阅读说明：本技术 丁香酚-槐糖脂纳米乳液及其制备方法与应用 (Eugenol-sophorolipid nano emulsion and preparation method and application thereof ) 是由 范琳琳 王英 刘小莉 周剑忠 王帆 张宏志 朱勇生 于 2021-08-06 设计创作，主要内容包括：本发明公开了一种丁香酚-槐糖脂纳米乳液及其制备方法与应用,其中,所述丁香酚-槐糖脂纳米乳液由丁香酚、槐糖脂和水组成,对蜡样芽孢杆菌(Bacilluscereus)、志贺氏菌(Shigella Castellani)、金黄色葡萄球菌(Staphylococcus aureus)中的至少一种微生物起抑制作用。本发明丁香酚-槐糖脂纳米乳液稳定性好,不仅可以使丁香酚增溶,而且槐糖脂与丁香酚起到协同抑菌效果,可以作为配料应用于防腐剂、抑菌涂膜、抗菌包装膜等领域中,且制备方法简单、不需要助表面活性剂,成本低,具有很大的市场前景。(The invention discloses a eugenol-sophorolipid nanoemulsion and a preparation method and application thereof, wherein the eugenol-sophorolipid nanoemulsion consists of eugenol, sophorolipid and water and plays a role in inhibiting at least one microorganism in bacillus cereus (Bacillus cereus), Shigella (Shigella Castellani) and Staphylococcus aureus (Staphylococcus aureus). The eugenol-sophorolipid nanoemulsion disclosed by the invention is good in stability, can be used for solubilizing eugenol, has a synergistic antibacterial effect with the sophorolipid, can be used as an ingredient to be applied to the fields of preservatives, antibacterial coating films, antibacterial packaging films and the like, is simple in preparation method, does not need a cosurfactant, is low in cost and has a great market prospect.)

1. The eugenol-sophorolipid nanoemulsion is characterized by comprising eugenol, sophorolipid and distilled water, wherein the total mass of the eugenol and the sophorolipid in each 1L of water is 0.5-10 g, and the mass ratio of the eugenol to the sophorolipid is 1: 9-9: 1.

2. The eugenol-sophorolipid nanoemulsion as claimed in claim 1, wherein the nanoemulsion is less than 500nm in particle size.

3. The method for preparing the eugenol-sophorolipid nanoemulsion as claimed in claim 1 or 2, comprising the steps of:

(1) dissolving eugenol and sophorolipid in formula ratio in organic solvent, drying solvent and drying;

(2) dropwise adding distilled water, and uniformly mixing by vortex to obtain the nano emulsion.

4. The method according to claim 3, wherein in the step (1), the organic solvent is selected from chloroform, acetone, ethyl acetate, methanol and ethanol.

5. The preparation method according to claim 3, wherein in the step (1), the solvent is dried by a nitrogen blower at a nitrogen flow rate of 1.2-2.5L/min and a temperature of 20-45 ℃ overnight in vacuum, so as to ensure no residue of the solvent.

6. The method according to claim 3, wherein in the step (1), the eugenol and the sophorolipid are mixed in a mass ratio of 1.5 to 9: 1.

7. The preparation method according to claim 3, wherein in the step (2), distilled water is added dropwise at a rate of 50-100 uL/time, and after each addition, the mixed solution is vortexed and mixed at 800-1500 rpm and is kept still, and the water addition frequency is controlled to enable the turbidity A of the mixed solution₆₀₀And (3) 0.01-1.0, thus obtaining the nano emulsion.

8. Use of the eugenol-sophorolipid nanoemulsion according to claim 1 or 2 for inhibiting the growth of microorganisms.

9. The use according to claim 8, wherein the microorganism is selected from Bacillus cereus, Shigella Shigella Castellani, Staphylococcus aureus.

10. The application according to claim 8, characterized in that it comprises the following steps: adding the eugenol-sophorolipid nano emulsion into a culture medium, inoculating at least one microorganism of Bacillus cereus, Shigella Castellani and Staphylococcus aureus, and allowing the onset time to be 1-2 h and the bacteriostatic aging to be 24-72 h.

