Polypeptide-modified phenolic essential oil nanoliposomes and a preparation method thereof

Abstract

This invention proposes a polypeptide-modified phenolic essential oil nanoliposome and its preparation method. The method includes: (1) sequentially subjecting soybean lecithin, cholesterol, and oregano essential oil to rotary evaporation, thin-film hydration, and ultrasonic dispersion to obtain an oregano essential oil nanoliposome suspension; (2) sequentially mixing and stirring the oregano essential oil nanoliposome suspension with polylysine to obtain the polypeptide-modified phenolic essential oil nanoliposome. This invention uses a specific mass ratio of soybean lecithin, cholesterol, and oregano essential oil, and a specific mass of polylysine, to self-assemble and form nanoliposomes that slowly release antibacterial and antioxidant active substances. These nanoliposomes can effectively inhibit the growth and reproduction of Escherichia coli and Staphylococcus aureus. At the same time, the main substrates of the nanoliposomes are all edible materials, which can be used for the functional delivery of active substances and the storage and preservation of fresh food, meeting people's pursuit of safe, nutritious, and healthy diets and the concept of green environmental protection.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a polypeptide-modified phenolic essential oil nanoliposome and its preparation method. Background Technology

[0002] Plant essential oils are fat-soluble natural compounds extracted from aromatic plants. They are volatile and rich in aroma, typically described as concentrated hydrophobic liquids containing volatile aromatic compounds. They are natural extracts composed of various monoterpenes, monoterpenes, sesquiterpenes, and other volatile compounds (esters, ketones, aromatic phenols, alcohols, aldehydes, ethers, hydrocarbons, coumarins, and organic acids). Oregano essential oil (OEO) is a pale yellow liquid extracted from oregano. It is a highly potent natural antibacterial agent, exhibiting some antibacterial activity against both Gram-positive and Gram-negative bacteria, making it one of the most broad-spectrum antibacterial pharmaceutical additives. Due to its high efficiency, non-toxicity, and non-irritation, it can effectively kill a variety of bacteria and fungi, and has wide applications in the pharmaceutical, chemical, and food industries. Furthermore, OEO also possesses antioxidant activity; its main compounds, cymene and γ-terpinene, can enhance its bio-antioxidant activity by inhibiting reactive oxygen species-producing enzymes (such as lipoxygenase and xanthine oxidase). The antibacterial and antioxidant activities of ozone (OEO) can be attributed to its high content of phenolic compounds, primarily carvacrol and thymol from the essential oils. However, OEO suffers from high volatility, poor water solubility, and sensitivity to light, oxygen, and temperature, which significantly limits its application as a food preservative and results in low bioavailability. Therefore, fully utilizing the advantages of OEO's excellent antioxidant and antibacterial activities to develop it into a stable and safe food preservative is of great significance.

[0003] Poly-L-lysine (PL) is a polypeptide derived from microbial metabolites that exhibits inhibitory effects against both bacteria and fungi. This inhibitory effect is primarily dependent on its molecular weight. PL's antibacterial mechanism relies on electrostatic interactions with the surface of microorganisms. By interfering with the transmission of cellular substances, energy, and information, it disrupts the cell membrane structure of microorganisms. Lysine residues that penetrate the cell can stimulate the production of reactive oxygen species within the microorganism, ultimately achieving a bactericidal effect. Furthermore, this antibacterial activity is also attributed to PL's ability to induce oxidative stress, DNA damage, and disrupt intracellular processes. PL was approved as a food additive by the U.S. Food and Drug Administration (FDA) in 2003. It possesses advantages such as being edible, non-toxic, harmless, having good stability, and biocompatibility, and shows broad application prospects in food storage and preservation.

[0004] Nanoliposomes (NLs) are self-assembling phospholipid carrier systems formed by dispersing lipids with polar groups in a solution medium. They are widely used to encapsulate hydrophilic substances in aqueous compartments or to encapsulate lipophilic and amphiphilic substances in concentric phospholipid layers. Due to their advantages such as biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and targeted sustained release, NLs can be used to encapsulate antibacterial drugs for active targeting of bacterial flora, making them a promising alternative to encapsulation-based drug delivery technology. However, the phospholipid membrane of NLs can be disrupted at low pH and in the presence of enzymes, leading to leakage of the encapsulated active substances. Therefore, electrostatic interactions can be used to form electrostatic bridges to stabilize the surface of NLs.

[0005] Currently, there is a lack of extensive research on the use of peptide-modified NLs for the encapsulation and delivery of phenolic essential oils. In addition, there is limited research on peptides as surface modifiers for NLs, and it remains to be explored whether they can enhance the stability of NLs and whether they can exert synergistic antibacterial and antioxidant effects with phenolic essential oils. Summary of the Invention

[0006] This application is based on the inventor's discoveries and understanding of the following problems:

[0007] There is a lack of extensive research on the encapsulation and delivery of phenolic essential oils using peptide-modified NLs. This invention employs a thin-film hydration ultrasonic dispersion method to prepare phenolic essential oil nanoliposomes. First, the inventors optimized the preparation process using single-factor and orthogonal experimental methods, obtaining oregano essential oil nanoliposomes with an encapsulation efficiency (up to 78%) far exceeding that of existing thin-film hydration ultrasonic dispersion methods. Then, different concentrations of peptides were used to modify the surface of the phenolic essential oil nanoliposomes through electrostatic self-assembly. The adsorption rate, particle size distribution, pH, turbidity, structure, environmental stability, antioxidant activity, and antibacterial activity of the peptide-modified phenolic essential oil nanoliposomes were studied. The inventors unexpectedly discovered that polylysine can effectively modify nanoliposomes encapsulated with oregano essential oil, enhancing the stability of the oregano essential oil nanoliposomes and exhibiting synergistic antibacterial and antioxidant effects with phenolic essential oils. Unexpectedly, peptide-modified phenolic essential oil nanoliposomes with high encapsulation efficiency, uniform particle size, effective inhibition of bacterial growth and reproduction, and strong antioxidant properties were obtained.

