A method for preparing high-encapsulation-rate nisin nanoliposomes by a stabilizer film hydration method and application thereof

Abstract

The present application belongs to the technical field of nisin nanoliposome preparation and application, and particularly relates to a method for preparing nisin nanoliposome by film hydration, which comprises the following steps: dissolving egg yolk lecithin, a stabilizer and a solvent to obtain a mixture, removing the solvent, dissolving nisin in a buffer solution, and then adding the mixture into the buffer solution to prepare nisin nanoliposome by conventional film hydration; wherein the stabilizer is one or more of curcumin, resveratrol or naringin. The present application selects a new stabilizer for preparing nanoliposome by film hydration, and improves the encapsulation efficiency of nanoliposome to nisin, especially when curcumin is used as the stabilizer, the prepared nisin nanoliposome (Cu-NLs) has good effect. The prepared Cu-NLs has the characteristics of small particle size, uniform particle size distribution, high absolute value of Zeta potential and good stability. In the milk preservation experiment, the Cu-NLs avoids the adverse effect of fat on nisin, and effectively protects the antibacterial activity of nisin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of Nisin nanoliposome preparation and application, and specifically relates to a method for preparing high-encapsulation-ratio Nisin nanoliposomes by a novel stabilizer thin film hydration method and application thereof. BACKGROUND

[0002] Bacteriocins are an important source of food biological preservatives due to their high safety and effective antibacterial activity. Among them, Nisin is the most representative bacteriocin, which has a strong inhibitory effect on Gram-positive pathogenic bacteria (such as Staphylococcus aureus, Yusuf, 2018). Nisin is listed as "Generally Recognized as Safe" (GRAS) by the U.S. Food and Drug Administration (FDA) (Ucar, Ozogul, Ozogul, Durmus, Koseker, & Kuley Boga, 2021); it has been widely studied and widely used. However, the antibacterial effect of bacteriocins may be reduced by the interaction with complex food ingredients such as fat and protein (Chollet, Sebti, Martial-Gros, & Degraeve, 2008). At present, the bioactive substances are encapsulated in nanocarriers by using encapsulation technology to protect their activity (Jayaprakash, Maudhuit, Gaiani, & Desobry, 2023; Lopez-Polo, Monasterio, Cantero-Lopez, & Osorio, 2021). Therefore, by encapsulating bacteriocins in nanoliposomes, it may be an effective strategy to improve the stability of their antibacterial activity in food (da Silva Malheiros, Daroit, & Brandelli, 2010).

[0003] There are several methods for preparing nanoliposomes, among which the thin film hydration method is relatively simple (Khorasani, Danaei, & Mozafari, 2018). In addition, the thin film hydration method allows the use of less toxic organic solvents (such as ethanol), which are miscible with water (Penoy, Grignard, Evrard, & Piel, 2021), so the safety of this method is relatively high compared to other preparation methods. However, during the preparation process, whether the bacteriocin in the aqueous phase can be effectively encapsulated into the nanoliposome mainly depends on the interaction between the bacteriocin and the phospholipid layer (Mozafari, Johnson, Hatziantoniou, & Demetzos, 2008). However, the phospholipid layer has defects such as easy accumulation, which leads to problems such as uneven size and uneven distribution of nanoliposomes, and even affects the encapsulation efficiency, limiting its application (Ajeeshkumar, Aneesh, Raju, Suseela, Ravishankar, & Benjakul, 2021). At present, it is crucial to add stabilizers to improve the defects of the phospholipid layer when preparing nanoliposomes. Since cholesterol is amphiphilic, it can be combined into the phospholipid layer, thereby adjusting its fluidity and improving the stability of the generated nanoliposomes (Bondu & Yen, 2022). Therefore, cholesterol is the most commonly used nanoliposome stabilizer (Thabet, Elsabahy, & Eissa, 2022). However, considering the health risks caused by a high-cholesterol diet (Gorjian, Raftani Amiri, Mohammadzadeh Milani, & Ghaffari Khaligh, 2021; H. Ma, 2004), therefore, screening safer compounds to replace cholesterol to improve the stability of nanoliposomes and improve the encapsulation efficiency has become the focus of research.