Technical Field

The invention belongs to an antibacterial technology, and particularly relates to eugenol-sophorolipid nanoemulsion as well as a preparation method and application thereof.

Background

Food safety has been a major public health problem worldwide, with health hazards caused by food-borne pathogens being of great concern. Common food-borne pathogenic bacteria include Escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Bacillus cereus (Bacillus cereus), Listeria monocytogenes (Listeria monocytogenes), Salmonella (Salmonella), and the like. The use of bacteriostatic agents is an effective method for effectively inhibiting and killing pathogenic microorganisms in food. With the pursuit of consumers for health products, green, safe and efficient natural bacteriostatic agents have become a necessary trend for the research and application development of the bacteriostatic agent field.

The clove essential oil extracted from clove buds is light yellow or colorless oily liquid, has special fragrant smell, and is an antibacterial medicine with wide application. The clove essential oil can obviously inhibit the growth and reproduction of bacteria and fungi. Eugenol is the main antibacterial active ingredient of clove essential oil. But the eugenol has poor water solubility and is easy to volatilize, the application effect in a water-containing system is not good, and the eugenol is a fat-soluble component and is easy to combine with components such as lipid and the like to influence the efficacy. The emulsion system composed of surfactant, water, oil phase or cosurfactant can increase oil-soluble components and stabilize activity.

The sophorolipid is a glycolipid surfactant obtained by microbial fermentation and transformation, has good emulsibility, surface/interface activity, biodegradability and the like, simultaneously has physiological activities such as bacteriostatic activity, tumor cell apoptosis induction and mutation induction and the like, and has potential application value in the fields of food, cosmetics, environmental protection, medicines, materials and the like. In particular to be used as an emulsifier for the construction of an emulsion system. Under the condition of no cosurfactant, the sophorolipid, the eugenol and the water can form a transparent or semitransparent dispersion system with isotropy and thermodynamic stability, and the dispersed phase particle is 1-200 nm. Because both the eugenol and the sophorolipid have antibacterial activity, the eugenol-sophorolipid nanoemulsion not only can solubilize the eugenol, but also has antibacterial synergy.

At present, no eugenol-sophorolipid nano emulsion, preparation thereof and application in inhibiting the growth of food-borne pathogenic bacteria are reported.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the prior art, the invention provides eugenol-sophorolipid nanoemulsion and a preparation method and application thereof.

The technical scheme is as follows: the eugenol-sophorolipid nanoemulsion comprises eugenol, sophorolipid and distilled water, wherein the total mass of the eugenol and the sophorolipid in each 1L of water is 0.5-10 g, and the mass ratio of the eugenol to the sophorolipid is 1: 9-9: 1.

Further, the particle size of the nano emulsion is less than 500nm, the turbidity A600 of the nano emulsion is measured to be 0.01-1.0, and the particle size can be further determined by a nano particle size analyzer.

The preparation method of the eugenol-sophorolipid nanoemulsion comprises the following steps of:

(1) dissolving eugenol and sophorolipid in formula ratio in organic solvent, drying solvent and drying;

(2) dropwise adding distilled water, and uniformly mixing by vortex to obtain the nano emulsion.

In the step (1), the organic solvent is selected from one of chloroform, acetone, ethyl acetate, methanol and ethanol.

In the step (1), a nitrogen blowing instrument is adopted for blow-drying, the nitrogen flow rate is 1.2-2.5L/min, the temperature is 20-45 ℃, and vacuum drying is carried out overnight, so that no solvent residue is ensured.

In the step (2), distilled water is gradually dripped into the mixture according to 50-100 uL, and after each addition, the mixed solution is uniformly mixed for 10min in a vortex manner under the condition of 800-1500 rpm, and is kept stand for 30min, and the water addition frequency is controlled to enable the turbidity A600 of the mixed solution to be 0.01-1.0, so that the nano emulsion is obtained.

The application of the eugenol-sophorolipid nanoemulsion in inhibiting the growth of microorganisms is also within the protection scope of the invention.

