A-type mannosylerythritol lipid nano-emulsion as well as preparation method and application of A-type mannosylerythritol lipid nano-emulsion
By preparing type A mannose erythritol nanoemulsion, using Tween 20 and glycerol monooleate as emulsifiers, the problem of difficult removal of Listeria monoethyl in food was solved, and effective inhibition and safety protection were achieved.
Patent Information
- Application Number
- CN202510681189.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-08-12
AI Technical Summary
Listeria monocytogenes is widely present in foods, with strong stress resistance and biofilm formation ability, making it difficult to completely remove. The existing antibacterial agents have limited effects in food safety protection.
A type mannose erythritol nanoemulsion is used, and MEL-A is encapsulated inside the emulsion particles through Tween 20 and glycerol monooleate as emulsifiers to form oil-in-water emulsion particles, which improves solubility and maintains antibacteriality, and is applied to food safety protection.
It has achieved effective inhibition of Listeria monocytogenes, reduced food safety risks, and is simple to operate and easy to industrially apply.
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Figure CN120458126A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of antibacterial agent preparation, in particular to a type A mannose erythritol lipid nanoemulsion and a preparation method and application thereof. Background Art
[0002] Listeria monocytogenes is a typical foodborne pathogen. Despite a relatively low annual incidence, the mortality rate following infection is as high as 2% to 30%. L. monocytogenes is highly resistant to stresses (low temperature, high salt, and low pH) and can be detected in a variety of foods, including vegetables, fruits, meat, dairy products, aquatic products, and ready-to-eat foods (RTEs). After ingestion of contaminated food, L. monocytogenes can penetrate the intestinal epithelial barrier and enter the lamina propria, then spread via the lymphatic and blood circulation to its target organs, namely the liver and spleen. Furthermore, L. monocytogenes is highly susceptible to forming biofilms in food processing environments and equipment. Biofilm formation significantly increases the bacteria's tolerance to biocides and promotes the bacteria's transition to a VBNC state, which prolongs their survival and makes complete removal difficult, leading to repeated food contamination. Summary of the Invention
[0003] In view of this, the object of the present invention is to provide a type A mannose erythritol lipid nanoemulsion and its preparation method and application. The type A mannose erythritol lipid nanoemulsion provided by the present invention has excellent antibacterial properties and can be used for food safety protection.
[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0005] The present invention provides a type A mannose erythritol lipid nanoemulsion, comprising the following components in percentage by weight:
[0006] 1-5% of type A mannose erythritol lipids, 0.1-0.75% of Tween 20, 0.1-0.75% of glycerol monooleate and the balance of water.
[0007] Preferably, the average particle size of the type A mannose-erythritol lipid nanoemulsion is 1 to 10 nm.
[0008] The present invention also provides a method for preparing the type A mannose erythritol lipid nanoemulsion described in the above technical solution, comprising the following steps:
[0009] Type A mannose erythritol lipid, Tween 20, glycerol monooleate and water are mixed, and shearing treatment and homogenization treatment are performed in sequence to obtain the type A mannose erythritol lipid nanoemulsion.
[0010] Preferably, the shearing treatment is performed at a rotation speed of 8000 to 12000 rpm and for a time of 5 to 10 minutes.
[0011] Preferably, the pressure of the homogenization treatment is 150-200 MPa and the temperature is 2-4°C.
[0012] Preferably, the homogenization process is performed 5 to 10 times.
[0013] Preferably, the time for each homogenization treatment is 120 to 300 seconds.
[0014] Preferably, the interval between two adjacent homogenization treatments is 30 to 60 seconds.
[0015] The present invention also provides the use of the type A mannose erythritol lipid nanoemulsion described in the above technical solution or the type A mannose erythritol lipid nanoemulsion prepared by the preparation method described in the above technical solution in antibacterial applications.
[0016] Preferably, the bacteria include Listeria monocytogenes.
[0017] The invention provides a type A mannose erythritol lipid nanoemulsion.
