chol ratio of 1:5; (B) temperature stability of liposomes with a PE:Chol ratio of 1:5; (C) salt ion stability of liposomes with a PE:Chol ratio of 1:5; (D) storage stability of liposomes with a PE:Chol ratio of 1:5.

[0029] Figure 6 This is a diagram of the digestive stability of simulated gastrointestinal fluids in Lips@WP; different letters in a–b indicate significant differences between groups.

[0030] Figure 7 The graph shows the in vitro antioxidant capacity analysis of Lips@WP, where: (A) color change and (B) scavenging rate represent the ABTS radical scavenging activity of Lips@WP; (C) color change and (D) scavenging rate represent the DPPH radical scavenging activity of Lips@WP; values ​​are expressed as mean ± standard deviation (n=3). * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] In the following examples and comparative examples, L-α-phosphatidylethanolamine (98%) was purchased from Adoma Life Sciences Co., Ltd. (Jingzhou, China), and cholesterol chloroformate (98%) was purchased from Shanghai Kaiwei Chemical Technology Co., Ltd. (Shanghai, China). ABTS (98%) and DPPH (98%) were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China) and Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), respectively. Vitamin C (Vc) and trypsin were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Pepsin (1:3000) was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Porcine bile salts were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPEMPEG-2000) was purchased from Jiangsu Aikon Biopharmaceutical R&D Co., Ltd. (Nanjing, China).

[0033] Example 1

[0034] Preparation of liposomes:

[0035] L-α-phosphatidylethanolamine (98%) and cholesterol chloroformate (98%) were sampled in proportion (5% by weight of stabilizer (DSPEMPEG-2000) was added to the lipid ethanol solution to improve the stability of the encapsulation system when preparing the stability sample). 1 mL of anhydrous ethanol was added and stirred evenly on a magnetic stirrer at 45 °C to completely dissolve L-α-phosphatidylethanolamine and cholesterol chloroformate. 1 mL of the solution was injected into the 45 °C peptide aqueous solution at a rate of 1 mL / min using a sterile syringe. Stirring was continued for 40-60 min. Then, the solution was rotary evaporated at 45 °C for 30-60 min to remove the anhydrous ethanol. Subsequently, sonication was performed for 60 min (150 W, working for 2 seconds and then stopping for 2 seconds to prevent aggregation) to obtain the liposome dispersion (Lips@WP).

[0036] Comparative Example 1

[0037] The difference from Example 1 is that wheat peptides are not added. Instead, the dissolved L-α-phosphatidylethanolamine and cholesterol chloroformate are injected into 45°C water using a 1 mL sterile syringe at a rate of 1 mL / min to obtain Blank lips.

[0038] Experimental Example

[0039] I. Structural Analysis of Lips@WP

[0040] The morphology of Lips and Lips@WP was observed using transmission electron microscopy (TEM) (HT7800, Hitachi High Technology Co., Ltd., Japan). The particle size, particle size distribution, polydispersity index (PDI), and zeta potential of Lips@WP were determined using a high-sensitivity particle size and potential analyzer (Malvern Instruments Limited, UK). The encapsulation efficiency (EE) and drug loading efficiency (DLE) of Lips@WP were determined using a microplate reader (MRP, INFINITE E PLEX, Tecan Austria GmbH, Austria). The structures of WP, Blank lips, WP+Lips, and Lips@WP were characterized using UV-Vis spectroscopy and X-ray diffraction (XRD) (Shimadzu, Japan).

[0041] II. Stability Analysis of Lips@WP

[0042] Lips@WP and WP dispersion (1 mg / mL) were stored for different periods of time (0, 1, 2, 3, 4, 5, 6, 7, and 14 days). Then, the solutions were centrifuged for 1 hour (150 rpm), and the supernatant was collected. The absorbance was measured using a microplate reader, and the retention rate was calculated using the 1-1 formula. The storage stability of Lips@WP was evaluated by comparing the results with those of WP.