[0008] Therefore, in a first aspect of the present invention, a method for preparing peptide-modified phenolic essential oil nanoliposomes is provided. According to an embodiment of the present invention, the method includes: (1) sequentially subjecting soybean lecithin, cholesterol, and oregano essential oil to rotary evaporation, thin-film hydration, and ultrasonic dispersion to obtain an oregano essential oil nanoliposome suspension; (2) sequentially mixing and stirring the oregano essential oil nanoliposome suspension with polylysine to obtain the peptide-modified phenolic essential oil nanoliposomes.

[0009] The inventors have optimized the method for preparing phenolic essential oil nanoliposomes. The polypeptide-modified phenolic essential oil nanoliposomes prepared according to the method of the present invention have a very high encapsulation rate of oregano essential oil. Furthermore, polylysine can adsorb onto the surface of the oregano essential oil nanoliposomes, forming a more stable complex, thereby further improving the encapsulation rate of the nanoliposomes. In addition, experimental verification shows that polylysine can exert synergistic antibacterial and antioxidant effects with oregano essential oil, thereby enhancing the antibacterial and antioxidant effects of the nanoliposomes. Since the components of the nanoliposomes are all edible materials, their application scenarios include medical, food, cosmetic, and skin care products, etc., and they have high application value.

[0010] According to embodiments of the present invention, the method for preparing the above-mentioned nanoliposomes may further include at least one of the following additional technical features:

[0011] According to an embodiment of the present invention, the mass ratio of soybean lecithin, cholesterol, and oregano essential oil is 10:(1-2):(4-8). This allows for the production of oregano essential oil nanoliposomes with an encapsulation rate significantly higher than that of phenolic essential oil nanoliposomes in the prior art.

[0012] According to an embodiment of the present invention, the mass ratio of soybean lecithin, cholesterol, and oregano oil is 10:(1.25-1.75):(5-7). This allows for the acquisition of phenolic essential oil nanoliposomes with further improved oregano oil encapsulation efficiency.

[0013] According to an embodiment of the present invention, the mass ratio of soybean lecithin, cholesterol and oregano oil is 10:1.5:5.

[0014] According to an embodiment of the present invention, prior to the rotary evaporation, the soybean lecithin, cholesterol, and oregano oil are pre-dissolved in anhydrous ethanol.

[0015] According to an embodiment of the present invention, the concentration of soybean lecithin is 10 mg / mL, and / or the concentration of cholesterol is 1.00–2.00 mg / mL, and / or the concentration of oregano essential oil is 4–8 mg / mL.

[0016] In some specific embodiments of the present invention, before rotary evaporation, 200 mg of soybean lecithin and 20-40 mg of cholesterol solid are weighed into a 50 mL round-bottom flask, and 80-160 mg of oregano essential oil is weighed and pre-dissolved in 20 mL of anhydrous ethanol. The concentration of oregano essential oil is 4-8 mg / mL. The oregano essential oil dissolved in anhydrous ethanol is transferred to the above-mentioned 50 mL round-bottom flask, and after ultrasonic dispersion together with soybean lecithin and cholesterol, rotary evaporation is performed.

[0017] According to embodiments of the present invention, the rotary evaporation temperature is 35°C to 45°C, and in some specific embodiments, the rotary evaporation temperature is preferably 40°C. The rotary evaporation evaporates the solvent using a rotary evaporator, leaving a dry lipid film.

[0018] According to an embodiment of the present invention, the film hydration is carried out in a surfactant and a PBS buffer solution with a pH of 6.4–8.0. During the preparation of nanoliposomes, soybean lecithin and cholesterol can form a lipid bilayer structure, which is the basic framework of the liposome. PBS (phosphate buffer) allows soybean lecithin and cholesterol to form a homogeneous lipid solution and maintains the pH of the solution within a relatively stable range. The charge state and hydration of the lipids are conducive to liposome formation. The pH range of PBS must also meet the requirements of being similar to the in vivo environment, which helps to improve the biocompatibility of liposomes and reduce potential damage to biological tissues. At a suitable pH, the hydrolysis or oxidation of lipid components can also be reduced, which is crucial for the formation, stability, and functionality of liposomes.

[0019] According to an embodiment of the present invention, the mass-to-volume ratio of the surfactant and the PBS buffer solution with a pH of 6.4 to 8.0 is 2 mg: 20 mL.

[0020] According to an embodiment of the present invention, the surfactant comprises polyvinylpyrrolidone.

[0021] According to an embodiment of the present invention, after ultrasonic dispersion and before step (2), the product after ultrasonic dispersion is subjected to pulverization and centrifugation in sequence.

[0022] According to an embodiment of the present invention, the pulverization process is carried out for 15 minutes at a power of 350W, a pulverization time of 10s, and a 5s interval.

[0023] According to an embodiment of the present invention, the centrifugation process is performed by centrifuging at 4000g for 5 minutes.

[0024] According to an embodiment of the present invention, the polylysine is pre-dissolved in a PBS buffer solution with a pH of 6.4 to 8.0, preferably in a PBS buffer solution with a pH of 7.0.

