Polypeptide modified phenolic essential oil nano-liposome and preparation method thereof

Abstract

The invention provides a polypeptide modified phenol essential oil nano-liposome and a preparation method thereof, and the method comprises the following steps: (1) successively carrying out rotary evaporation, film hydration and ultrasonic dispersion on soybean lecithin, cholesterol and origanum vulgare essential oil to obtain an origanum vulgare essential oil nano-liposome suspension; and (2) sequentially mixing the origanum vulgare essential oil nano-liposome suspension and polylysine, and stirring to obtain the polypeptide modified phenolic essential oil nano-liposome. Soybean lecithin, cholesterol and origanum vulgare essential oil in a specific mass ratio and polylysine in a specific mass are self-assembled to form the nano-liposome in which antibacterial and antioxidant active substances are slowly released, growth and reproduction of escherichia coli and staphylococcus aureus can be effectively inhibited, meanwhile, main base materials of the nano-liposome are edible materials, and the nano-liposome has a good antibacterial and anti-oxidation effect. The product can be used for functional delivery of active substances and storage and preservation of fresh food, and meets the requirements of people for safe, nutritional and healthy diet and green and environment-friendly ideas.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a polypeptide-modified phenolic essential oil nanoliposome and a preparation method thereof. Background Art

[0002] Plant essential oils are fat-soluble natural compounds extracted from aromatic plants. They are volatile and have rich aromas. They are usually described as concentrated hydrophobic liquid substances containing volatile aromatic compounds. They are natural extracts composed of different monoterpenes, monoterpenes, sesquiterpenes and other volatiles (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 very potent natural antibacterial agent. It has a certain antibacterial effect on both Gram-positive and Gram-negative bacteria. It is the most broad-spectrum antibacterial drug additive. Due to its high efficiency, non-toxicity and non-irritation, it can effectively kill a variety of bacteria and molds and has a wide range of applications in the fields of medicine, chemical industry and food. In addition, OEO also has certain antioxidant activity. Its main compounds, para-cymene and γ-terpinene, can promote its biological antioxidant activity by inhibiting active oxygen-producing enzymes (such as lipoxygenase and xanthin oxidase). The antibacterial and antioxidant activities of OEO can be attributed to the presence of a large number of phenolic compounds, mainly carvacrol and thymol in essential oils. However, OEO has defects such as high volatility, poor water solubility, and sensitivity to light, oxygen, and temperature, which greatly limits its application as a food preservative and has problems such as low bioavailability. Therefore, it is of great significance to make full use of the advantages of OEO's good antioxidant and antibacterial activities and develop it into a stable and safe food preservative.

[0003] Poly-L-lysine (PL) is a polypeptide produced by microbial metabolites. It has an inhibitory effect on both bacteria and fungi, and this inhibitory effect mainly depends on its molecular weight. The antibacterial mechanism of PL depends on the electrostatic interaction with the surface of microorganisms. It disrupts the cell membrane structure of microorganisms by interfering with the transmission of cellular substances, energy and information. Lysine residues that penetrate cells can stimulate the production of reactive oxygen in microorganisms, ultimately achieving a bactericidal effect. In addition, this antibacterial activity is also attributed to the ability of PL 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 has the advantages of being edible, non-toxic, good stability and biocompatibility, and has broad application prospects in food storage and preservation.

[0004] Nanoliposomes (NLs) are a self-assembled phospholipid carrier system, which is formed by lipids with polar groups dispersed 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. NLs can be used to encapsulate antimicrobial drugs to actively target the bacterial community due to their advantages such as biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and targeted sustained release, and are one of the good alternatives to encapsulation-based drug delivery technology. However, the phospholipid membrane of NLs will be destroyed in the presence of low pH and enzymes, resulting in the leakage of the encapsulated active substances. Therefore, the surface of NLs can be stabilized by forming electrostatic bridges through electrostatic interactions.