[0043] 1 mL of Lips@WP dispersion (1 mg / mL) and WP solution (1 mg / mL) were uniformly dispersed in 3 mL of NaCl solution of different concentrations (0.01, 0.02, 0.05, 0.1, and 0.2 M). After standing for 2 h, the solutions were centrifuged, and the absorbance was measured. The retention rate of WP was calculated to assess their salt ion stability. To study pH stability, 3 mL of pH solution (pH = 2, 4, 6, and 8) was added to 1 mL of the sample, and the peptide retention rates of free WP and Lips@WP were measured. The mixed samples were placed in a shaker (37°C, 100 rpm) and shaken for 6 h. The samples were then ultracentrifuged for 1 h, and the absorbance of WP was measured using a microplate reader. The absorbance was substituted into a standard curve to calculate the concentration (C) of WP. This step was repeated 3 times for each sample.

[0044] The formula for calculating the retention rate of WP is as follows:

[0045] (1-1)

[0046] In addition, to study temperature stability, liposome samples were heated in water baths at 20°C, 40°C, 60°C and 80°C for 10 min, then centrifuged at ultraspeed for 1 h. The supernatant was taken and its absorbance was measured by microplate reader (MRP) and its concentration C was calculated. The peptide retention rate was determined according to formula 1-1.

[0047] III. Stability Analysis of Simulated Gastrointestinal Fluids in Lips@WP

[0048] The stability of Lips@WP and WP in the gastrointestinal tract was analyzed. To study digestive stability, 1 mL of WP and Lips@WP sample dispersions were diluted to 4 mL with simulated gastric juice (containing 0.32 mg / mL pepsin) and incubated at 37°C on a shaker at 120 rpm for 2 h. Samples were collected every 30 minutes during incubation, then incubated in a water bath (70°C, 3 min) and ultracentrifuged. After the simulated gastric juice experiment, the samples were adjusted to neutral (pH=7), and then incubated for another 4 h with intestinal fluid (containing 0.32 mg / mL pancreatin and 0.02 mg / mL porcine bile salts). Samples were collected every 1 h during incubation, then incubated in a water bath (70°C, 3 min) and ultracentrifuged. The WP content was then determined by microplate reader (MRP), and this step was repeated 3 times for each sample. The cumulative release of WP was calculated for quantitative analysis.

[0049] IV. In vitro antioxidant activity of Lips@WP

[0050] To assess the scavenging activity of DPPH free radicals, a series of solutions containing different concentrations (12.5, 25, 50, 100, 300, and 600 μg / mL) of free WP, Vc, Blank lips, and Lips@WP were prepared. Subsequently, 0.4 mL of DPPH ethanol solution (0.25 mM) was mixed with 0.6 mL of each sample, and the mixture was reacted at room temperature in the dark for 30 min. The absorbance of the reaction solution at 517 nm was then measured using a multi-mode microplate reader. The DPPH free radical scavenging activity was calculated according to Equation 1-2:

[0051] (1-2)

[0052] Where Ac is the absorbance of the DPPH solution without WP, and As is the absorbance of WP in the presence of WP.

[0053] The ABTS radical scavenging activity of Lips@WP was determined: ABTS•+ solutions were prepared by mixing ABTS stock solution (2 mM) with potassium persulfate (2.45 mM) and reacting at room temperature in the dark for 12–16 h. The ABTS solution was diluted with PBS (pH=7.4) to achieve an absorbance of 0.70 (±0.02) at 734 nm. Next, the obtained ABTS solution was mixed with different concentrations (12.5, 25, 50, 100, 300, and 600 μg / mL) of free WP, Vc, Blank lips, and Lips@WP dispersions, and allowed to stand at room temperature in the dark for 30 min. Finally, the absorbance was measured at 734 nm. The scavenging rate was calculated using Equations 1-3.

[0054] (1-3)

[0055] As and Ac are the absorbances of the sample and control solutions, respectively. Each experiment was performed in triplicate.

[0056] Data analysis: All experimental measurements were performed three times, and data are expressed as mean ± standard deviation (SD). Two-way ANOVA was used to determine statistical significance. P < 0.05 was considered significant.