Wherein the microorganism is selected from Bacillus cereus (Bacillus cereus), Shigella (Shigella Castellani), and Staphylococcus aureus (Staphylococcus aureus).

The application comprises the following steps: adding the eugenol-sophorolipid nano emulsion into a culture medium, inoculating at least one microorganism of Bacillus cereus, Shigella Castellani and Staphylococcus aureus, and allowing the onset time to be 1-2 h and the bacteriostatic aging to be 24-72 h.

Further, the concentration of the eugenol-sophorolipid nano emulsion is more than 0.5 g/L.

Has the advantages that: compared with the prior art, the method has the following advantages: (1) the nano emulsion only consists of eugenol, sophorolipid and water, does not need to add cosurfactant, has high stability, can solubilize the eugenol, has the synergistic effect of the sophorolipid and the eugenol, has obvious bacteriostatic effect on common food-borne pathogenic bacteria, and has broad spectrum. (2) The invention has simple operation method and low cost, is suitable for the fields of preservatives, bacteriostatic coating films, antibacterial packaging films and the like, and has wide market prospect.

Drawings

FIG. 1 shows the particle size change of eugenol-sophorolipid (ratio 9:1) nanoemulsion stored at 4 deg.C, 25 deg.C, and 37 deg.C for 15 days;

FIG. 2 shows the inhibition results of different mass ratios of nano-emulsion prepared from eugenol and sophorolipid on Bacillus cereus (A), Staphylococcus aureus (B) and Shigella (C) after 24h culture;

FIG. 3 shows the growth of Bacillus cereus treated with eugenol-sophorolipid (ratio 9:1) nanoemulsion for 72 h;

FIG. 4 is a graph of the morphology of Bacillus cereus in the control (untreated) group cultured for 2 h;

FIG. 5 shows the form of Bacillus cereus cultured for 2h in the nanoemulsion-treated group of eugenol-sophorolipid (ratio 9: 1);

FIG. 6 shows the morphology of sophorolipid in a single group of Bacillus cereus cultured for 2 h;

FIG. 7 is a diagram of the morphology of eugenol single component treated group Bacillus cereus cultured for 2 h;

FIG. 8 shows the morphology of positive control nisin-treated groups of Bacillus cereus cultured for 2 h.

Detailed Description

The present application will be described in detail with reference to specific examples.

Sophorolipid is a glycolipid type biosurfactant produced by metabolism of saccharomycetes, has excellent surface/interface activity, emulsibility, stability and biodegradability, is safe and non-toxic, has physiological activities such as bacteriostatic activity, tumor cell apoptosis induction and variation induction and the like, and has potential application value in the fields of food, cosmetics, environmental protection, medicines, materials and the like.

Acid type and lactone type sophorolipid structural formula

The sophorolipid can be obtained by fermentation, can be a fermentation liquor extract or a concentrate, can be a pure product obtained by separation and purification of an organic solvent, and can be liquid, semisolid, solid paste or powder.

The fermentation preparation method of sophorolipid comprises the following steps:

candida globulifera is activated by YPD culture medium (g/L, glucose 15.0, peptone 20.0, yeast powder 10.0), and the activated liquid seed is inoculated to 100mL seed culture medium (g/L, glucose 20.0, yeast powder 3.0, KH) at 2%₂PO₄0.3，MgSO₄·7H₂O 0.3，Na₂HPO₄0.3) in a 300mL Erlenmeyer flask, and incubated at 30 ℃ for 10 hours at 200 rpm. The activated seed solution was inoculated into a flask containing 100mL of the fermentation medium at an inoculum size of 2%, and cultured at 30 ℃ for 7 days at 200 rpm. And after the fermentation is finished, extracting for 3 times by adopting equal volume of ethyl acetate to obtain the sophorolipid.

Sophorolipids are also commercially available as CAS 148409-20-5.

Eugenol is commercially available as CAS 97-53-0.