[0005] Currently, there is no extensive research on the use of peptide surface-modified NLs for the encapsulation and delivery of phenolic essential oils. In addition, there are few studies on peptides as surface modifiers of NLs. Whether they can enhance the stability of NLs and whether they can exert synergistic antibacterial and antioxidant effects with phenolic essential oils remains to be explored. Summary of the invention

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

[0007] There is no extensive research on the use of polypeptide surface modified NLs to encapsulate and deliver phenolic essential oils. The present invention adopts a thin film hydration ultrasonic dispersion method to prepare phenolic essential oil nanoliposomes. First, the inventor optimizes its preparation process by single factor and orthogonal test method, and obtains oregano essential oil nanoliposomes with an encapsulation rate (up to 78%) far higher than the existing thin film hydration ultrasonic dispersion method. Then, the surface of the phenolic essential oil nanoliposomes is modified with polypeptides of different concentrations by electrostatic self-assembly. After studying the adsorption rate, particle size distribution, pH, turbidity, structure, environmental stability, antioxidant activity and antibacterial activity of the polypeptide-modified phenolic essential oil nanoliposomes, the inventor unexpectedly finds that polylysine can effectively modify the nanoliposomes encapsulating oregano essential oil, enhance the stability of the oregano essential oil nanoliposomes and play a synergistic antibacterial and antioxidant effect with the phenolic essential oil, and unexpectedly obtains polypeptide-modified phenolic essential oil nanoliposomes with high encapsulation rate, uniform particle size, effective inhibition of bacterial growth and reproduction, and strong antioxidant performance.

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

[0009] The inventors optimized the method for preparing phenolic essential oil nanoliposomes. The polypeptide-modified phenolic essential oil nanoliposomes prepared by the method of the embodiment of the present invention have a very high oregano essential oil encapsulation rate, and polylysine can be adsorbed on the surface of the oregano essential oil nanoliposomes to form a more stable complex, thereby further improving the encapsulation rate of the nanoliposomes. In addition, it has been verified by experiments that polylysine can exert a synergistic antibacterial and antioxidant effect 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, cosmetics, skin care products and other fields, and their application value is very high.

[0010] According to an embodiment of the present invention, the method for preparing the nanoliposome 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). Thus, oregano essential oil encapsulation efficiency can be obtained, which is much 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 essential oil is 10:(1.25-1.75):(5-7). Thus, phenolic essential oil nanoliposomes with further improved oregano essential oil encapsulation efficiency can be obtained.

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

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

[0015] According to an embodiment of the present invention, the concentration of the soybean lecithin is 10 mg / mL, and / or the concentration of the cholesterol is 1.00-2.00 mg / mL, and / or the concentration of the 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, 80-160 mg of the oregano essential oil is weighed and pre-dissolved in 20 mL of anhydrous ethanol, the concentration of the 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 rotary evaporation is performed after ultrasonic dispersion together with soybean lecithin and cholesterol.

[0017] According to an embodiment of the present invention, the temperature of the rotary evaporation is 35° C. to 45° C. In some specific embodiments, the temperature of the rotary evaporation is preferably 40° C. The rotary evaporation evaporates the solvent through a rotary evaporator, leaving a layer of 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 value of 6.4 to 8.0. In the process of preparing nanoliposomes, soybean lecithin and cholesterol can form a lipid bilayer structure, which is the basic skeleton of liposomes. PBS (phosphate buffered saline) allows soybean lecithin and cholesterol to form a uniform lipid solution, and maintains the pH value of the solution within a relatively stable range. The charge state and hydration of lipids are conducive to the formation of liposomes. The pH value range of PBS must also meet the needs close to the internal environment of the organism, which helps to improve the biocompatibility of liposomes and reduce potential damage to biological tissues. At a suitable pH value, the hydrolysis or oxidation of lipid components can also be reduced, which is crucial to the formation, stability and functionality of liposomes.

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

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

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

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

[0023] According to an embodiment of the present invention, the centrifugation is performed at a speed of 4000 g 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 value of 6.4 to 8.0, and is preferentially dissolved in a PBS buffer solution with a pH value of 7.0.