[0004] In addition, as an important component of the phospholipid layer of nanoliposomes, the interaction between the stabilizer and the bacteriocin is also worth paying attention to. By screening new stabilizers used in the preparation process, and using the thin film hydration method to prepare bacteriocin nanoliposomes with high safety and high encapsulation efficiency. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a method for preparing Nisin nanoliposomes by thin film hydration method. The present application uses Nisin as a model bacteriocin and adopts thin film hydration method to prepare Nisin-loaded nanoliposomes. Three compounds (curcumin, resveratrol and naringin) naturally having amphiphilic groups are used as potential stabilizers of Nisin nanoliposomes. Screening of new stabilizers can improve the characteristics of Nisin nanoliposomes and increase the encapsulation efficiency. The particle size, PDI, Zeta potential and encapsulation efficiency of Nisin nanoliposomes are measured for screening. Subsequently, the interaction between the new stabilizer (curcumin) and Nisin is studied by Fourier exchange infrared spectroscopy (FT-IR) and molecular docking technology to clarify the mechanism of improving the encapsulation efficiency of Nisin nanoliposomes. Finally, the inhibitory effect of Nisin nanoliposomes on Staphylococcus aureus in milk is also evaluated.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] A method for preparing Nisin nanoliposomes by thin film hydration method, first, egg yolk lecithin, stabilizer and solvent are mixed and dissolved, then the solvent is removed to obtain a mixture; then Nisin is dissolved in a buffer solution, and then added to the above mixture to prepare Nisin nanoliposomes by conventional thin film hydration method; the stabilizer is one or more of curcumin, resveratrol or naringin.

[0008] Preferably, the mixing ratio of egg yolk lecithin and stabilizer is 10-20:1.

[0009] Preferably, Nisin is dissolved in PBS buffer solution to obtain a nisin solution with a concentration of 0.5-1 mg / mL.

[0010] Preferably, the pH value of the PBS buffer solution is 6-7.

[0011] Preferably, the mass ratio of Nisin to stabilizer is 1:0.01-2.

[0012] Preferably, the stabilizer is curcumin or resveratrol.

[0013] Compared with the prior art, the beneficial effects of the present application are reflected in:

[0014] The application screens a new stabilizer which can be used for preparing nanoliposomes by thin film hydration method, improves the encapsulation efficiency of nanoliposomes on Nisin, and especially, the nanoliposomes (Cu-NLs) prepared by using curcumin as the stabilizer have the best effect. The prepared Cu-NLs have the characteristics of small particle size, uniform particle size distribution, high absolute value of zeta potential, good stability and the like. In the milk preservation experiment, the Cu-NLs avoid the adverse effect of fat on Nisin, and effectively protect the activity of Nisin. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 (a) NL and (b, c) Cu-NL.

[0016] Figure 2 (a) Cu-BL and (b) Cu-NL.

[0017] Figure 3 (a) Cu-BL and (b) Cu-NL.

[0018] Figure 4 (a) Cu-BL and (b) Cu-NL.

[0019] Figure 5 (a) Cu-BL and (b) Cu-NL.

[0020] Figure 6 (a) Cu-BL and (b) Cu-NL. DETAILED DESCRIPTION

[0021] The application will be described in detail below with specific examples, but the scope of the application is not limited. It should be understood that those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes should belong to the protection scope of the appended claims of the application.

[0022] The preparation was carried out according to the conventional conditions or the conditions recommended by the manufacturer, if the specific conditions were not mentioned in the examples. The reagents or instruments used were conventional products which can be purchased on the market, if the manufacturers were not mentioned.