[0057] V. Experimental Results:

[0058] Structure and Characterization of Lips@WP: The Lips@WP liposome system is constructed based on the synergistic assembly effect of phosphatidylethanolamine (PE) and cholesterol (Chol). Its core innovation lies in utilizing the amphiphilic characteristics of the phospholipid bilayer to achieve efficient WP encapsulation. By systematically regulating the mass ratio of PE to Chol, the complex correlation mechanism between liposome physical properties and encapsulation efficiency was revealed. Transmission electron microscopy (TEM) results showed the typical morphology of Lips and Lips@WP. Figure 1 (AB). Clearly, blank liposomes without wheat peptide loading readily aggregate and precipitate in solution. Conversely, after loading with wheat peptide, the liposomes exhibit uniformly dispersed spherical particles. Furthermore, the particle size of the wheat peptide-loaded liposomes is larger than that of the blank liposomes, demonstrating the successful liposome loading of wheat peptide. However, liposomes composed of different ratios of phospholipids and cholesterol exhibit varying particle size and distribution for wheat peptide loading. Figure 1(D) The particle size fluctuated with changes in the PE:Chol ratio. As the PE:Chol ratio decreased from 6:1 to 1:6, the particle size and PDI of Lips@WPs generally showed a trend of first decreasing, then increasing, and then decreasing again. This synchronous decrease or increase in particle size and PDI essentially reflects the balance between increased rigidity and decreased fluidity of the lipid bilayer induced by cholesterol: moderate cholesterol can optimize membrane fluidity to promote peptide insertion, but excessive cholesterol may cause differences in lipid packing density, leading to uneven liposome size. At a PE:Chol ratio of 1:5, both particle size and PDI reached relatively high values ​​(particle size approximately 280 nm, PDI approximately 0.3), while at a PE:Chol ratio of 1:2, both particle size and PDI were at their minimum (particle size approximately 130 nm, PDI 0.20).

[0059] The results in Table 1 show that the 1:5 ratio exhibits unique advantages in encapsulation efficiency and loading capacity—the encapsulation efficiency reaches 84.31±0.01%, and the loading rate reaches 69.96±0.07%, significantly better than other ratio combinations. In Lips@WP with a PE:Chol ratio of 1:5, as the relative content of encapsulated wheat peptides increases, the liposome particle size distribution becomes more uniform, and the PDI is relatively smaller (…). Figure 1 E). Results combining the encapsulation efficiency and loading rate of wheat peptides using liposomes with different ratios indicated that liposomes with a peptide loading capacity of 30% exhibited the best encapsulation capacity for wheat peptides when the PE:Chol ratio was 1:5. Furthermore, from... Figure 1 The zeta potential of Blank lips with a PE:Chol ratio of 1:5 (as shown in Figure F) is approximately -18 mV, while that of Lips@WP (WP 30%) is approximately -21 mV. After encapsulation with wheat peptides, the absolute value of the zeta potential of the liposomes increases. This increase in absolute value indicates that the encapsulation of wheat peptides increases the negative charge on the liposome surface, enhances the electrostatic repulsion between particles, which is beneficial to the stability and uniform distribution of the liposome system and reduces liposome aggregation. These results prove that WP was successfully encapsulated in the liposomes. Furthermore, the zeta potential distribution map (…) Figure 1 C) It can be seen that after loading wheat peptides, the surface properties of liposomes are changed, the absolute value of the zeta potential increases, and the electrostatic repulsion is enhanced, which helps to improve the stability of peptide-loaded liposomes in solution; Blank lips have less surface charge, so their stability is relatively weak.

[0060] Table 1. Effects of different phospholipid and cholesterol ratios and different WP concentrations on particle size, PDI, EE, and LE of Lips@WP liposomes.

[0061]

[0062] from Figure 2The particle size distribution results of Lips@WP liposomes with different phospholipid to cholesterol ratios showed that at ratios of 1:2, 1:4, 1:5, and 1:6, the particle size distribution of liposomes differed with increasing cholesterol content. This indicates that changes in the cholesterol ratio significantly affect the particle size and distribution of liposomes. At ratios of 2:1, 4:1, 5:1, and 6:1, the particle size distribution of liposomes also differed with increasing phospholipid ratio, but the effect of the phospholipid ratio on the particle size distribution was relatively smaller compared to that of cholesterol. For a 1:5 ratio with WP concentrations of 10%, 30%, 50%, and 70%, the particle size distribution of Lips@WP liposomes changed with increasing WP concentration. The peak parameters differed at different ratios, indicating that even with the same phospholipid to cholesterol ratio, changes in concentration also affect the particle size distribution and size of liposomes. In summary, the cholesterol ratio is the main influencing factor on particle size distribution, followed by the phospholipid ratio; optimizing the concentration at the optimal ratio can improve particle size uniformity. This provides a precise basis for the design of nanocarriers.