[0025] Polylysine is a cationic polypeptide. Due to the positive charge on its molecules, it can modify the surface of nanoliposomes through electrostatic adsorption, forming a stable complex. Furthermore, the inventors discovered that this modification can enhance the encapsulation effect of nanoliposomes on oregano oil, and synergistically enhance the antibacterial and antioxidant effects of oregano oil.

[0026] According to an embodiment of the present invention, the concentration of polylysine is 0-5 mg / mL.

[0027] According to an embodiment of the present invention, the concentration of polylysine is 3-5 mg / mL.

[0028] According to an embodiment of the present invention, the polylysine is [C6H] 12 N2O] n , where n is 25 to 35.

[0029] According to an embodiment of the present invention, the volume ratio of the oregano essential oil nanoliposome suspension to polylysine is 0.8 to 1.2.

[0030] According to an embodiment of the present invention, the stirring time is 30 to 50 minutes.

[0031] According to an embodiment of the present invention, the stirring time is 40 minutes.

[0032] In a second aspect, the present invention provides a polypeptide-modified phenolic essential oil nanoliposome. According to an embodiment of the present invention, the nanoliposome is prepared using the method described in the first aspect. The polypeptide-modified phenolic essential oil nanoliposome prepared by the method of the embodiments of the present invention has a very high encapsulation rate of oregano essential oil, and polylysine can adsorb onto the surface of the oregano essential oil nanoliposome, forming a more stable complex, thereby further improving the encapsulation rate of the liposome. Furthermore, experimental verification shows that polylysine can exert synergistic antibacterial and antioxidant effects with oregano essential oil, thus enhancing the antibacterial and antioxidant effects of the nanoliposome. Since the components of this nanoliposome are all edible materials, its application scenarios include medical, food, cosmetic, and skincare fields, demonstrating high application value.

[0033] According to embodiments of the present invention, the above-mentioned polypeptide-modified phenolic essential oil nanoliposomes may further include at least one of the following additional technical features:

[0034] According to an embodiment of the present invention, oregano essential oil is encapsulated in the nanoliposomes, and polylysine is modified on the surface of the nanoliposomes.

[0035] According to an embodiment of the present invention, the particle size of the nanoliposomes is 350-500 nm, preferably 420-500 nm.

[0036] According to an embodiment of the present invention, the PDI of the liposomes is 0.160 to 0.210.

[0037] According to an embodiment of the present invention, the zeta potential of the liposome is -15 to 15 mV, preferably 7 to 12 mV.

[0038] In a third aspect, the present invention provides a medicine, preservative, cosmetic, or skincare product. According to an embodiment of the invention, the medicine, preservative, cosmetic, or skincare product comprises peptide-modified phenolic essential oil nanoliposomes prepared using the method described in the first aspect or peptide-modified phenolic essential oil nanoliposomes described in the second aspect. As mentioned above, the peptide-modified phenolic essential oil nanoliposomes prepared using the method described in the first aspect or the peptide-modified phenolic essential oil nanoliposomes described in the second aspect have a very high encapsulation rate of oregano essential oil. Furthermore, polylysine can adsorb onto the surface of the oregano essential oil nanoliposomes, forming a more stable complex, thereby further improving the encapsulation rate of the liposomes. In addition, experiments have verified that polylysine can synergistically exert antibacterial and antioxidant effects with oregano essential oil, enhancing the antibacterial and antioxidant effects of the nanoliposomes. Since the components of these nanoliposomes are all edible materials, their applications include medical, food, cosmetic, and skincare products, demonstrating high application value. Drugs, preservatives, cosmetics, and skincare products containing the peptide-modified phenolic essential oil nanoliposomes can effectively inhibit bacterial growth and proliferation, such as Escherichia coli and Staphylococcus aureus. Furthermore, they can effectively release antioxidant effects.

[0039] In a fourth aspect, the present invention proposes the use of polypeptide-modified phenolic essential oil nanoliposomes prepared by the method described in the first aspect or the polypeptide-modified phenolic essential oil nanoliposomes described in the second aspect in the preparation of a medicament for the prevention, relief, and / or treatment of diseases caused by pathogenic bacteria. Medicinal products containing the polypeptide-modified phenolic essential oil nanoliposomes can effectively inhibit bacterial growth and proliferation, such as Escherichia coli and Staphylococcus aureus, and also have an effective sustained-release antioxidant effect.

[0040] According to an embodiment of the present invention, the pathogenic bacteria include at least one of Escherichia coli and Staphylococcus aureus.

[0041] Furthermore, polylysine-modified nanoliposomes can achieve active targeting through further chemical or biomolecular modifications. For example, targeting specific cells or tissues can be achieved by binding polylysine to specific ligands (such as folic acid, antibodies, or peptides). This strategy is particularly promising in cancer treatment because it can increase drug concentration in tumor tissues while reducing damage to normal tissues.

[0042] In a fifth aspect, the present invention proposes the use of polypeptide-modified phenolic essential oil nanoliposomes prepared by the method described in the first aspect or the polypeptide-modified phenolic essential oil nanoliposomes described in the second aspect in the preparation of a preservative for fresh food, wherein the preservative is used to inhibit bacteria. As mentioned above, since the components of the nanoliposomes are all edible materials, they can be used in the food industry. The preservative containing the polypeptide-modified phenolic essential oil nanoliposomes can effectively inhibit the growth and proliferation of bacteria in fresh food, and can be used for the storage and preservation of fresh food, such as fruits, vegetables, and meat. Thus, it can replace preservatives and can also effectively provide sustained-release antioxidant effects, meeting people's pursuit of safe, nutritious, and healthy diets, and conforming to the concept of green environmental protection.

[0043] According to an embodiment of the present invention, the bacteria include at least one of Escherichia coli and Staphylococcus aureus.