[0018] Mannosylerythritol lipid-A (MEL-A) is an amphiphilic biosurfactant containing a hydrophilic group primarily composed of 4-O-β-D-pyranose-erythritol and a hydrophobic group primarily composed of fatty acid chains. MEL-A exhibits broad-spectrum antimicrobial properties against a variety of microorganisms, including bacteria, fungi, and yeast. However, its water solubility is low. The present invention utilizes Tween 20 and glycerol monooleate as emulsifiers to encapsulate MEL-A within emulsion particles, forming oil-in-water emulsion particles. This improves the solubility of MEL-A without affecting the antimicrobial properties of Mannosylerythritol lipid-A, making it promising for use in food safety protection. Furthermore, the Tween 20 and glycerol monooleate of the present invention are both food-grade emulsifiers, reducing the safety risks of Mannosylerythritol lipid-A nanoemulsions as food antimicrobial agents in the food industry. The results of transcriptomic analysis showed that MEL-A nanoemulsion inhibited the growth of Listeria monocytogenes mainly by significantly downregulating the phosphotransferase system (PTS), resulting in insufficient bacterial carbohydrate supply.
[0019] The present invention also provides a method for preparing the type A mannose-erythritol lipid nanoemulsion described in the above technical solution. The preparation method provided by the present invention is simple to operate and easy to industrialize. Furthermore, the present invention produces a type A mannose-erythritol lipid nanoemulsion having a nanometer-scale particle size and a concentrated particle size distribution by sequentially performing shearing and homogenization treatments. Data from the examples show that the resulting MEL-A nanoemulsion has an average particle size of 4.582 nm and an aggregation index (PDI) of 0.248. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a physical picture of the MEL-A nanoemulsion obtained in Example 1;
[0021] Figure 2 The particle size distribution diagram of the MEL-A nanoemulsion obtained in Example 1;
[0022] Figure 3 This is the particle size distribution diagram of the MEL-A emulsion obtained in Comparative Example 1;
[0023] Figure 4 This is the particle size distribution diagram of the MEL-A nanoemulsion obtained in Comparative Example 2;
[0024] Figure 5 This is a graph showing the effect of the MEL-A nanoemulsion obtained in Example 1 on the bacterial activity of Listeria monocytogenes;
[0025] Figure 6 This is a graph showing the effect of the MEL-A emulsion obtained in Comparative Example 1 on the bacterial activity of Listeria monocytogenes;
[0026] Figure 7 This is a graph showing the effect of the MEL-A emulsion obtained in Comparative Example 2 on the bacterial activity of Listeria monocytogenes;
[0027] Figure 8 This is the volcano plot of differentially expressed genes after treatment with the MEL-A nanoemulsion obtained in Example 1;
[0028] Figure 9 Bubble chart of KEGG enrichment analysis of differentially expressed genes. DETAILED DESCRIPTION
[0029] The present invention provides a type A mannose erythritol lipid nanoemulsion, comprising the following components in percentage by weight:
[0030] 1-5% of type A mannose erythritol lipids, 0.1-0.75% of Tween 20, 0.1-0.75% of glycerol monooleate and the balance of water.
[0031] Unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.
[0032] The present invention provides a type A mannose erythritol lipid nanoemulsion comprising 1-5% of type A mannose erythritol lipid (MEL-A), preferably 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% by weight. In the present invention, the type A mannose erythritol lipid has the following structure:
[0033]
[0034] In the present invention, the preparation method of the type A mannose erythritol lipid preferably comprises the following steps:
[0035] The aphid yeast P. aphidis DSM 70725 is inoculated into a culture medium and fermented to obtain the type A mannose erythritol lipid.
[0036] In the present invention, the P. aphidis DSM 70725 bacterium is preferably an activated P. aphidis DSM 70725 bacterium; in the present invention, the preparation method of the activated P. aphidis DSM 70725 bacterium is not specifically limited, and operations well known to those skilled in the art can be used.