[0063] In addition, through XRD patterns ( Figure 3 Analysis A) reveals significant differences in diffraction characteristics between Lips@WP liposomes and purely physically mixed WP+Lips, free WP, and blank liposomes. This difference stems from the liposome encapsulation process inducing the formation of a supramolecularly ordered assembly structure. Wheat peptide molecules couple with the lipid bilayer through non-covalent interactions (such as hydrogen bonds, hydrophobic interactions, and electrostatic attraction), promoting the transformation of the liposomes from a disordered liquid crystalline phase to a more stable layered phase. In contrast, the simply mixed WP+Lips only exhibits superimposed characteristic diffraction peaks of the two components, lacking the synergistic effect of structural optimization, confirming the crucial role of the encapsulation process in enhancing the system's orderliness. Ultraviolet spectra (...) Figure 3 B) It is evident that wheat peptides (WP) exhibit a strong absorption peak at 200-210 nm (absorbance 3.42 au), originating from electronic transitions of peptide bonds and aromatic amino acids; blank liposomes show lower absorbance in the same wavelength range (3.01 au), mainly due to acyl chain vibrations. The physical mixture (WP+Lips) shows a λ peak at 205 nm. max The absorbance was 3.15 au, lower than that of free WP at 210 nm (3.42 au) despite containing the same concentration of WP. This significant deviation from the simple mixing behavior can be attributed to light scattering by the liposome particles, which reduces the intensity of transmitted light and thus the measured absorbance. Conversely, Lips@WP showed a λ at 203 nm. maxThe absorbance was 2.99 au, showing a 7 nm blue shift compared to free WP, but only a 1 nm red shift compared to blank liposomes. This significant blue shift is not only much larger than the shift between free WP and the physical mixture, but also indicates a significant increase in the electronic excitation energy of the chromophore after encapsulation, reflecting a significant reduction in the polarity of its surrounding microenvironment. Compared to the strong absorption of free WP at 210 nm, the degree of blue shift and the decrease in absorbance of the Lips@WP absorption band suggest a more thorough migration of peptide bonds and aromatic amino acid residues from the aqueous phase to the hydrophobic core of the lipid bilayer, and an effective weakening of the hydrogen bonding between the chromophore and the solvent. Meanwhile, the 1 nm red shift compared to blank lips indicates that the absorption peak of the encapsulation system is not simply superimposed from the two components, but is further modulated by ground-state intermolecular interactions on the basis of spectral overlap. In summary, this non-additive spectral behavior provides quantitative evidence for the hydrophobic interactions, π-π stacking, and interfacial rearrangement between WP and the lipid bilayer, confirming the stable incorporation of peptides into liposomes. This spectral perturbation is consistent with the formation of hydrogen bonds and hydrophobic interactions between peptide and lipid components, which alters the electronic environment of the chromophores in WP.

[0064] By analyzing Fourier transform infrared spectra ( Figure 3 Further analysis (C) revealed that the characteristic protein peaks of wheat peptide (WP) (such as amide I and II bands) and the fatty acid chain CH peak and ester C=O peak of blank liposomes (Blank lips) were significantly shifted and broadened in the Lips@WP encapsulation system. This indicates that the two undergo interfacial coupling through non-covalent interactions such as hydrogen bonding and hydrophobic interactions, forming a supramolecular structure. In contrast, the physical mixture WP+Lips only exhibited a simple superposition of characteristic peaks without changes in peak position or shape, demonstrating specific molecular interactions during the encapsulation process, rather than simple mixing. This spectral difference, complementary to the XRD and UV spectral results, together elucidates the structural evolution and interaction nature of wheat peptide encapsulated in liposomes.