[0044] In a sixth aspect, the present invention provides a method for preserving fresh food. According to an embodiment of the invention, the method includes applying to the fresh food a polypeptide-modified phenolic essential oil nanoliposome prepared using the method described in the first aspect or the polypeptide-modified phenolic essential oil nanoliposome described in the second aspect. The method according to the embodiments of the present invention can effectively preserve and prevent spoilage of fresh food, meeting people's pursuit of safe, nutritious, and healthy diets, while also conforming to the concept of green environmental protection.

[0045] In a seventh aspect of the invention, a method for in vitro inhibition of pathogenic bacteria is provided. According to an embodiment of the invention, the method is for non-therapeutic purposes and includes contacting tissues and / or cells with polypeptide-modified phenolic essential oil nanoliposomes prepared using the method described in the first aspect or the polypeptide-modified phenolic essential oil nanoliposomes described in the second aspect. As previously mentioned, the polypeptide-modified phenolic essential oil nanoliposomes can effectively inhibit the growth of bacteria, such as *Escherichia coli* and *Staphylococcus aureus*, and therefore can exert an antibacterial effect in tissues and / or cells, and can be used for scientific research.

[0046] According to an embodiment of the present invention, the pathogenic bacteria include at least one of Escherichia coli and Staphylococcus aureus.

[0047] Compared with the prior art, the present invention has at least the following beneficial effects:

[0048] (1) The method for preparing polypeptide-modified phenolic essential oil nanoliposomes of the present invention uses soybean lecithin, cholesterol and oregano essential oil in a specific mass ratio, and polylysine at a specific concentration. The charged self-assembly of polypeptides and phospholipids is used to form nanoliposomes. The raw materials are easy to obtain and the preparation process is simple and efficient.

[0049] (2) The encapsulation efficiency of oregano essential oil in the polypeptide-modified phenolic essential oil nanoliposomes is much higher than that of the existing thin-film hydration ultrasonic dispersion method, reaching up to 78%. Furthermore, it can effectively inhibit bacterial growth and reproduction, such as Escherichia coli and Staphylococcus aureus, and can facilitate the slow release of antioxidant active substances. Simultaneously, since the main substrates of the nanoliposomes are edible materials, they can be used for the functional delivery of active substances, such as treating diseases caused by pathogenic bacteria or for antioxidant purposes, as well as for the storage and preservation of fresh food, meeting people's pursuit of safe, nutritious, and healthy diets, while also conforming to the concept of green environmental protection.

[0050] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0051] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 This is a graph showing the effect of OEO concentration, cholesterol concentration, and PBS buffer solution pH on the encapsulation efficiency of OEO-NLs in Example 1 of the present invention.

[0053] Figure 2 The figures show the detection results of the appearance (Figure A), PL adsorption rate (Figure B), pH value and turbidity (Figure C) of PL / OEO-NLs prepared with different concentrations of PL according to Example 1 of the present invention.

[0054] Figure 3 The infrared spectra of OEO, PL, empty NLs (without OEO and PL) and PL / OEO-NLs at different concentrations are shown in Example 1 of the present invention.

[0055] Figure 4 The figure shows the storage stability test results of OEO-NLs and PL / OEO-NLs prepared with different concentrations of PL in Example 1 of the present invention at 25°C.

[0056] Figure 5 The graph shows the DPPH free radical scavenging rate detection results of PL / OEO-NLs prepared with different concentrations of PL according to Example 1 of the present invention;

[0057] Figure 6The figure shows the killing effect of PL / OEO-NLs prepared with different concentrations of PL according to Example 1 of the present invention on E. coli O157:H7 and S. aureus. Detailed Implementation

[0058] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0059] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "multiple" or "a plurality of" means at least two, two types, such as two, two, three, three, etc., unless otherwise explicitly specified.

[0060] In this document, the terms “comprising,” “having,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0061] In this document, the term “optionally” generally means that an event or condition described below may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0062] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0063] In this application, thin-film hydration and ultrasonic dispersion is a commonly used technique for preparing liposomes. It combines the methods of thin-film hydration and ultrasonic dispersion. The advantages of thin-film hydration and ultrasonic dispersion are its simplicity and low cost. However, it also has some limitations, such as lower encapsulation efficiency compared to other methods, and the possibility that ultrasonic treatment may damage certain sensitive biomolecules. Therefore, when using this method, it is necessary to optimize the conditions according to the intended use of the liposomes and the characteristics of the encapsulated substance. For example, factors such as the concentration of the lipid solution, the characteristics of the encapsulating substance, the ionic strength of PBS, temperature, and pH value can all affect the formation and properties of liposomes.

[0064] In this application, Dynamic Light Scattering (DLS), also known as Photon Correlation Spectroscopy (PCS) or Quasi-elastic Light Scattering (QELS), is a physical characterization technique used to measure the particle size distribution in a solution or suspension. It obtains information by measuring the changes in light intensity over time, which are caused by the Brownian motion of tiny particles in the solution.

[0065] In this application, electrophoretic light scattering (ELS) is a technique for determining the zeta potential of particles. The zeta potential is an important parameter characterizing the stability of particles in fluids, reflecting the nature of the particle surface charge. ELS indirectly determines the zeta potential by measuring the electrophoretic mobility of particles under an applied electric field. Combining ELS with dynamic light scattering technology allows for the simultaneous measurement of particle size and zeta potential in the same experiment, providing more comprehensive information for particle characterization. This combined technique enables a more accurate assessment of particle stability and interactions.

[0066] The present invention will now be described with reference to specific embodiments. It should be noted that conventional methods, devices, reagents and materials used in the embodiments will not be described in detail here. These embodiments are merely descriptive and do not limit the present invention in any way.