[0025] Polylysine is a cationic polypeptide. Due to the positive charge on its molecule, it can modify the surface of nanoliposomes through electrostatic adsorption to form a stable complex. In addition, the inventors found that this modification can be used to enhance the encapsulation effect of nanoliposomes on oregano essential oil, and to synergize with oregano essential oil to exert antibacterial and antioxidant effects.

[0026] According to an embodiment of the present invention, the concentration of polylysine is 0 to 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 treatment time is 30 to 50 minutes.

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

[0032] In the second aspect of the present invention, the present invention proposes a polypeptide-modified phenolic essential oil nanoliposome. According to an embodiment of the present invention, the nanoliposome is prepared by the method described in the first aspect. The polypeptide-modified phenolic essential oil nanoliposome prepared by the method of the embodiment of the present invention has a very high oregano essential oil encapsulation rate, and polylysine can be adsorbed on the surface of the oregano essential oil nanoliposome to form a more stable complex, thereby further improving the encapsulation rate of the liposome. In addition, it has been experimentally verified that polylysine can play a synergistic antibacterial and antioxidant effect with oregano essential oil, so that the antibacterial and antioxidant effects of the nanoliposome are enhanced, and since the components of the nanoliposome are all edible materials, its application scenarios include medical, food, cosmetics, skin care products and other fields, and the application value is very high.

[0033] According to an embodiment of the present invention, the above-mentioned polypeptide-modified phenolic essential oil nanoliposomes may also 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 nanoliposome, and polylysine is modified on the surface of the nanoliposome.

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

[0036] According to an embodiment of the present invention, the PDI of the liposome 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 the third aspect of the present invention, the present invention provides a medicine, a preservative, a cosmetic or a skin care product. According to an embodiment of the present invention, the medicine, the preservative, the cosmetic or the skin care product comprises the polypeptide-modified phenolic essential oil nanoliposome prepared by the method described in the first aspect or the polypeptide-modified phenolic essential oil nanoliposome described in the second aspect. As mentioned above, the 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 have a very high oregano essential oil encapsulation rate, and polylysine can be adsorbed on the surface of the oregano essential oil nanoliposomes to form a more stable complex, thereby further improving the encapsulation rate of the liposomes. In addition, it has been experimentally verified that polylysine can exert a synergistic antibacterial and antioxidant effect 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, cosmetics, skin care products and other fields, and their application value is very high. The drugs, preservatives, cosmetics and skin care products containing the polypeptide-modified phenolic essential oil nanoliposomes can effectively inhibit bacterial growth and proliferation, for example, Escherichia coli and Staphylococcus aureus. In addition, they can also effectively release antioxidant effects.

[0039] In the fourth aspect of the present invention, the present invention proposes the use of the 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 drug, wherein the drug is used to prevent, alleviate and / or treat related diseases caused by pathogenic bacteria. The drug containing the polypeptide-modified phenolic essential oil nanoliposomes can effectively inhibit the growth and proliferation of bacteria, such as Escherichia coli and Staphylococcus aureus, and can also effectively slow-release antioxidant effects.

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

[0041] In addition, polylysine-modified nanoliposomes can also be actively targeted by further chemical or biomolecular modification. For example, targeting to specific cells or tissues can be achieved by combining polylysine with specific ligands (such as folic acid, antibodies or peptides). This strategy is particularly promising in tumor treatment because it can increase the concentration of drugs in tumor tissues while reducing damage to normal tissues.

[0042] In the fifth aspect of the present invention, the present invention proposes the use of the 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 fresh food preservatives, and the preservatives are used to inhibit bacteria. As mentioned above, since the components of the nanoliposomes are all edible materials, they can be used in the field of food. The preservatives containing the polypeptide-modified phenolic essential oil nanoliposomes can effectively inhibit the growth and proliferation of bacteria in fresh food, and are used for the storage and preservation of fresh food, such as fruits, vegetables, and meat. Therefore, preservatives can be replaced, and antioxidant effects can be effectively sustained, which meets people's pursuit of safe, nutritious, and healthy diets, and conforms to the green environmental protection concept.