[0065] Stability analysis of Lips@WP: To investigate the stability of liposomes loaded with wheat peptides, experiments were conducted on three influencing factors: pH, temperature, and ions. Firstly, liposomes with different ratios of phospholipids and cholesterol (…) were analyzed. Figure 4 The stability of AC under multiple coupled factors varied significantly. A 5:1 ratio showed balanced performance in high-temperature, high-salt, and moderately alkaline environments, while a 1:2 ratio was superior in acidic and low-salt environments, providing a basis for component selection in targeted design. Based on the above conclusions, further experiments were conducted using the optimal phospholipid to cholesterol ratio (1:5). The pH stability results ( Figure 5A) It can be seen that in acidic environments (pH 4-6), the WP retention rate of Lips@WP is significantly higher than that of free WP, reflecting the buffering effect of liposomes on acidic environments; under alkaline conditions (pH 6-8), although the retention rate shows a decreasing trend, Lips@WP still maintains a high level. This indicates that the liposome bilayer components can improve the acid and alkali resistance of WP.

[0066] From temperature stability ( Figure 5 B) The results showed that at room temperature (20-60℃), there was no significant difference in the retention rates of Lips@WP and free WP (both >90%); however, at high temperature (100℃), the difference was significant, with the retention rate of Lips@WP (84%) being significantly higher than that of free WP (20%), indicating that liposomes have a barrier effect on thermal degradation.

[0067] Furthermore, from the stability of NaCl ions ( Figure 5 C) It can be seen that the retention rate of Lips@WP remains stable at over 80% under 10-200mM conditions, and is significantly higher than that of WP; this indicates that the phospholipid bilayer of the liposome encapsulates the peptide chain, forming a physical barrier and reducing Na+. + Direct electrostatic adsorption of peptide chains. The positive charge on the liposome surface can react with Na+. + Competition reduces ion penetration efficiency, which is consistent with research on the "ion shielding effect" of liposomes in drug delivery. Experimental results show that liposomes inhibit the salting-out effect.

[0068] from Figure 5 The storage stability results for D showed that the encapsulation efficiency of liposomes decreased to varying degrees under different storage conditions with prolonged storage time, but the overall trend was: Fresh group > 4℃ storage group > 25℃ > 37℃ storage group. Combined with the phospholipid-cholesterol ratio (the optimal ratio of 1:5 was used here), this further demonstrates the synergistic effect of appropriate component ratios and storage conditions on the long-term stability of liposomes. The essence of this stability difference is that temperature affects the thermal motion and phase transition of lipid molecules, thereby altering the integrity of the liposome membrane structure and ultimately affecting the encapsulation and retention capacity of wheat peptides. This provides a crucial reference for the practical application of liposomes as a wheat peptide delivery system: for long-term storage of this liposome drug delivery system, a low-temperature environment (e.g., 4℃) is more conducive to maintaining its encapsulation efficiency and protecting wheat peptides, ensuring the effectiveness of active substance delivery.

[0069] Lips@WP simulated gastrointestinal fluid stability analysis: such as Figure 6As shown, in the simulated gastrointestinal digestion stability experiment, the cumulative release rate of wheat peptides (WP) was significantly higher than that of Lips@WP liposomes. From a molecular interaction perspective, liposomes, as carriers, possess an amphiphilic phospholipid bilayer, allowing them to form complexes with wheat peptides through hydrophobic interactions and hydrogen bonds. Furthermore, the hydrophobic tails of phospholipid molecules interact hydrophobically with hydrophobic amino acid residues (such as leucine and isoleucine) in WP molecules, while the polar groups at the head of the phospholipid molecule (such as -PO3) also interact hydrophobically. 2- Lipomers (such as -COOH and -NH2) can form hydrogen bonds or ionic bonds with the polar functional groups of wheat peptides. These interactions encapsulate WP within liposomes or bind to their surface, thus delaying WP release in a simulated gastrointestinal fluid environment. Consequently, the cumulative release rate of Lips@WP liposomes is lower than that of free WP. Related studies have also shown that liposomes have excellent encapsulation and sustained-release effects on bioactive peptides. Through various intermolecular interactions between liposomes and peptide molecules, the release behavior of peptides in gastrointestinal fluid can be effectively regulated, which is consistent with the results observed in this experiment that the cumulative release rate of peptide-encapsulated liposomes is lower than that of wheat peptides.