[0067] Example 1: Preparation of Oregano Oil Nanoliposomes (OEO-NLs)

[0068] This embodiment provides a method for preparing peptide-modified phenolic essential oil nanoliposomes, which mainly includes the following steps:

[0069] 1. Single-factor experiment on the preparation of oregano essential oil nanoliposomes

[0070] This experiment was used to preliminarily screen important parameters and conditions in the method for preparing oregano essential oil nanoliposomes. The procedure is as follows:

[0071] First, single-factor experiments were conducted on the OEO-NLs preparation process: The effects of OEO concentration (4, 5, 6, 7, 8 mg / mL), cholesterol concentration (1.00, 1.25, 1.50, 1.75, 2.00 mg / mL), and pH value of the PBS buffer solution (6.4, 6.8, 7.2, 7.6, 8.0) on the encapsulation efficiency of OEO-NLs were investigated.

[0072] OEO-NLs were prepared using a thin-film hydration ultrasonic dispersion method: 200 mg of soybean lecithin (purchased from Beijing Solarbio Biotechnology Co., Ltd.) and 20 mg, 25 mg, 30 mg, 35 mg and 40 mg of cholesterol (purchased from Shanghai Maclean Biotechnology Co., Ltd.) were weighed and added to 50 mL round-bottom flasks respectively. Then, 80 mg, 100 mg, 120 mg, 140 mg and 160 mg of OEO (purchased from Shanghai Maclean Biotechnology Co., Ltd.) dissolved in 20 mL of anhydrous ethanol (purchased from Jingchun Reagent) were transferred to the round-bottom flasks containing soybean lecithin and cholesterol. The mixture was fully dispersed under ultrasonic conditions in a water bath. Then, the organic solvent was evaporated under reduced pressure in a rotary evaporator at 40 °C until a smooth lipid film appeared on the inner wall of the round-bottom flask. Then, 2 mg of polyvinylpyrrolidone (purchased from Beijing Solarbio Biotechnology Co., Ltd.) surfactant and 20 mL of PBS buffer (pH 6.4, 6.8, 7.2, 7.6 and 8.0) were added to the round-bottom flask and dispersed thoroughly under ultrasonic conditions in a water bath. The resulting mixture was then poured into a centrifuge tube and pulverized for 15 min in an ultrasonic cell disruptor (10 s working time / 5 s interval, 350 W power). The resulting product was then centrifuged at 4000 g for 5 min to remove the upper free oregano oil and the lower impurities, thus obtaining the OEO-NLs suspension. The OEO concentration, cholesterol concentration and pH value of the PBS buffer solution were set as shown in Table 1.

[0073] Table 1: Factors and levels in the single-factor experiment for NLs preparation

[0074]

[0075] The encapsulation efficiency of the oregano essential oil nanoliposomes prepared in the above groups was detected. The specific detection steps are as follows:

[0076] (1) Preparation of OEO standard curve: To determine the optimal wavelength for OEO measurement, a UV spectrophotometer was used with anhydrous ethanol as a blank to scan 0.5 mg / mL OEO ethanol solution in the wavelength range of 200 nm to 600 nm. OEO ethanol solutions with concentrations of 0.075, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, and 0.22 mg / mL were prepared, and absorbance A was measured at the optimal measurement wavelength with ethanol as a blank. A standard curve of A versus concentration C was fitted using data processing software. Using anhydrous ethanol as a blank control, the optimal measurement wavelength for OEO was observed to be 276 nm. After measuring the absorbance A of a series of OEO ethanol solutions of different concentrations at 276 nm, a linear regression was performed on absorbance A versus OEO concentration C, yielding the standard equation: A = 8.31104C + 0.03254, with a correlation coefficient R. 2=0.99833, and the linear relationship is good in the concentration range of 0.075 mg / mL to 0.22 mg / mL.

[0077] (2) Determination of OEO-NLs encapsulation efficiency: First, take 2 mL of the 15 OEO-NLs prepared by the above method into a 2 mL centrifuge tube and centrifuge at 12000 rpm for 15 min. Then, remove the supernatant, add anhydrous ethanol to the precipitate in the centrifuge tube to 2 mL, sonicate in a 40℃ water bath for 10 min, vortex mix for 2 min, and make up to 100 mL with anhydrous ethanol. Take 2 mL of the diluted solution into a 2 mL centrifuge tube, centrifuge at 12000 rpm for 15 min, let stand, take the supernatant, and measure the absorbance of the supernatant at 276 nm using a UV spectrophotometer. Calculate the concentration of free OEO (C0) using the OEO standard curve regression equation. NE ), calculate the encapsulation ratio according to formula (1):

[0078]

[0079] In equation (1): C N Encapsulation efficiency (%) of OEO-NLs; C NE Content of OEO encapsulated by OEO-NLs (mg); C NT Add the total OEO content (mg) to OEO-NLs.

[0080] The encapsulation efficiency results of the single-factor experiment are as follows Figure 1 As shown, OEO-NLs obtained with OEO contents of 5 mg / mL, 6 mg / mL, and 7 mg / mL, cholesterol contents of 1.25 mg / mL, 1.50 mg / mL, and 1.75 mg / mL, and PBS buffer solutions with pH values ​​of 6.8, 7.0, and 7.2 all exhibited good encapsulation rates, exceeding 60%.