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

[0044] In the sixth aspect of the present invention, the present invention proposes a method for preserving fresh food. According to an embodiment of the present invention, the method comprises applying the 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 to the fresh food. The method according to the embodiment of the present invention can effectively preserve and prevent fresh food, meet people's pursuit of safe, nutritious and healthy diet, and conform to the green environmental protection concept.

[0045] In the seventh aspect of the present invention, the present invention proposes a method for inhibiting pathogenic bacteria in vitro. According to an embodiment of the present invention, the method is used for non-therapeutic purposes, and the method comprises contacting tissues and / or cells with the 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. As mentioned above, the polypeptide-modified phenolic essential oil nanoliposomes can effectively inhibit the growth of bacteria, such as Escherichia coli and Staphylococcus aureus, and therefore, it can play an antibacterial role 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, as well as polylysine at a specific concentration, and utilizes the charged properties of polypeptides and phospholipids to self-assemble to form nanoliposomes. The raw materials are easily available, and the preparation process is simple and efficient.

[0049] (2) The encapsulation rate 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, up to 78%, and can effectively inhibit the growth and reproduction of bacteria, such as Escherichia coli and Staphylococcus aureus, and can slowly release antioxidant active substances. At the same time, 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 performing antioxidant activities, as well as for the storage and preservation of fresh food, meeting people's pursuit of safe, nutritious and healthy diets, and conforming to the green environmental protection concept.

[0050] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0052] Figure 1 This is a result diagram showing the effects of OEO concentration, cholesterol concentration and pH value of PBS buffer solution on the encapsulation efficiency of OEO-NLs in Example 1 of the present invention;

[0053] Figure 2 The test results of the appearance (Figure A), PL adsorption rate (Figure B), pH value and turbidity (Figure C) of PL / OEO-NLs prepared with different PL concentrations in Example 1 of the present invention are shown;

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

[0055] Figure 4 This is a graph showing the storage stability test results of OEO-NLs and PL / OEO-NLs prepared with different concentrations of PL at 25°C according to Example 1 of the present invention;

[0056] Figure 5 This is a graph showing the DPPH radical scavenging rate test results of PL / OEO-NLs prepared with different concentrations of PL in Example 1 of the present invention;

[0057] Figure 6This is a graph showing the killing effect of PL / OEO-NLs prepared with different concentrations of PL in Example 1 of the present invention on E. coli O157:H7 and S. aureus. DETAILED DESCRIPTION

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

[0059] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" and "multiple" is at least two or two, such as two, two, three, three, etc., unless otherwise clearly and specifically defined.

[0060] In this document, the terms “include”, “have” or “comprises” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0061] As used herein, the term "optionally" generally means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0062] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0063] In this application, the thin film hydration ultrasonic dispersion method is a common technique for preparing liposomes, which combines the thin film hydration method and the ultrasonic dispersion method. The advantages of the thin film hydration ultrasonic dispersion method are simple operation and low cost, but it also has some limitations, such as the encapsulation rate is not as high as other methods, and ultrasonic treatment may cause damage to some sensitive biological molecules. Therefore, when using this method, it is necessary to optimize the conditions according to the purpose of the liposome and the characteristics of the encapsulated substance. For example, the concentration of the lipid solution, the characteristics of the encapsulated substance, the ionic strength of the PBS, the temperature, the pH value and other factors may affect the formation and characteristics of the liposome.

[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 method for measuring the particle size distribution in a solution or suspension. It obtains information by measuring the fluctuation of light intensity over time, which is 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. Zeta potential is an important parameter for characterizing the stability of particles in a fluid, which reflects the nature of the surface charge of the particles. Electrophoretic light scattering indirectly determines the Zeta potential by measuring the electrophoretic mobility of particles under an applied electric field. Electrophoretic light scattering combined with dynamic light scattering technology can simultaneously measure the size and Zeta potential of particles in the same experiment, which provides more comprehensive information for the characterization of particles. Through this combined technology, the stability and interaction of particles can be more accurately evaluated.