[0023] Example 1 Preparation of Nisin nanoliposomes

[0024] Nanoparticles were prepared by thin film hydration method using cholesterol, curcumin, resveratrol and naringin as stabilizers, respectively (Lopes, Pinilla, & Brandelli, 2017). Egg yolk lecithin (70 mg), stabilizer (5 mg) and absolute ethanol (5 mL) were placed in a brown flask, sealed and vortexed until the solids were completely dissolved. Then the ethanol was removed by rotary evaporation at 30 °C, leaving a highly viscous organic gel at the bottom of the flask. Nisin was dissolved in PBS buffer (0.01 mol / mL, pH 6.8) to obtain a Nisin solution (0.5 mg / mL). Then the Nisin solution (5 mL) was added to the brown flask, and the resulting mixture was sonicated until the viscous organic gel was completely dispersed in the solution. Sonication (37 kHz) was repeated for the size reduction of the nanoparticles, using 1 minute of sonication followed by 1 minute of rest, for 10 consecutive cycles (Sarabandi, Jafari, Mohammadi, Akbarbaglu, Pezeshki, & Khakbaz Heshmati, 2019). Then the nanoparticle suspension was passed 11 times through a 200-nanometer polycarbonate membrane using a liposome extruder (LiposoFast, Avestin, Canada) to obtain cholesterol Nisin nanoparticles (Ch-NLs), curcumin Nisin nanoparticles (Cu-NLs), resveratrol Nisin nanoparticles (Re-NLs) and naringin Nisin nanoparticles (Na-NLs), respectively. The same procedure was also used to prepare Nisin nanoparticles (NLs) without the addition of stabilizers. NLs were used as a blank control, and Ch-NLs were used as a positive control.

[0025] Performance test:

[0026] 1. Size, PDI and Zeta potential of Nisin nanoparticles

[0027] The particle size, polydispersity index (PDI) and Zeta potential of the nanoparticles were measured using a nanoparticle analyzer (NANO ZSE, Malvern, UK). The prepared nanoparticle suspension was diluted with ultrapure water to 10 -1 concentration, and the average particle size and PDI were determined by dynamic light scattering (DLS), while the Zeta potential was determined three times at 25 °C by laser Doppler microelectrophoresis (Jiao et al., 2020; Zhang, Gu, et al., 2022).

[0028] 2. Encapsulation efficiency of Nisin nanoparticles

[0029] Nisin was dissolved in PBS buffer (0.01 mol / mL, pH=6.8) to obtain a gradient concentration of Nisin solution. Then, the absorbance of Nisin solution at 220 nm was measured by a UV spectrophotometer (U-3900H, Hitachi, Ltd., Japan), and a Nisin-absorbance standard curve was fitted and established. (Lopes et al., 2017).

[0030] The prepared Nisin nanoliposome suspension was immediately added into an Ultra-15 centrifugal filter device (10 kDa, MWCO, Millipore, USA), and centrifuged at 9,000 rpm for 10 minutes at 4 °C using a refrigerated centrifuge (UNIVERSAL 320 R, Hettich, German), and the filtrate was collected. Subsequently, the prepared Nisin nanoliposome suspension was first added into an Ultra-15 centrifugal filter device (10 kDa, Millipore, USA), and then centrifuged at 9,000 rpm for 10 minutes at 4 °C using a refrigerated centrifuge (UNIVERSAL 320 R, Hettich, German), and the filtrate was collected. Subsequently, the absorbance of the filtrate at 220 nm was measured by a UV spectrophotometer, and the Nisin concentration in the filtrate was calculated in combination with the standard curve. Subsequently, the encapsulation efficiency of the nanoliposome was calculated according to formula (1).

[0031]

[0032] 3、 Nisin nanoliposome morphology structure observation

[0033] The morphology and structure of Cu-NL and NL were observed by transmission electron microscopy (TEM). The prepared NL and Cu-NL suspension was diluted with ultrapure water, then dropped on a 200-mesh copper grid with carbon film support, negatively stained with 2% uranyl acetate solution for 30 s, and naturally air-dried. After the sample preparation was completed, the transmission electron microscope (JEM1400, JEOL, Japan) was used to observe the sample at a voltage of 80 kV.

[0034] 4、 Nanoliposome thermodynamic analysis

[0035] Blank nanoliposomes were prepared to study the interaction of curcumin with phospholipids and Nisin. The same method described in section 2.2 was used to prepare blank nanoliposomes, but without adding nisin in PBS buffer (0.01 mol / mL, pH=6.8). Blank nanoliposomes (BLs, without stabilizer), cholesterol blank nanoliposomes (Ch-BLs, using cholesterol as stabilizer) and curcumin blank nanoliposomes (Cu-BLs, using curcumin as stabilizer) were prepared.

[0036] Differential scanning calorimetry (DSC; 214 Polyma, NETZSCH, Germany) was used to determine the thermodynamic changes during phase transition of Cu-NLs, Cu-BLs, Ch-BLs and BLs. Each sample (3 mg) was placed in an aluminum crucible, which was then sealed and placed in a nitrogen atmosphere with an empty aluminum crucible as a reference. The sample temperature was adjusted to 25 °C and held for 5 min, then cooled to -40 °C at a rate of 10 °C / min -1 and held for 10 min. Finally, the sample was heated to 121 °C at a rate of 5 °C / min -1 and held for 10 min.