[0070] Evaluation of the in vitro antioxidant activity of Lips@WP: The in vitro antioxidant activity of Lips@WP was evaluated by its ability to scavenge ABTS and DPPH free radicals. Figure 7 As shown in AD, the color of samples treated with different concentrations of Lips@WP (12.5-600 μg / mL) also changed for ABTS radical scavenging activity. With increasing concentration, the sample color changed from light green to light pink. The ABTS radical scavenging rate increased from 14.93±0.009% to 97.61±0.015%, and the ABTS radical scavenging activity of Lips@WP was significantly different from that of WP, Vc, and Blanklips. With increasing Lips@WP concentration, the scavenging effect on DPPH radicals also significantly increased. At a concentration of 600 μg / mL, the DPPH radical scavenging activity of Lips@WP reached 83.58±0.038%, and the sample color changed from purple to light brown. These results indicate that Lips@WP has significant in vitro antioxidant activity. Furthermore, to exclude the contribution of the encapsulation material, we evaluated the antioxidant activity of blank liposomes. There were no significant differences in DPPH and ABTS clearance activities among blank liposomes of different concentrations, confirming that Lips@WP is solely attributable to the encapsulated WP.

[0071] In summary, this invention systematically investigates the use of liposomes as a carrier for the effective loading of wheat peptide (WP), and studies the physicochemical properties, structural characteristics, and in vitro antioxidant activity of Lips@WP. It confirms that utilizing liposomes as a nanocarrier, with its lipid bilayer barrier protection, significantly enhances the stability of wheat peptide, effectively prevents WP degradation, and further strengthens the antioxidant efficacy of WP through the lipid bilayer structure. This innovative delivery strategy provides a scientifically feasible solution for overcoming the stability bottleneck and activity protection challenges of WP during oral delivery. It lays the technical foundation for the in-depth application of WP in food fortification, pharmaceutical functional ingredient delivery, and health product development.

[0072] The embodiments and experimental examples described in this specification are presented in a progressive manner. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving the stability and bioavailability of wheat peptides, characterized in that, Includes the following steps: (1) Weigh the raw materials according to the mass ratio of L-α-phosphatidylethanolamine to cholesterol chloroformate 1:5, add anhydrous ethanol, and stir magnetically at 45°C until completely dissolved to form a homogeneous lipid ethanol solution. (2) Use a sterile syringe to inject the lipid ethanol solution prepared in step (1) into a wheat peptide aqueous solution preheated at 45°C, control the amount of peptide to be 30% of the total mass of lipid raw materials, and stir at 45°C for 40-60 min to form colostrum. (3) After ultrasonic treatment of colostrum, anhydrous ethanol is removed to obtain peptide liposome dispersion, thus completing the liposome encapsulation of wheat peptides.

2. The method for improving the stability and bioavailability of wheat peptides according to claim 1, characterized in that, The purity of the L-α-phosphatidylethanolamine is ≥98%, and the purity of the cholesterol chloroformate is ≥98%.

3. The method for improving the stability and bioavailability of wheat peptides according to claim 1, characterized in that, In step (1), distearylphosphatidylethanolamine-polyethylene glycol 2000 is added as a stabilizer, wherein the amount of distearylphosphatidylethanolamine-polyethylene glycol 2000 added is 5% of the mass of the lipid ethanol solution.

4. The method for improving the stability and bioavailability of wheat peptides according to claim 1, characterized in that, The concentration of the wheat peptide aqueous solution in step (2) is 0.1 mg / mL, and the volume ratio of the lipid ethanol solution to the wheat peptide aqueous solution is 1:

9.

5. The method for improving the stability and bioavailability of wheat peptides according to claim 1, characterized in that, In step (2), the injection rate of the lipid ethanol solution is 1 mL / min.

6. The method for improving the stability and bioavailability of wheat peptides according to claim 1, characterized in that, In step (3), the ultrasonic power is 150w, the working time is 2s and the stopping time is 2s, and the processing time is 60min.

7. The method for improving the stability and bioavailability of wheat peptides according to claim 1, characterized in that, In step (3), ethanol is removed by rotary evaporation at 45°C for 30-60 minutes.

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