[0066] The present invention is described below with reference to specific examples. It should be noted that conventional methods, equipment, reagents, and materials in the art used in the examples are not described in detail herein. These examples are merely illustrative and do not limit the present invention in any way.

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

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

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

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

[0071] Firstly, a single factor experiment of the preparation process of OEO-NLs was carried out: a single factor experiment was used to study the effects of factors such as 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 solvent PBS buffer solution (6.4, 6.8, 7.2, 7.6, 8.0) on the encapsulation efficiency of OEO-NLs.

[0072] OEO-NLs were prepared by thin film hydration ultrasonic dispersion method: 200 mg soybean lecithin (purchased from Beijing Solebow Biotechnology Co., Ltd.) and 20 mg, 25 mg, 30 mg, 35 mg and 40 mg cholesterol (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) were weighed and added into a 50 mL round-bottom flask, respectively. Then, 80 mg, 100 mg, 120 mg, 140 mg and 160 mg of OEO (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) dissolved in 20 mL of anhydrous ethanol (purchased from Jingchun Reagent) were transferred into the round-bottom flask containing soybean lecithin and cholesterol, and the mixture was fully dispersed under water bath ultrasonic conditions. 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 polyvinyl pyrrolidone (purchased from Beijing Solebow 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 the mixture was fully dispersed under water bath ultrasound. The obtained mixture was poured into a centrifuge tube and crushed in an ultrasonic cell crusher (working 10 s / interval 5 s, power 350 W) for 15 min. The obtained product was then centrifuged at 4000 g for 5 min to remove the free oregano essential oil in the upper layer and the impurities in the lower layer to obtain an OEO-NLs suspension, wherein 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 of single factor experiment for NLs preparation

[0074]

[0075] The oregano essential oil nanoliposomes prepared in the above groups were tested for encapsulation efficiency, and the specific testing steps were as follows:

[0076] (1) Preparation of OEO standard curve: To determine the optimal measurement wavelength of OEO, an ultraviolet 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 respectively, and the absorbance A was measured at the optimal measurement wavelength with ethanol as a blank, and the standard curve of A versus concentration C was fitted using data processing software. Using anhydrous ethanol as a blank control, it was observed that the optimal measurement wavelength of OEO was 276 nm. After measuring the absorbance A of a series of OEO ethanol solutions with different concentrations at 276 nm, a linear regression was performed using the absorbance A versus the OEO concentration C to obtain the standard equation: A=8.31104C+0.03254, with a correlation coefficient R 2=0.99833, with a good linear relationship in the concentration range of 0.075 mg / mL to 0.22 mg / mL.

[0077] (2) Determination of OEO-NLs encapsulation efficiency: First, 2 mL of the 15 kinds of OEO-NLs prepared by the above method were taken into a 2 mL centrifuge tube and centrifuged at 12000 rpm for 15 min. Then, the supernatant was removed, and anhydrous ethanol was added to the precipitate in the centrifuge tube to make up to 2 mL. The solution was ultrasonicated in a water bath at 40°C for 10 min, vortexed and mixed for 2 min, and the volume of the solution was made up to 100 mL with anhydrous ethanol. 2 mL of the diluted solution was taken from the solution and placed in a 2 mL centrifuge tube. The solution was centrifuged at 12000 rpm for 15 min, and allowed to stand. The supernatant was taken and the absorbance of the supernatant at 276 nm was measured by a UV spectrophotometer, and the concentration of free OEO (C) was calculated by the regression equation of the OEO standard curve. NE ), and the encapsulation efficiency was calculated according to formula (1):

[0078]

[0079] In formula (1): C N is the encapsulation efficiency of OEO-NLs (%); C NE is the content of OEO encapsulated by OEO-NLs (mg); C NT is the total OEO content (mg) added to OEO-NLs.