[0037] In the present invention, the formula of the culture medium is preferably: soybean oil 80.0 mL / L, glucose 30.0 g / L, MgSO4·7H2O 0.3 g / L, KH2PO4 0.3 g / L, NaNO3 3.0 g / L, and yeast extract 3.0 g / L.
[0038] In the present invention, the fermentation is preferably carried out in a fermenter. In the present invention, the fermentation time is preferably 7 days.
[0039] After fermentation, the present invention preferably further comprises: mixing the obtained crude fermentation product with an equal volume of ethyl acetate, separating by extraction, and collecting the organic layer; removing the solvent from the organic layer to obtain crude mannose erythritol lipid; sequentially washing the crude mannose erythritol lipid with methanol and cyclohexane to obtain a crude mannose erythritol lipid; and purifying the crude mannose erythritol lipid with a silica gel column to obtain the type A mannose erythritol lipid. In the present invention, the method for removing the solvent from the organic layer is preferably vacuum filtration. The present invention does not specifically limit the operation of the vacuum filtration, as long as the ethyl acetate can be completely removed. In the present invention, the number of methanol washes is preferably one, and the number of cyclohexane washes is preferably one; the methanol wash and cyclohexane wash can remove residual oil and fatty acids. In the present invention, the mobile phase A for silica gel column purification is preferably dichloromethane, the mobile phase B is preferably a mixture of ethyl acetate and methanol, the volume ratio of ethyl acetate to methanol in the mixture of ethyl acetate and methanol is preferably 1:1, and the volume ratio of mobile phase A to mobile phase B is preferably 1:1.
[0040] In the present invention, Mannosylerythritol lipid-A (MEL-A) is a biological surfactant with amphiphilic properties, which contains a hydrophilic group mainly composed of 4-O-β-D-pyranose-erythritol and a hydrophobic group mainly composed of fatty acid chains.
[0041] In terms of mass percentage, the type A mannose erythritol lipid nanoemulsion provided by the present invention includes Tween 200.1-0.75%, specifically preferably 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7% or 0.75%.
[0042] In terms of mass percentage, the type A mannose erythritol lipid nanoemulsion provided by the present invention includes 0.1% to 0.75% of glycerol monooleate, specifically preferably 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7% or 0.75%.
[0043] Calculated by weight percentage, the type A mannose erythritol lipid nanoemulsion provided by the present invention includes a balance of water, and the water is preferably deionized water.
[0044] In the present invention, although MEL-A has broad-spectrum antimicrobial properties against a variety of microorganisms (including bacteria, fungi, and yeast), it has low water solubility. The present invention uses Tween 20 and glycerol monooleate as emulsifiers to encapsulate MEL-A to form an oil-in-water emulsion, which improves MEL-A's solubility in water and thus enhances its application effect.
[0045] In the present invention, the average particle size of the type A mannose erythritol lipid nanoemulsion is preferably 1 to 10 nm, and more preferably 4.582 nm.
[0046] The present invention also provides a method for preparing the type A mannose erythritol lipid nanoemulsion described in the above technical solution, comprising the following steps:
[0047] Type A mannose erythritol lipid, Tween 20, glycerol monooleate and water are mixed, and shearing treatment and homogenization treatment are performed in sequence to obtain the type A mannose erythritol lipid nanoemulsion.
[0048] In the present invention, the shearing treatment is preferably carried out at a rotation speed of 8000 to 12000 rpm, more preferably 10000 rpm, for a time of 5 to 10 min, more preferably 5 min, 6 min, 7 min, 8 min, 9 min or 10 min. In the present invention, the shearing treatment is preferably carried out in a high-speed shearing machine.