[0037] 5. FT-IR analysis of nanoliposomes

[0038] Cu-NLs, Cu-BLs, egg yolk lecithin, Nisin and curcumin were mixed with KBr powder separately and pressed into thin slices using a tablet press at a pressure of 12.5 MPa for more than 1 min. These thin slices were then scanned using an FT-IR spectrometer (Tensor 27, Bruker, USA) in the range of 4000 to 400 cm-1.

[0039] 6. XRD analysis of nanoliposomes

[0040] The X-ray diffraction (XRD) system (D8 Advance, Bruker, Germany) was set at 40 kV and 40 mA to analyze Cu-NLs, Cu-BLs, egg yolk lecithin, Nisin and curcumin. Scanning was performed from 10° to 90° 2Q at a rate of 10° / min -1 (Sun et al., 2022).

[0041] 7. Simulation analysis of the binding mode of curcumin and Nisin

[0042] The interaction between curcumin and nisin was further investigated using molecular docking simulations. The X-ray crystal structure of nisin (PDB ID: 5XHB) was obtained from the RCSB protein database (http: / / www.rcsb.org). The structure of curcumin (PubChem CID: 969516) was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov). Molecular docking was performed using AutoDock Vina 4.2 software, with nisin set as the rigid acceptor and curcumin as the flexible ligand. Finally, the docking result with the lowest binding energy was selected as the optimal model (Li, Yang, Wang, Ma, & Peng, 2023), and the binding mode between curcumin and nisin was visualized using Discovery Studio 4.5.

[0043] 8. Inhibitory effect of Nisin nanoliposomes on Staphylococcus aureus in whole and skim milk.

[0044] The inhibitory effects of Cu-NLs and free Nisin on Staphylococcus aureus in liquid whole milk (fat = 4.0 g / 100 mL, protein = 3.2 g / 100 mL) and skim milk (fat = 0 g / 100 mL, protein = 3.2 g / 100 mL) were evaluated using a colony counting method (Li et al., 2023). A Staphylococcus aureus suspension (2 × 10⁶ CFU / min, 100 μL) was added to tubes containing either skim or whole milk (10 mL). Then, Cu-NL (1 mL), Cu-BL (1 mL), Nisin solution (0.5 mg / min, 1 mL), or sterile water (1 mL) were added. All tubes were then stored at 4 °C, and Staphylococcus aureus counts were performed at 0, 1, 3, 5, and 7 days. The cultures were then diluted decimal, divided into three aliquots, and inoculated onto plates containing colony count agar and incubated at 37 °C for 24 hours.

[0045] 9. Data processing and statistical analysis

[0046] Results were analyzed using one-way ANOVA with SPSS 22 (IBM, USA). Values ​​were considered statistically significant if p < 0.05. Images were processed using ImageJ, while linear fitting and plotting were performed using Origin 9.65 (Origin Lab, USA). Molecular docking models were visualized and analyzed using Discovery Studio 4.5 (Biovia, USA).

[0047] Test results

[0048] 1. Particle size, PDI, zeta potential and encapsulation efficiency (EE) of Nisin nanoliposomes

[0049] Particle size, PDI and zeta potential are three important indicators for evaluating the uniformity and stability of nanoliposomes. Size refers to the average diameter of nanoliposomes. Smaller nanoliposomes are beneficial for better delivery of active substances to various parts of the food system, improving their utilization (Weiss, Takhistov, & McClements, 2006). As shown in Table 1, all Nisin nanoliposomes were less than 200 nm. Compared with the size of NLs (162.63 nm), Ch-NLs (153.93 nm), Re-NLs (153.73 nm) and Cu-NLs (155.00 nm) were significantly smaller, but Na-NLs (173.03 nm) were larger. PDI reflects the particle size distribution of nanoliposomes, and the smaller the value, the better the uniformity of nanoliposomes (da Silva et al., 2014). The PDI of Re-NLs (0.137), Ch-NLs (0.131) and Cu-NLs (0.121) was significantly lower than that of NLs (0.199), but the PDI of Na-NLs (0.200) did not decrease. Therefore, Cu-NLs and Re-NLs were similar to Ch-NLs in terms of size and PDI, with no significant difference.