[0080] The results of the single factor experiment encapsulation efficiency are as follows Figure 1 As shown, when the OEO content is 5 mg / mL, 6 mg / mL, and 7 mg / mL, the cholesterol content is 1.25 mg / mL, 1.50 mg / mL, and 1.75 mg / mL, and the pH value of the PBS buffer solution is 6.8, 7.0, and 7.2, the OEO-NLs obtained have good encapsulation efficiency, which is above 60%.

[0081] 2. Orthogonal experiment for the preparation of oregano essential oil nanoliposomes (OEO-NLs)

[0082] This experiment is used to further screen the important parameter conditions in the method of preparing oregano essential oil nanoliposomes based on Experiment 1. The operation is as follows:

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

[0084] Table 2 shows that the highest encapsulation efficiency was in the test group No. 2 (OEO concentration 5.0 mg / mL, cholesterol concentration 1.50 mg / mL, PBS buffer solution pH 7.0), followed by No. 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. After comprehensive consideration, the test condition No. 2 was selected as the best preparation process. From the range analysis with encapsulation efficiency as the evaluation index, it can be seen that the order of factors affecting the encapsulation efficiency of NLs is: 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] In this example, polylysine (i.e., PL) was modified on the basis of the OEO-NLs prepared in Example 1 to prepare PL / OEO-NLs. The specific experimental operation is as follows:

[0090] Based on the results of the orthogonal test in Example 1, the OEO-NLs prepared by the optimal preparation process conditions screened by the orthogonal test in Table 2: OEO concentration 5.0 mg / mL, cholesterol concentration 1.50 mg / mL, PBS buffer solution pH 7.0 were modified with PL (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), wherein 20, 40, 60, 80, and 100 mg of PL were dissolved in 20 mL of PBS buffer solution with a pH value of 7.0 to obtain PL solution modification of different concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL), and the above-mentioned PL solutions of equal volumes of different concentrations were separately added to the suspension containing OEO-NLs prepared under the optimal preparation process conditions obtained in Table 2, and stirred for 40 minutes with a magnetic stirrer to obtain the following appearance: Figure 2 (A) PL / OEO-NLs shown.

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

[0092] The obtained polypeptide-modified phenolic essential oil nanoliposomes were tested for adsorption rate, pH value and turbidity. The testing steps are as follows:

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

[0094]

[0095] In formula (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: The pH value of PL / OEO-NLs was measured using an acidity meter. The turbidity of PL / OEO-NLs was measured using an ultraviolet spectrophotometer with a measurement wavelength of 600 nm and a cuvette thickness of 1 cm. PL / OEO-NLs were diluted 10 times with ultrapure water.

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

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

[0099] The particle size, PDI and Zeta potential of the above-mentioned polypeptide-modified phenolic essential oil nanoliposomes prepared under the conditions of OEO concentration 5 mg / mL, cholesterol concentration 1.50 mg / mL and PBS buffer solution pH 7.0 and PL solution concentrations of 1, 2, 3, 4 and 5 mg / mL were tested. The detection steps were as follows: OEO-NLs and each group of PL / OEO-NLs dispersion systems were diluted 10 times by volume with ultrapure water before testing. The average particle size and PDI were determined by dynamic light scattering technology, and the Zeta potential was determined by electrophoretic light scattering. The samples were balanced in the instrument for 2 minutes before testing, and each sample was measured at least 3 times.

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

[0101] Table 3 Average particle size, PDI and Zeta potential of PL / OEO-NLs with 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 infrared spectroscopy detection

[0105] The OEO, PL, empty NLs (without OEO and PL), OEO-NLs and PL / OEO-NLs with different concentrations were tested by Fourier infrared spectrometer using a tabletting method. The testing steps were 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, ground evenly, and pressed into tablets under a pressure of 15-20 MPa. The sample was placed in a sample chamber and the infrared absorption spectrum of the sample was measured at a wavelength of 400-4000 cm -1 , resolution 4cm -1 , each image was scanned 64 times cumulatively.

[0106] Figure 3 It shows that OEO was successfully encapsulated inside the nanoliposomes, PL was successfully modified on the surface of the nanoliposomes, and OEO encapsulation and PL modification had little effect on the internal structure of PL / OEO-NLs.