[0049] In the present invention, the pressure of the homogenization treatment is preferably 150-200 MPa, more preferably 180-195 MPa, specifically preferably 150 MPa, 155 MPa, 160 MPa, 165 MPa, 170 MPa, 175 MPa, 180 MPa, 185 MPa, 190 MPa, 193 MPa, 195 MPa or 200 MPa, and the temperature is preferably 2-4°C, more preferably 3°C. In the present invention, the number of homogenization treatments is preferably 5-10 times, specifically preferably 5 times, 6 times, 7 times, 8 times, 9 times or 10 times; the time for each homogenization treatment is preferably 120-300 s, specifically preferably 120 s, 150 s, 200 s, 250 s or 300 s; the interval between two adjacent homogenization treatments is preferably 30-60 s, specifically preferably 30 s, 40 s, 50 s or 60 s. In the present invention, the homogenization treatment is preferably carried out on a high-pressure homogenizer.
[0050] The present invention also provides the use of the type A mannose erythritol lipid nanoemulsion described in the above technical solution or the type A mannose erythritol lipid nanoemulsion prepared by the preparation method described in the above technical solution in antibacterial applications.
[0051] In the present invention, the bacteria preferably include Listeria monocytogenes.
[0052] In the present invention, when the type A mannose erythritol lipid nanoemulsion is used in the antibacterial field, the type A mannose erythritol lipid nanoemulsion is used in the form of a type A mannose erythritol lipid nanoemulsion solution. The concentration of the type A mannose erythritol lipid nanoemulsion solution is preferably 0.010 to 2.500 mg / mL, more preferably 0.010 to 0.313 mg / mL, and specifically preferably 0.313 mg / mL. The type A mannose erythritol lipid nanoemulsion has the best antibacterial property.
[0053] In the present invention, the antibacterial agent is preferably an antibacterial agent for the food field.
[0054] The following examples describe in detail the type A mannose erythritol lipid nanoemulsion provided by the present invention, its preparation method and application, but they should not be construed as limiting the scope of protection of the present invention.
[0055] Example 1
[0056] To produce MEL-A, a suspension of activated P. aphidis DSM 70725 bacteria was inoculated into a fermentation tank containing the following medium: 80.0 mL / L soybean oil, 30.0 g / L glucose, 0.3 g / L MgSO₄·7H₂O, 0.3 g / L KH₂PO₄, 3.0 g / L NaNO₃, and 3.0 g / L yeast extract. After 7 days of fermentation, a crude fermentation product was obtained. This product was mixed with an equal volume of ethyl acetate, and the organic layer was separated by extraction. The ethyl acetate was then removed from the organic layer by vacuum filtration to obtain crude mannoerythritol lipids. The crude mannoerythritol lipids were then washed twice with methanol and cyclohexane to remove residual oil and fatty acids, resulting in a highly pure crude mannoerythritol lipid. Finally, the product was separated and purified by silica gel column using mobile phase A: dichloromethane-mobile phase B (ethyl acetate-methanol, volume ratio of 1:1) as the mobile phase, wherein the volume ratio of mobile phase A to mobile phase B was 1:1, to obtain high-purity MEL-A.
[0057] Preparation of MEL-A nanoemulsion: 50 g / kg of MEL-A was used as the substrate, along with 7.5 g / kg of Tween 20 and 7.5 g / kg of glycerol monooleate, and finally 935 g / kg of water. The mixture was then treated with a high-speed shear at 10,000 rpm for 10 minutes to obtain a crude emulsion. The crude emulsion was placed in a second container and subjected to a high-pressure homogenizer. The mixture was homogenized for 6 cycles at 193 MPa and 3°C, with each homogenization lasting 300 seconds and a 60-second interval between homogenizations, to obtain the MEL-A nanoemulsion.
[0058] Comparative Example 1
[0059] 50 g / kg of MEL-A was used as the substrate, and 950 g / kg of water was added directly without adding an emulsifier. This was then treated with a high-speed shear at 10,000 rpm for 10 minutes to obtain a crude emulsion. The crude emulsion was placed in a second container and placed in a high-pressure homogenizer. The process was repeated six times at a pressure of 193 MPa and a temperature of 3°C, with each homogenization lasting 300 seconds and a 60-second interval between homogenizations, to obtain a MEL-A nanoemulsion.