[0050] In addition, zeta potential reflects the charge on the surface of nanoliposomes. Higher absolute values indicate greater electrostatic repulsive forces between nanoliposomes, making them less likely to aggregate together (Li et al., 2023). Compared with the zeta potential of NLs (-34.17 mV), the zeta potential of Cu-NLs (-47.10 mV), Re-NLs (-45.00 mV), Ch-NLs (-39.07 mV) and Na-NLs (-36.73 mV) was more negative. In addition, the absolute values of the zeta potential of Cu-NLs and Re-NLs were significantly higher than those of Ch-NLs, indicating that the Cu-NL and Re-NL systems were more uniform and stable.

[0051] Encapsulation efficiency (EE) is an important parameter to determine the loading amount of bioactive ingredients. Table 1 shows the EE of NLs, Cu-NLs, Re-NLs, Na-NLs and Ch-NLs. Compared with the EE of NLs (62.74%), the EE values of Cu-NLs (95.94%), Re-NLs (92.55%), Ch-NLs (90.65%) and Na-NLs (64.58%) were significantly higher, indicating that the addition of stabilizers can improve the encapsulation efficiency of nanoliposomes. However, the EE of Na-NLs was significantly lower than that of Ch-NLs, indicating that naringin is not suitable to replace cholesterol as a stabilizer. Cu-NLs (95.94%) and Re-NLs (92.55%) with curcumin and resveratrol as stabilizers, respectively, were found to be significantly more effective than Ch-NLs, with Cu-NLs having the highest EE.

[0052] So far, Nisin is the most studied bacteriocin, but the EE prepared by Malheiros is only 34% (da Silva Malheiros et al., 2010). Pinilla et al. used the film hydration method to prepare Nisin nanoliposomes using cholesterol as a stabilizer, and the highest EE was only 80.7% (Pinilla, Reque, & Brandelli, 2020).

[0053] Cu-NLs with curcumin as a stabilizer had the highest EE and also had good characteristics (particle size, PDI and Zeta potential). Therefore, curcumin was selected as a potential new stabilizer for preparing Nisin nanoliposomes with good performance and high EE.

[0054] Table 1 Size, PDI, Zeta potential and EE of Nisin nanoliposomes

[0055]

[0056] Note: Different letters in the same column indicate significant differences at the level of P < 0.05

[0057] 2、 Nisin nanoliposomes morphology

[0058] The microstructure of NLs and Cu-NLs, as well as the distribution of Nisin in nanoliposomes were observed by TEM Figure 1 ). The particle size of NLs (160.04 nm, Figure 1 a) and Cu-BLs (155.24 nm, Figure 1 b,c) were consistent with the results obtained by DLS method. From Figure 2It can be seen that NLs and Cu-NLs have a spherical morphology. Notably, a double-layer structure similar to that reported by Amjadi, Almasi, Hamishehkar, Alisadeh Khaledabad, & Lim (2022) can be seen, with the outer layer being the phospholipid layer (light grey) and the inner layer being the homogeneous water layer containing Nisin (dark grey). The inner layer of Cu-NLs is similar to that of NLs, which is the water core containing Nisin (dark grey). However, compared with NLs, Nisin (black) is more concentrated at the edge of Cu-NLs. This is speculated to be due to the interaction between curcumin (as a nanoliposome stabilizer) and Nisin, causing Nisin in the solution to concentrate at the edge of the phospholipid bilayer, resulting in the high encapsulation efficiency of Cu-NLs.

[0059] 3. Thermodynamic results of nanoliposomes

[0060] Differential scanning calorimetry (DSC) is an effective method for studying the thermal behavior of phospholipid bilayers and the stability of nanoliposomes (Demetzos, 2008). The thermodynamic changes in the phase transition process of nanoliposomes are related to the molecular arrangement of the phospholipid bilayer (Demetzos, 2008; Klopfenstein, de Kruyff, Verkleij, Demel, & van Deenen, 1974). The thermodynamic data of BLs, Ch-BLs, Cu-BLs, and Cu-NLs are shown in Figure 2 The enthalpy of phase transition (△E) of phospholipids is strongly influenced by intermolecular interactions. The enthalpy of phase transition of Cu-BLs (18.089 J / g) is lower than that of BLs (21.475 J / g). In addition, the △E of Cu-BLs (18.089 J / g) is reduced compared to BL (21.475 J / g), which is due to the insertion of curcumin and the interaction between curcumin and phospholipid molecules, causing changes in the molecular arrangement of the phospholipid layer.