[0081] 2. Orthogonal Experiment for the Preparation of Oregano Essential Oil Nanoliposomes (OEO-NLs)

[0082] This experiment was conducted to further screen important parameters and conditions in the preparation of oregano essential oil nanoliposomes based on Experiment 1. The procedure is as follows:

[0083] Based on the single-factor results, orthogonal experiments were conducted to determine the preparation conditions of OEO-NLs using OEO concentrations of 5 mg / mL, 6 mg / mL, and 7 mg / mL, cholesterol concentrations of 1.25 mg / mL, 1.50 mg / mL, and 1.75 mg / mL, and the pH values ​​of the PBS buffer solution of 6.8, 7.0, and 7.2. The final evaluation was based on the encapsulation efficiency. The detection steps for the encapsulation efficiency are described in Experiment 1. The experimental groups are shown in Table 2.

[0084] Table 2 shows that the experimental group with the highest encapsulation efficiency was group 2 (OEO concentration 5.0 mg / mL, cholesterol concentration 1.50 mg / mL, PBS buffer solution pH 7.0), followed by group 5 (OEO concentration 6.0 mg / mL, cholesterol concentration 1.50 mg / mL, PBS buffer solution pH 6.8), with encapsulation efficiencies of 78.26% and 73.63%, respectively. Considering all factors, experimental conditions 2 were selected as the optimal preparation process. Range analysis using encapsulation efficiency as the evaluation index showed that the order of factors affecting the encapsulation efficiency of NLs was: OEO concentration (R = 7.71) > cholesterol concentration (R = 5.03) > PBS buffer solution pH (R = 3.65).

[0085] Table 2: Results of orthogonal experiments for NLs preparation

[0086]

[0087]

[0088] Example 2: Preparation of peptide-modified phenolic essential oil nanoliposomes (PL / OEO-NLs)

[0089] This embodiment modifies the OEO-NLs prepared in Example 1 with polylysine (PL) to prepare PL / OEO-NLs. The specific experimental procedures are as follows:

[0090] Based on the results of the orthogonal experiment in Example 1, OEO-NLs prepared under the optimal preparation conditions (OEO concentration 5.0 mg / mL, cholesterol concentration 1.50 mg / mL, PBS buffer solution pH 7.0) obtained from the orthogonal experiment in Table 2 were modified with PL (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.). Specifically, 20, 40, 60, 80, and 100 mg of PL were dissolved in 20 mL of PBS buffer solution at pH 7.0 to obtain PL solutions of different concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL). Equal volumes of the above-mentioned PL solutions of different concentrations were added separately to the suspensions containing OEO-NLs prepared under the optimal preparation conditions obtained in Table 2, and the mixture was stirred with a magnetic stirrer for 40 min to obtain the product with the appearance shown above. Figure 2 (A) shows the PL / OEO-NLs.

[0091] 1. Detection of adsorption rate, pH value and turbidity of peptide-modified phenolic essential oil nanoliposomes

[0092] The obtained peptide-modified phenolic essential oil nanoliposomes were subjected to adsorption rate, pH value, and turbidity measurements. The detection steps were as follows:

[0093] (1) PL adsorption rate: The PL adsorption rate was quantitatively analyzed using the BCA method with a micro-protein detection kit (purchased from ELISA). After centrifuging PL / OEO-NLs at 12000 r / min for 15 min, the content of free PL in the supernatant was measured. The formula for calculating the PL adsorption rate is as follows:

[0094]

[0095] In equation (2), A is the PL adsorption rate (%), C0 is the initial concentration of PL (mg / mL), and C1 is the concentration of free PL (mg / mL).

[0096] (2) pH value and turbidity: pH value of PL / OEO-NLs was measured using a pH meter; turbidity of PL / OEO-NLs was measured using a UV spectrophotometer with a wavelength of 600 nm and a cuvette thickness of 1 cm. PL / OEO-NLs were diluted 10 times with ultrapure water.

[0097] Figure 2 The results show that the PL adsorption rate is the highest in PL / OEO-NLs when the PL concentration is 3 mg / mL, the pH value of the system is between 6.58 and 6.89, the average particle size of PL / OEO-NLs gradually increases with the increase of PL concentration, and the turbidity increases accordingly.

[0098] 2. Detection of particle size, PDI and Zeta potential of peptide-modified phenolic essential oil nanoliposomes

[0099] OEO-NLs prepared under conditions of 5 mg / mL OEO, 1.50 mg / mL cholesterol, and PBS buffer solution pH 7.0, and PL solutions with concentrations of 1, 2, 3, 4, and 5 mg / mL, were used to prepare the aforementioned peptide-modified phenolic essential oil nanoliposomes. Particle size, PDI, and Zeta potential were measured. The detection steps were as follows: Before testing, OEO-NLs and each PL / OEO-NLs dispersion system were diluted 10 times with ultrapure water. The average particle size and PDI were determined using dynamic light scattering (DLS), and the Zeta potential was determined using electrophoretic light scattering (ESL). Samples were equilibrated in the instrument for 2 minutes before testing, and each sample was measured at least three times.

[0100] The results in Table 3 show the average particle size, PDI, and Zeta potential of PL / OEO-NLs prepared with different concentrations of reagents under the optimal preparation process conditions. It can be seen that the average particle size of PL / OEO-NLs is between 333.8 nm and 473.7 nm, and the system has good dispersibility. Positively charged PL was successfully adsorbed on the surface of OEO-NLs.