[0060] Comparative Example 2
[0061] 50g / kg of MEL-A was used as the substrate, 10g / kg of soy lecithin was added, and finally 940g / kg of water was added. The mixture was then treated with a high-speed shear at 10,000 rpm for 10 minutes to obtain a crude emulsion. The crude emulsion was placed in a second container and placed in a high-pressure homogenizer. The mixture was homogenized six times at a pressure of 193 MPa and a temperature of 3°C, with each homogenization lasting 300 seconds and a 60-second interval between homogenizations, to obtain a MEL-A nanoemulsion.
[0062] Performance Characterization
[0063] 1. Appearance and particle size distribution of MEL-A nanoemulsion
[0064] Figure 1 This is a physical picture of the MEL-A nanoemulsion obtained in Example 1, as shown Figure 1 As shown in FIG, MEL-A nanoemulsion appears as a milky white emulsion when observed with the naked eye.
[0065] The average particle size and potential of the MEL-A nanoemulsions obtained in Example 1 and Comparative Examples 1 and 2 were measured using a Zetasizer Nano particle size potential analyzer. Figures 2 to 4 shown.
[0066] Figure 2 This is the particle size distribution diagram of the MEL-A nanoemulsion obtained in Example 1, Figure 2 The average particle size of the nanoemulsion was 4.582 nm, and the aggregation index (PDI) was 0.248. The small PDI value indicates that the particle size distribution of the MEL-A nanoemulsion is relatively uniform.
[0067] Figure 3 The particle size distribution diagram of the MEL-A emulsion obtained in Comparative Example 1 is shown in FIG. Figure 3 As shown in FIG. 3 , the average particle size of the obtained MEL-A emulsion is 3106 nm, and the aggregation index (PDI) is 0.351. These average particle size and PDI are much larger than those obtained when 7.5 g / kg of Tween 20 and 7.5 g / kg of glycerol monooleate are used as emulsifiers.
[0068] Figure 4 The particle size distribution diagram of the MEL-A nanoemulsion obtained in Comparative Example 2 is shown in FIG. Figure 4 As shown in FIG, the average particle size of the obtained MEL-A nanoemulsion was 44.15 nm, and the aggregation index (PDI) was 0.527. Both the average particle size and the aggregation index were greater than those when 7.5 g / kg of Tween 20 and 7.5 g / kg of glycerol monooleate were used as emulsifiers.
[0069] 2. Antibacterial activity of MEL-A nanoemulsion against Listeria monocytogenes
[0070] First, the MEL-A nanoemulsion obtained in the examples and comparative examples was prepared into a 5 mg / mL stock solution, and then the MEL-A nanoemulsion was added to the BHI culture medium using a two-fold serial dilution method to obtain final concentrations of 0, 0.005, 0.010, 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.250, and 2.500 mg / mL, respectively. Listeria monocytogenes was cultured in BHI at 37°C to the logarithmic phase, then washed three times with phosphate buffered saline (PBS) and diluted to obtain a final concentration of 1.0×10 6 CFU / mL of bacterial liquid. Then, 1mL of bacterial liquid was inoculated into 50mL of BHI medium containing different concentrations of MEL-A nanoemulsion. BHI medium without MEL-A nanoemulsion was set as the control group, and 3 parallels were set up for each group. The conical flask was shaken and cultured in a shaker (37°C, 180rpm). After 12 hours, the bacterial growth was observed and the OD600nm value was measured with a microplate reader. The results are shown in Figure 2. Figures 5 to 7 shown.
[0071] Figure 5 The effect of the MEL-A nanoemulsion obtained in Example 1 on the bacterial activity of Listeria monocytogenes is shown in FIG. Figure 5 As shown, the MEL-A nanoemulsion obtained in Example 1 can significantly inhibit the growth of Listeria monocytogenes in the concentration range of 0.010 to 2.500 mg / mL, and the antibacterial effect increases with the increase of MEL-A nanoemulsion concentration in the concentration range of 0.010 to 0.313 mg / mL. When the concentration of MEL-A nanoemulsion reaches 0.313 mg / mL, the antibacterial effect is most significant, and can inhibit more than 50% of bacterial growth. When the concentration of MEL-A nanoemulsion exceeds 0.313 mg / mL, the effect gradually weakens.