[0061] The peak of the phase transition temperature (Tpeak) indicates the fluidity of the phospholipid layer (Zhang, Gu et al., 2022). As the temperature increases, the phospholipid layer changes from solid to liquid, and the fluidity increases (Demetzos, 2008). The Tpeak of Cu-BLs (7.68°C) is significantly lower than that of BLs (16.07°C). This indicates that the Cu-BL phospholipid layer has better fluidity in practical applications, which is beneficial for the release of encapsulated active substances.

[0062] As a non-crystalline substance, lecithin has a wide phase transition temperature range rather than a specific phase transition temperature. The phase transition temperature range of Ch-BLs (-2.66 to 23.14 °C) is narrower than that of BLs (-20.78 to 29.82 °C) because the interaction between cholesterol and phospholipid bilayers eliminates the pre-transition phase of the latter (Matsingou & Demetzos, 2007). The phase transition temperature range of Cu-BLs (-16.97 to 26.84 °C) is also narrower than that of BLs. Curcumin contains some chemical groups similar to cholesterol (such as carbonyl); therefore, the interaction of curcumin with phospholipids can be similar to that of cholesterol. So, the changes in temperature range and enthalpy value indicate that curcumin has changed the structure of the phospholipid bilayer in Cu-BLs.

[0063] Similarly, Cu-NLs loaded with Nisin also have a wide phase transition temperature range (-14.03 to 27.01 °C) and a low Tpeak(7.50 °C). Therefore, the phospholipid layer of Cu-NL also has good fluidity. The DSC results show that the addition of curcumin changes the structure of the phospholipid bilayer, and Cu-NL based on the Cu-BL system is also ideal.

[0064] 4. FT-IR analysis of nanoliposomes

[0065] To confirm the presence of curcumin and Nisin in Cu-BL and Cu-NL, respectively, and to study the interaction between curcumin and Nisin, FT-IR spectroscopy was performed. The FT-IR spectra of egg yolk lecithin, curcumin, Cu-BL, Nisin, and Cu-NL are shown in Figure 3 The spectrum of egg yolk lecithin contains six characteristic peaks ( Figure 3 a): olefinic =C-H stretch of unsaturated acyl chains (3010 cm -1 ), antisymmetric (2924 cm 1 ) and symmetric stretch (2855 cm -1 ) of ester chains -CH2-, symmetric and asymmetric stretch of C=O on ester chains (1735 and 1238 cm -1 ), and symmetric stretch of P=O double bond of phosphoric acid (1091 cm -1 ; Li et al., 2023). These six peaks are also evident in the Cu-BL spectrum, and these common absorption peaks indicate that egg yolk lecithin is the main component of Cu-BL.

[0066] The peak at 3420 cm -1 in egg yolk lecithin and the peak at 3504 cm -1(Chen, Zou, Niu, Liu, Peng, & Liu, 2015) are caused by -OH stretching, and the superposition of these two peaks makes the corresponding absorption peak of Cu-BLs shift to 3450 cm -1 . The absorption peak of Cu-BLs at 1,633 cm-1 is formed by the superposition of the stretching vibration of the unsaturated acyl chain C=C double bond (1,643 cm -1 ) of egg yolk lecithin and the benzene ring C=C double bond (1,622 cm -1 ; Chen et al., 2015) of curcumin. In addition, the three characteristic peaks of curcumin--the stretching vibration of the benzene ring (1,595 cm -1 ; Chen et al., 2015), the mixed vibration of the C=O double bond of the carbonyl group and the C=C double bond (1,508 cm -1 ), and the C-O stretching vibration of the benzene ring (1,276 cm -1 ; Hu et al., 2021) are also observed in the spectrum of Cu-BLs. These absorption peaks confirm that curcumin is inserted into the egg yolk lecithin to form Cu-BLs.