[0101] Table 3. Average particle size, PDI, and Zeta potential of PL / OEO-NLs at different concentrations

[0102] PL concentration (mg / mL) Average particle size / nm PDI Zeta potential / mV 0 <![CDATA[333.8±2.70 f ]]> <![CDATA[0.213±0.007 a ]]> <![CDATA[-43.10±4.20 d ]]> 1 <![CDATA[364.5±2.54 e ]]> <![CDATA[0.202±0.017 a ]]> <![CDATA[-12.93±1.08 c ]]> 2 <![CDATA[401.3±3.69 d ]]> <![CDATA[0.187±0.019 ab ]]> <![CDATA[6.27±0.13 b ]]> 3 <![CDATA[421.2±2.60 c ]]> <![CDATA[0.168±0.002 b ]]> <![CDATA[7.06±0.27 ab ]]> 4 <![CDATA[456.9±2.29 b ]]> <![CDATA[0.192±0.002 ab ]]> <![CDATA[9.92±0.92 ab ]]> 5 <![CDATA[473.7±2.35 a ]]> <![CDATA[0.204±0.011 a ]]> <![CDATA[11.25±0.69 a ]]>

[0103] Note: Different superscript letters in the same column indicate significant differences (P<0.05).

[0104] 3. Fourier transform infrared spectroscopy detection

[0105] Fourier transform infrared spectroscopy (FTIR) was used to detect the above-mentioned OEO, PL, empty NLs (without OEO and PL), OEO-NLs, and PL / OEO-NLs at different concentrations using a pellet method. The detection procedure was as follows: 200 mg of dry potassium bromide (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) and 1-2 mg of sample powder were mixed in an agate mortar and ground evenly. The mixture was then pressed into pellets under a pressure of 15-20 MPa. The samples were placed in a sample chamber, and the infrared absorption spectra of the samples were measured at wavelengths of 400-4000 cm⁻¹. -1 4cm resolution -1 Each image was scanned a total of 64 times.

[0106] Figure 3 This indicates that OEO was successfully embedded inside the nanoliposomes, and PL was successfully modified on the surface of the nanoliposomes. Furthermore, the OEO embedding and PL modification had little impact on the internal structure of PL / OEO-NLs.

[0107] 4. Stability testing of peptide-modified phenolic essential oil nanoliposomes

[0108] The stability of OEO-NLs prepared under the conditions of OEO concentration of 5 mg / mL, cholesterol concentration of 1.50 mg / mL, and PBS buffer solution pH of 7.0, and the above-mentioned peptide-modified phenolic essential oil nanoliposomes prepared with PL solutions of concentrations of 1, 2, 3, 4, and 5 mg / mL were tested. The test steps were as follows: OEO-NLs and PL / OEO-NLs samples were placed in a constant temperature and humidity chamber and stored at 25°C for 30 days. On days 0, 5, 10, 15, 20, 25, and 30, the average particle size, PDI, Zeta potential, and adsorption rate of OEO-NLs and PL / OEO-NLs were determined by the methods in Experiment 2 and Experiment 1 (1) above.

[0109] The results are as follows Figure 4 As shown, the physicochemical properties of PL / OEO-NLs remained relatively stable within 30 days.

[0110] 5. Detection of DPPH free radical scavenging rate of peptide-modified phenolic essential oil nanoliposomes

[0111] OEO-NLs prepared under conditions of OEO concentration of 5 mg / mL, cholesterol concentration of 1.50 mg / mL, and PBS buffer solution pH of 7.0, and phenolic essential oil nanoliposomes prepared with PL solutions of concentrations of 1, 2, 3, 4, and 5 mg / mL were subjected to DPPH free radical scavenging rate detection (purchased from Fuzhou Feijing Biotechnology Co., Ltd.). The detection steps were as follows: 0.1 mmol / L DPPH methanol solution (purchased from Jingchun Reagent) was mixed with the sample solution in equal volume and shaken well. The reaction was carried out at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm. Ultrapure water was used as a blank control. The DPPH free radical scavenging rate was calculated according to formula (3):

[0112]

[0113] In formula (3), A2 is the absorbance of an equal volume mixture of sample solution and DPPH solution; A1 is the absorbance of an equal volume mixture of sample solution and anhydrous methanol; and A0 is the absorbance of an equal volume mixture of ultrapure water and DPPH solution.

[0114] The results are as follows Figure 5 As shown, PL surface modification enhances the original antioxidant activity of OEO-NLs. The DPPH radical scavenging rate of OEO-NLs modified with 3 mg / mL PL increased from 70.47% to 82.80% without modification. The DPPH radical scavenging rate of OEO-NLs modified with 4–5 mg / mL PL was slightly higher than that of OEO-NLs modified with 3 mg / mL PL.

[0115] 6. Detection of the killing effect of peptide-modified phenolic essential oil nanoliposomes on E. coli O157:H7 and S. aureus

[0116] The killing effects of OEO-NLs prepared under conditions of OEO concentration of 5 mg / mL, cholesterol concentration of 1.50 mg / mL, and PBS buffer solution pH 7.0, and the above-mentioned peptide-modified phenolic essential oil nanoliposomes prepared with PL solutions at concentrations of 1, 2, 3, 4, and 5 mg / mL on E. coli O157:H7 (purchased from the UK National Culture Collection Center) and S. aureus (purchased from the China General Microbiological Culture Collection Center) were investigated. The detection procedure was as follows: First, PL / OEO-NLs of different concentrations were sterilized under UV light for 4 h. Then, 1 mL of each sample was mixed with 5 mL of bacterial solution and incubated at 37℃ / 220 rpm for 12 h with shaking. A pure bacterial solution without any sample was set up as a blank control. After incubation, 2 mL of bacterial solution was aspirated into a sterile centrifuge tube, centrifuged at 4℃ and 4000 rpm for 4 min, the supernatant was discarded, and the bacterial cells were resuspended in PBS buffer solution (purchased from Beijing Solarbio Biotechnology Co., Ltd.), and washed twice. After washing, the bacterial suspension was appropriately diluted, and three suitable dilution gradients were selected for inverted plate operation. After the agar (purchased from Beijing Aoboxing Biotechnology Co., Ltd.) medium solidified, the plates were incubated upside down at 37℃ for 12 hours before counting. The results are expressed as log... 10 (N0 / N) represents the initial number of viable cells (CFU / mL) and the number of viable cells after 12 hours (CFU / mL).