[0072] Figure 6 The effect of the MEL-A emulsion obtained in Comparative Example 1 on the bacterial activity of Listeria monocytogenes is shown in FIG. Figure 6 As shown in the results, the MEL-A emulsion without emulsifier failed to inhibit the growth of Listeria monocytogenes in the concentration range of 0.005-0.625 mg / mL. Only when the concentration of the nanoemulsion reached 1.250 mg / mL could the growth of Listeria monocytogenes be inhibited, and the antibacterial effect was significantly weakened.
[0073] Figure 7 The effect of the MEL-A emulsion obtained in Comparative Example 2 on the bacterial activity of Listeria monocytogenes is shown in FIG. Figure 7As shown in the results, when soybean lecithin was used as an emulsifier, the MEL-A nanoemulsion was unable to inhibit the growth of Listeria monocytogenes within the concentration range of 0.005-0.313 mg / mL. Only when the concentration of the nanoemulsion reached 0.625 mg / mL could it inhibit the growth of Listeria monocytogenes, and the antibacterial effect was significantly weakened.
[0074] MEL-A was dispersed in water at a concentration of 0.010 to 2.500 mg / mL to form MEL-A dispersions of different concentrations. The effects of MEL-A dispersions of different concentrations on the bacterial activity of Listeria monocytogenes were measured. The results showed that when MEL-A dispersions were directly dispersed in water without being prepared into nanoemulsions, there was no antibacterial effect.
[0075] 3. Effect of MEL-A nanoemulsion on gene expression of Listeria monocytogenes
[0076] The test steps are:
[0077] (1) Preparation of transcriptome sequencing samples: approximately 1×10 6 CFU of Listeria monocytogenes was inoculated into 50 mL of BHI medium. The experimental group was added with the MEL-A nanoemulsion obtained in Example 1 at a concentration of 0.313 mg / mL, and the control group was added with an equal volume of sterile water. The culture was cultured in a shaker (37°C, 180 rpm) for 12 h, then removed and centrifuged at 4000 rpm for 5 min. The supernatant was discarded and the cells were quickly frozen in liquid nitrogen for storage. Three replicates were performed in each group.
[0078] (2) RNA extraction and detection: RNA samples from the experimental group and the control group were extracted according to the instructions of the Takara RNA extraction kit, and the RNA was tested for contamination, content, and integrity by agarose gel electrophoresis and Agilent 2100 bioanalyzer.
[0079] (3) Construction and sequencing analysis of the on-machine library: The construction and sequencing of the on-machine library were completed by Beijing Novogene Technology Co., Ltd. The sequencing platform was Illumina HiSeq PE150, and the sequencing generated 150 bp paired-end reads.
[0080] (4) Quality control and alignment analysis of off-machine data: The raw reads obtained from the machine were processed by removing adapters, removing reads with uncertain bases, removing low-quality reads, and calculating Q20, Q30, and GC content to obtain high-quality clean reads. Bowtie2 software was then used to perform genomic localization analysis on the filtered high-quality clean reads.
[0081] (5) Quantification of gene expression and identification of differentially expressed genes: The number of reads mapped to each gene was calculated using HTSeq software (v0.6.1), the FPKM value of each gene was calculated based on the gene length, and the number of reads mapped to each gene was counted. The DESeq2 R package (1.20.0) was used to identify differentially expressed genes between the experimental and control groups. Genes that simultaneously satisfied |log2(FoldChange)|>1 and p<0.05 were identified as differentially expressed genes. The results are shown in Figure 2. Figure 8 As shown, Figure 8 This is the volcano plot of differentially expressed genes under the treatment of MEL-A nanoemulsion obtained in Example 1, as shown in Figure 8 As shown, differentially expressed genes (DEGs) were screened based on p < 0.05 and |log2 Fold Change| > 1. Compared with the control group, a total of 528 differentially expressed genes were identified in the MEL-A nanoemulsion-treated group, including 311 upregulated genes and 217 downregulated genes. The results showed that there were significant differences in gene expression between the MEL-A nanoemulsion-treated group and the control group.