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

[0108] The stability of the above-mentioned polypeptide-modified phenolic essential oil nanoliposomes prepared by 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 7.0 and PL solutions with concentrations of 1, 2, 3, 4 and 5 mg / mL was tested. The testing steps are as follows: the OEO-NLs and PL / OEO-NLs samples were placed in a constant temperature and humidity chamber, stored at 25°C for 30 days, and the average particle size, PDI, Zeta potential and adsorption rate of the OEO-NLs and PL / OEO-NLs were measured on the 0th, 5th, 10th, 15th, 20th, 25th and 30th days respectively by the methods of the above-mentioned Experiment 2 and Experiment 1(1).

[0109] The results are as follows Figure 4 As shown in the figure, 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] The above-mentioned peptide-modified phenolic essential oil nanoliposomes prepared under the conditions of OEO concentration of 5 mg / mL, cholesterol concentration of 1.50 mg / mL, and PBS buffer solution pH 7.0 and PL solutions with concentrations of 1, 2, 3, 4, and 5 mg / mL were tested for DPPH (purchased from Fuzhou Feijing Biotechnology Co., Ltd.) free radical scavenging rate. The detection steps are as follows: 0.1mmol / LDPPH methanol (purchased from Jingchun Reagent) solution and sample solution were mixed in equal volumes and shaken. The reaction was carried out at room temperature in the dark for 30 minutes, and the absorbance was measured at 517nm, with ultrapure water as the blank control. The DPPH free radical scavenging rate was calculated according to formula (3):

[0112]

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

[0114] The results are as follows Figure 5 As shown in the figure, PL surface modification can improve 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% of unmodified OEO-NLs to 82.80%. The DPPH radical scavenging rate of OEO-NLs modified with 4-5 mg / mL PL was slightly increased compared with that of OEO-NLs modified with 3 mg / mL PL.

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

[0116] The 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 value of 7.0 and the above-mentioned polypeptide-modified phenolic essential oil nanoliposomes prepared by PL solutions with concentrations of 1, 2, 3, 4, and 5 mg / mL were tested for their killing effects on E. coli O157:H7 (purchased from the British National Culture Collection) and S. aureus (purchased from the China General Microbiological Collection Center). The detection steps are as follows: first, PL / OEO-NLs of different concentrations were sterilized under ultraviolet for 4 hours for use. Then, 1 mL of different samples were mixed with 5 mL of bacterial solution and placed in 37°C / 220 rpm shaking culture for 12 hours. A pure bacterial solution group without any sample was set as a blank control. After the culture was completed, 2 mL of bacterial solution was taken into a sterile centrifuge tube, centrifuged at 4°C and 4000 rpm for 4 minutes, and the supernatant was discarded. The bacterial cells were resuspended in PBS buffer solution (purchased from Beijing Solebow Biotechnology Co., Ltd.), and washed twice. After the washed bacterial suspension is appropriately diluted, three appropriate gradient dilutions are selected for inverted plate operation. After the agar medium (purchased from Beijing Aoboxing Biotechnology Co., Ltd.) solidifies, invert and culture at 37°C for 12 hours before counting. The results are expressed as log 10 (N0 / N), where N0 is the initial viable cell number (CFU / mL) and N is the viable cell number after 12 h (CFU / mL).

[0117] The results are as follows Figure 6 As shown in the figure, the killing effect of 3 mg / mL PL-modified OEO-NLs on E. coli O157:H7 and S. aureus was 1.73 log higher than that of unmodified OEO-NLs. 10 CFU / mL and 2.05log 10 CFU / mL increased to 3.63log 10 CFU / mL and 4.29log 10 CFU / mL, the killing effect of 4-5 mg / mL PL-modified OEO-NLs on E. coli O157:H7 and S. aureus also increased exponentially.

[0118] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "embodiment" or "specific embodiment" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without contradiction.