[0067] Figure 3 b shows a broad peak at 3,439 cm -1 , which corresponds to the N-H stretching vibration of the free amino group of Nisin (Lopes, Barreto Pinilla, & Brandelli, 2019). The strong and broad absorption peak near 3420 cm-1 in the spectrum of Cu-BLs is attributed to -OH stretching. In the spectrum of Cu-NLs, these absorption peaks are mixed and red-shifted to 3,410 cm -1 ; Li, Li, Huang, Luo, & Mei, 2021; Li et al., 2023) due to the formation of hydrogen bonds between the amino group (-NH2) and the hydroxyl group (-OH). This confirms that Nisin is incorporated into the Cu-BL system to form Cu-NLs.

[0068] Nisin has two characteristic absorption peaks, from the stretching of the C=O group of amide I (1,651 cm -1 ) and the deformation of N-H (1,541 cm -1 ) and the vibration of the N-H group of amide II (Khan, Salmieri, Fraschini, Bouchard, Riedl, & Lacroix, 2014). In Cu-NLs, the C=O stretching peak of the amide I band of Nisin is red-shifted to 1,631 cm -1 , and the benzene ring stretching vibration of curcumin (1,595 cm -1) and N-H vibration of Amide II band of Nisin (1,541 cm -1 ) were also slightly red-shifted and weakened. These changes in absorption peak position and intensity indicated the presence of hydrophobic interactions between Nisin and curcumin (Chao Song et al., 2023).

[0069] 5. XRD analysis of nanoliposomes

[0070] The XRD diffractograms of egg lecithin, curcumin, Cu-BL, Nisin and Cu-NLs were compared (Fig. Figure 4 ) to verify the insertion of curcumin into Cu-BL and the loading of Nisin bacteriocin into Cu-NLs. As shown in Fig. Figure 4 a, the XRD diffractogram of egg lecithin had a broad peak at 2q 10-30°, indicating its non-crystalline nature. In contrast, the XRD diffractogram of curcumin contained a series of sharp peaks in the range of 2q = 10-30°, indicating its crystalline nature (Huang et al., 2017). In the XRD diffractogram of Cu-BLs, the broad peak corresponding to egg lecithin decreased and the sharp peaks of curcumin became significantly weaker, indicating that curcumin was inserted into the phospholipid layer and changed the crystalline structure of the phospholipid. The results of XRD and DSC combined together strongly indicated that the addition of curcumin changed the structure of the phospholipid layer.

[0071] As shown in Fig. Figure 4 b, the XRD spectrum of Nisin contained some sharp characteristic peaks, indicating its crystalline nature. Notably, the two main characteristic peaks of Nisin (2q = 31.67° and 45.41°) could still be observed in the XRD pattern of Cu-NL, but these peaks became significantly weaker, indicating that the crystallinity of Nisin was reduced in Cu-NL.

[0072] 7. Simulation of curcumin binding with Nisin

[0073] Figure 5 The best simulation molecular docking results of curcumin-Nisin system are shown. The three-dimensional view ( Figure 5 a) shows that curcumin bends and folds into the Nisin binding pocket. Curcumin interacts with 20 amino acid residues, including THR-36, ANS-37, GLU-115, LYS-118 and GLY-119. Among them, the two-dimensional view ( Figure 5b) The three amino acid residues (GLU-115, LYS-118, GLY-119) form regular hydrogen bonds (dark green) with curcumin, with lengths of 2.08, 2.98, and 2.68 Å, respectively. THR-36, ANS-37, and ALA-123 form irregular hydrogen bonds (π-donor hydrogen bonds, light green) with curcumin, with lengths of 4.08, 3.63, and 3.59 Å, respectively. LYS-122, ALA-123, and LEU-225 form hydrophobic interactions (π-alkyl, pink) with curcumin, which is consistent with the FTIR observation. It is worth noting that ALA-123 is involved in both hydrogen bonding and hydrophobic interactions between curcumin and Nisin. In addition, 12 amino acid residues, including TYR-35, PHE-46, and GLU-75, are involved in the binding of whey protein to curcumin through van der Waals forces (green).

[0074] The hydrogen bonds, hydrophobic interactions, and van der Waals forces between curcumin and Nisin were calculated and simulated using AutoDock Vina 4.2. This is similar to the results obtained by Racz et al. (2023) in AutoDock Vina 4.2 simulations, which revealed the presence of hydrogen bonds, hydrophobic interactions, and π-π stacking between curcumin and β-lactoglobulin. Liu et al. studied the interactions between curcumin and ovalbumin using AutoDock and found hydrogen bonds and van der Waals forces (Liu, Cai, Ying, Fu, Xiong, & Le, 2018).