[0082] (6) KEGG enrichment analysis: KOBAS software was used to perform KEGG enrichment analysis on differentially expressed genes. KEGG enrichment-related bubble charts and bar charts were drawn using the Novogene cloud platform (https: / / magic.novogene.com). The results are shown in the figure below. Figure 9 shown. Figure 9 This is a bubble chart of KEGG enrichment analysis of differentially expressed genes. KEGG (Kyoto Encyclopedia of Genes and Genomes) is a comprehensive database that integrates genomic, chemical and system functional information. With the help of KEGG enrichment analysis, we can deeply explore the key metabolic pathways of differentially expressed genes. Figure 9As shown in the figure, the pathway with the most significant enrichment (padj < 0.05) of differentially expressed genes is the phosphotransferase system (PTS). The PTS system exists in true bacteria and a few archaea, but not in animals and plants. The PTS system contains two cytoplasmic phosphotransferase proteins (EI and HPr) and a species-dependent, variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC, IID). The PTS system has catalytic and regulatory functions, which can catalyze the transport and phosphorylation of various sugars and sugar derivatives, and also performs a variety of regulatory functions related to carbon, nitrogen and phosphate metabolism, chemotaxis, potassium transport and the virulence of certain pathogens. The first step of the phosphorylation cascade catalyzed by PTS is the reaction of EI with phosphoenolpyruvate (PEP) The PTS system is a key target for several antimicrobial agents. In addition to chemical antimicrobial agents, physical sterilization methods such as pulsed magnetic fields and high hydrostatic pressure can also lead to significant downregulation of the PTS system in Listeria monocytogenes. Therefore, the inhibition of PTS sugar transport and sugar phosphorylation by MEL-A nanoemulsion, which leads to insufficient carbohydrate supply to the bacteria, is an important reason for its inhibition of L. monocytogenes growth.
[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A type A mannose erythritol lipid nanoemulsion comprising the following components in percentage by weight: 1-5% of type A mannose erythritol lipids, 0.1-0.75% of Tween 20, 0.1-0.75% of glycerol monooleate and the balance of water.
2. The type A mannose erythritol lipid nanoemulsion according to claim 1, characterized in that The average particle size of the type A mannose erythritol lipid nanoemulsion is 1 to 10 nm.
3. The method for preparing the type A mannose erythritol lipid nanoemulsion according to claim 1 or 2, characterized in that: The following steps are involved: Type A mannose erythritol lipid, Tween 20, glycerol monooleate and water are mixed, and shearing treatment and homogenization treatment are performed in sequence to obtain the type A mannose erythritol lipid nanoemulsion.
4. The preparation method according to claim 3, characterized in that The shearing treatment is performed at a rotation speed of 8000 to 12000 rpm and for a time of 5 to 10 minutes.
5. The preparation method according to claim 3, characterized in that The pressure of the homogenization treatment is 150-200 MPa and the temperature is 2-4°C.
6. The preparation method according to claim 3 or 5, characterized in that The number of times of the homogenization treatment is 5 to 10 times.
7. The preparation method according to claim 6, characterized in that The time for each homogenization treatment is 120 to 300 seconds.
8. The preparation method according to claim 6, characterized in that The interval between two adjacent homogenization treatments was 30 to 60 seconds.
9. Use of the type A mannose erythritol lipid nanoemulsion according to claim 1 or 2 or the type A mannose erythritol lipid nanoemulsion prepared by the preparation method according to any one of claims 3 to 8 in antibacterial applications.
10. The use according to claim 9, characterized in that The bacteria include Listeria monocytogenes.