[0119] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary 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 comprises: (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) The oregano essential oil nanoliposome suspension and polylysine are sequentially mixed and stirred to obtain the polypeptide-modified phenolic essential oil nanoliposome.

2. The method according to claim 1, characterized in that The mass ratio of soybean lecithin, cholesterol and oregano essential oil is 10:(1-2):(4-8); Optionally, the mass ratio of the soybean lecithin, cholesterol and oregano essential oil is 10:(1.25-1.75):(5-7); Optionally, the mass ratio of the soybean lecithin, cholesterol and oregano essential oil is 10:1.5:

5.

3. The method according to claim 1, characterized in that Before the rotary evaporation, the soybean lecithin, cholesterol and oregano essential oil are pre-dissolved in anhydrous ethanol; Optionally, the concentration of the soybean lecithin is 10 mg / mL, and / or The concentration of cholesterol is 1.00-2.00 mg / mL, and / or The concentration of the oregano essential oil is 4-8 mg / mL.

4. The method according to claim 1, characterized in that The temperature of the rotary evaporation is 35°C to 45°C, preferably 40°C; Optionally, the film hydration is performed in a surfactant and a PBS buffer solution with a pH value of 6.4 to 8.0; Optionally, the mass volume ratio of the surfactant to the PBS buffer solution having a pH value of 6.4 to 8.0 is 2 mg: 20 mL; Optionally, the surfactant comprises polyvinylpyrrolidone.

5. The method according to claim 1, characterized in that After the ultrasonic dispersion and before the step (2), the product after the ultrasonic dispersion is subjected to a pulverizing treatment and a centrifugal treatment in sequence; Optionally, the pulverization process is performed for 15 minutes at a power of 350 W, pulverization for 10 seconds, and a rest period of 5 seconds; Optionally, the centrifugation is performed at 4000 g for 5 min; Optionally, the polylysine is pre-dissolved in a PBS buffer solution with a pH value of 6.4 to 8.0, preferably in a PBS buffer solution with a pH value of 7.0; Optionally, the concentration of the polylysine is 0 to 5 mg / mL; Optionally, the concentration of the polylysine is 3 to 5 mg / mL; Optionally, the volume ratio of the oregano essential oil nanoliposome suspension to polylysine is 0.8 to 1.2; Optionally, the stirring treatment time is 30 to 50 minutes; Optionally, the stirring treatment time is 40 minutes.

6. A polypeptide-modified phenolic essential oil nanoliposome, characterized in that: The nanoliposome is prepared by the method according to any one of claims 1 to 5.

7. The nanoliposome according to claim 6, characterized in that Oregano essential oil is encapsulated in the nanoliposomes, and polylysine is modified on the surface of the nanoliposomes; Optionally, the particle size of the nanoliposome is 350 to 500 nm, preferably 420 to 500 nm; Optionally, the PDI of the nanoliposome is 0.160 to 0.210; Optionally, the zeta potential of the nanoliposome is -15 to 15 mV, preferably 7 to 12 mV.

8. A medicine or preservative, characterized in that: The invention comprises a polypeptide-modified phenolic essential oil nanoliposome prepared by the method described in any one of claims 1 to 5 or a polypeptide-modified phenolic essential oil nanoliposome prepared by any one of claims 6 to 7.

9. Use of the polypeptide-modified phenolic essential oil nanoliposomes prepared by the method according to any one of claims 1 to 5 or the polypeptide-modified phenolic essential oil nanoliposomes according to any one of claims 6 to 7 in the preparation of a drug for preventing, alleviating and / or treating related diseases caused by pathogenic bacteria; Optionally, the pathogenic bacteria include at least one of Escherichia coli and Staphylococcus aureus.

10. Use of the polypeptide-modified phenolic essential oil nanoliposomes prepared by the method according to any one of claims 1 to 6 or the polypeptide-modified phenolic essential oil nanoliposomes according to any one of claims 7 to 8 in the preparation of a fresh food preservative, wherein the preservative is used to inhibit bacteria; Optionally, the bacteria include at least one of Escherichia coli and Staphylococcus aureus.

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