[0117] The results are as follows Figure 6 As shown, the killing effect of OEO-NLs modified with 3 mg / mL PL on E. coli O157:H7 and S. aureus was 1.73 log [value missing] compared to unmodified OEO-NLs. 10 CFU / mL and 2.05 log 10 CFU / mL increased to 3.63 log 10 CFU / mL and 4.29 log 10 CFU / mL and 4–5 mg / mL PL-modified OEO-NLs also showed a multiple increase in their killing effect on E. coli O157:H7 and S. aureus.

[0118] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "embodiment," or "specific embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0119] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing polypeptide-modified phenolic essential oil nanoliposomes, characterized in that, The method includes: (1) Soybean lecithin, cholesterol and oregano essential oil were subjected to rotary evaporation, thin film hydration and ultrasonic dispersion in sequence to obtain oregano essential oil nanoliposome suspension; (2) The oregano essential oil nanoliposome suspension was mixed and stirred with polylysine in sequence to obtain the polypeptide-modified phenolic essential oil nanoliposomes. The concentration of the soybean lecithin is 10 mg / mL. The cholesterol concentration is 1.00~2.00 mg / mL. The concentration of the oregano essential oil is 4-8 mg / mL. The concentration of the polylysine is 3-5 mg / mL. The mass ratio of soybean lecithin, cholesterol and oregano oil is 10:(1~2):(4~8). The volume ratio of the oregano essential oil nanoliposome suspension to polylysine is 0.8~1.

2. The membrane hydration was carried out in a surfactant and a PBS buffer solution with a pH of 6.8–7.

2.

2. The method according to claim 1, characterized in that, The mass ratio of soybean lecithin, cholesterol, and oregano oil is 10:(1.25~1.75):(5~7).

3. The method according to claim 1, characterized in that, The mass ratio of soybean lecithin, cholesterol, and oregano oil is 10:1.5:

5.

4. The method according to claim 1, characterized in that, Prior to the rotary evaporation, the soybean lecithin, cholesterol, and oregano oil were dissolved in anhydrous ethanol.

5. The method according to claim 1, characterized in that, The temperature of the rotary evaporation is 35℃~45℃.

6. The method according to claim 1, characterized in that, The mass-to-volume ratio of the surfactant and the PBS buffer solution with a pH of 6.8-7.2 is 2 mg: 20 mL.

7. The method according to claim 1, characterized in that, The surfactant includes polyvinylpyrrolidone.

8. The method according to claim 1, characterized in that, The temperature of the rotary evaporation is 40°C.

9. The method according to claim 1, characterized in that, After ultrasonic dispersion and before step (2), the product after ultrasonic dispersion is subjected to pulverization and centrifugation in sequence.

10. The method according to claim 1, characterized in that, The polylysine was pre-dissolved in a PBS buffer solution with a pH of 6.8–7.

2.

11. The method according to claim 1, characterized in that, The polylysine is preferentially dissolved in a PBS buffer solution with a pH of 7.

0.

12. The method according to claim 1, characterized in that, The stirring process takes 30 to 50 minutes.

13. The method according to claim 9, characterized in that, The pulverization process was carried out for 15 minutes at a power of 350W, with a pulverization time of 10 seconds and a 5-second interval.

14. The method according to claim 9, characterized in that, The centrifugation process involved centrifuging at 4000 g for 5 min.

15. The method according to claim 1, characterized in that, The stirring process takes 40 minutes.

16. A polypeptide-modified phenolic essential oil nanoliposome, characterized in that, The nanoliposomes are prepared using the method described in any one of claims 1 to 5, wherein, The oregano essential oil is encapsulated in the nanoliposomes, and polylysine is modified on the surface of the nanoliposomes. The nanoliposomes have a particle size of 350~500 nm. The PDI of the nanoliposomes is 0.160~0.

210. The zeta potential of the nanoliposomes is -15 to 15 mV.

17. The nanoliposomes according to claim 16, characterized in that, The particle size of the nanoliposomes is 420~500 nm.

18. The nanoliposomes according to claim 16, characterized in that, The zeta potential of the nanoliposomes is 7~12 mV.

19. A drug or preservative, characterized in that, The present invention comprises polypeptide-modified phenolic essential oil nanoliposomes prepared by the method according to any one of claims 1 to 15 or polypeptide-modified phenolic essential oil nanoliposomes according to any one of claims 16 to 18.

20. Use of polypeptide-modified phenolic essential oil nanoliposomes prepared by the method according to any one of claims 1 to 15, or polypeptide-modified phenolic essential oil nanoliposomes according to any one of claims 16 to 18, in the preparation of a medicament for the prevention, relief, and / or treatment of related diseases caused by pathogenic bacteria.

21. The use according to claim 20, characterized in that, The pathogenic bacteria include at least one of Escherichia coli and Staphylococcus aureus.

22. Use of polypeptide-modified phenolic essential oil nanoliposomes prepared by the method according to any one of claims 1 to 15 or polypeptide-modified phenolic essential oil nanoliposomes according to any one of claims 16 to 18 in the preparation of a fresh food preservative, wherein the preservative is used to inhibit bacteria.

23. The use according to claim 22, characterized in that, The bacteria include at least one of Escherichia coli and Staphylococcus aureus.

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