[0075] It has been reported that the binding between curcumin and proteins mainly occurs through hydrophobic interactions, with hydrogen bonds and van der Waals forces involved in some cases (Tang, 2020). Zhang et al. (2020) confirmed the hydrogen bonds and hydrophobic interactions between curcumin and myosin through circular dichroism and molecular dynamics simulations. Rajabi et al. confirmed the presence of hydrogen bonds and van der Waals forces between curcumin and bovine trypsin using thermal denaturation and molecular docking (Rajabi, Farhadian, Shareghi, Asgharzadeh, & Momeni, 2019). Therefore, the interactions between curcumin and Nisin revealed in this study are similar to the above reports.

[0076] It is worth noting that the hydrogen bonding and hydrophobic interactions between Nisin and curcumin were also confirmed by FT-IR. These two interaction modes, possibly with additional van der Waals forces, were considered to be responsible for the aggregation of Nisin to the phospholipid layer and the high encapsulation efficiency of Nisin-curcumin nanoliposomes. In addition, the simulation results showed that the energy of Nisin binding with curcumin was -6.7 kcal. This is similar to previous reports on the binding of curcumin with other proteins (Racz et al., 2023), with a binding energy of less than 0 kmol / mol - 1 , indicating that the interaction between nisin and curcumin is spontaneously generated (Guo, Gan, Cheng, Cui, & Yi, 2023).

[0077] 8. Antimicrobial activity of Nisin nanoliposomes in skim and whole milk

[0078] Staphylococcus aureus is a common foodborne pathogen. It produces toxins (Lin et al., 2023) that cause food spoilage and human food poisoning, and is a common Gram-positive contaminant in egg and dairy products (Zhang, Wang, et al., 2022). Nisin is a safe natural preservative with strong inhibitory effect on many Gram-positive bacteria, including those that cause food spoilage and pathogenic bacteria (Kirazli & Tunca, 2022). However, Nisin often interacts with food ingredients such as fat, thereby reducing its antimicrobial activity (Chollet et al., 2008). To verify the protective effect of Cu-NLs on the bacterial inhibitory activity of Nisin, the inhibitory effect of Nisin and Cu-NLs on Staphylococcus aureus in whole milk and skim milk was evaluated.

[0079] Figure 6 The growth curve of Staphylococcus aureus in milk at 4°C. In the absence of Nisin, the number of colonies in the Cu-BL treatment group was less than that in the negative control group (sterile water) due to the inhibitory effect of curcumin (Ma, Moser, Han, Leonhard, Schneider-Stickler, & Tan, 2020). However, the growth trend of Staphylococcus aureus in the sterile water and Cu-BL groups was the same, both reaching a peak on the 3rd day, and the growth of Staphylococcus aureus was not affected Figure 6 a) After 7 d, the growth of Staphylococcus aureus was inhibited after the addition of Nisin or Cu-NLs in milk, and the number of bacteria continued to decrease. On the 3rd day, the number of Staphylococcus aureus colonies in the Nisin group in whole milk rebounded, and continued to decrease in skim milk Figure 6b), this is mainly because Nisin is adsorbed by fat, making it unable to inhibit the growth of S. aureus. Most importantly, in whole milk, the total number of S. aureus in the Cu-NL group did not rebound, and the growth of bacteria was always inhibited, which indicated that Cu-NL protected Nisin in fat-containing milk.

[0080] The above embodiments of the present application are merely examples for clearly illustrating the technical solutions of the present application, and are not intended to limit the specific embodiments of the present application. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Nisin nanoliposomes, characterized in that, The egg yolk lecithin, stabilizer and solvent are mixed and dissolved first, and then the solvent is removed to obtain a mixture; then Nisin is dissolved in a buffer solution, and then added into the above mixture to prepare Nisin nanoliposomes by a conventional film hydration method; the stabilizer is curcumin; the mass ratio of the egg yolk lecithin and the stabilizer is 10-20:1; wherein the Nisin is dissolved in a PBS buffer solution to obtain a Nisin solution with a concentration of 0.5-1 mg / mL; the pH value of the PBS buffer solution is 6-7; and the mass ratio of Nisin to the stabilizer is 1:0.01-2.

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