Example 1 preparation of eugenol-sophorolipid nanoemulsion

Weighing eugenol and sophorolipid according to the mass ratio of 9:1, dissolving in chloroform, blow-drying the solvent by a nitrogen blowing instrument, and drying overnight in vacuum to completely remove the solvent. Gradually adding distilled water dropwise according to 100 μ L, mixing the mixture at 800rpm for 10min, standing and balancing for 30 min. The water addition amount is controlled until the ratio of the mixed components of the eugenol and the sophorolipid is 0.5g/L, the turbidity A600 is 0.2, and the average grain diameter of the emulsion is 137nm by adopting a laser nanometer particle size analyzer.

Example 2

Weighing eugenol and sophorolipid according to the mass ratio of 8:2, dissolving in chloroform, blow-drying the solvent by a nitrogen blowing instrument, and drying overnight in vacuum to completely remove the solvent. Gradually adding distilled water dropwise according to 100 μ L, mixing the mixture at 800rpm for 10min, standing and balancing for 30min after each addition. The water addition amount is controlled until the ratio of the mixed components of the eugenol and the sophorolipid is 0.5g/L, the turbidity A600 is 0.8, and the average grain diameter of the emulsion is measured to be 175nm by adopting a laser nanometer particle size analyzer.

Example 3

Weighing eugenol and sophorolipid according to the mass ratio of 1:9, dissolving in chloroform, blow-drying the solvent by a nitrogen blowing instrument, and drying overnight in vacuum to completely remove the solvent. Gradually adding distilled water dropwise according to 50 μ L, mixing the mixture at 800rpm for 10min, standing and balancing for 30min after each addition. The water addition amount is controlled until the ratio of the mixed components of the eugenol and the sophorolipid is 10g/L, the turbidity A600 is 1.0, and the average grain diameter of the emulsion is 301nm by adopting a laser nanometer particle size analyzer.

Example 4

Weighing eugenol and sophorolipid according to the mass ratio of 9:1, dissolving in chloroform, blow-drying the solvent by a nitrogen blowing instrument, and drying overnight in vacuum to completely remove the solvent. Gradually adding distilled water dropwise according to 100 μ L, mixing the mixture at 1000rpm for 10min, standing and balancing for 30 min. The water addition amount is controlled until the ratio of the mixed components of the eugenol and the sophorolipid is 0.5g/L, the turbidity A600 is 0.01, and the average grain diameter of the emulsion is measured to be 52nm by adopting a laser nanometer particle size analyzer.

Example 5

Weighing eugenol and sophorolipid according to the mass ratio of 6:4, dissolving in chloroform, blow-drying the solvent by a nitrogen blowing instrument, and drying overnight in vacuum to completely remove the solvent. Gradually adding distilled water dropwise according to 100 μ L, mixing the mixture at 1000rpm for 10min, standing and balancing for 30min after each addition. The water addition amount is controlled until the ratio of the mixed components of the eugenol and the sophorolipid is 0.5g/L, the turbidity A600 is 1.0, and the average particle size of the emulsion is 189nm by adopting a laser nanometer particle size analyzer.

Stability determination of eugenol-sophorolipid nanoemulsion

Storing freshly prepared eugenol-sophorolipid nanoemulsion at the temperature of 4, 25 and 37 ℃, measuring the average particle size every 3d, and evaluating the stability of the eugenol-sophorolipid nanoemulsion. As shown in FIG. 1, the particle size distribution of the prepared eugenol-sophorolipid nanoemulsion is below 200nm, and the prepared eugenol-sophorolipid nanoemulsion is transparent liquid, has small particle size change under the temperature conditions of 4, 25 and 37 ℃, and has good storage stability.

Example 6 bacteriostatic methods and assays

(1) Preparation of the bacterial suspension

Selecting 1-2 rings of the microorganism preserved on the inclined plane by using an inoculating ring, inoculating the microorganism into an LB culture medium, culturing for 12 hours in a full-temperature shaking table with the rotating speed of 37 ℃ and 180r/min, taking 1mL of bacterial liquid out of a centrifuge tube, centrifuging for 3-5 min at the conditions of 8000-15000 rpm and 4 ℃, collecting the bacterial body, washing and resuspending the bacterial body for at least 3 times by using sterile physiological saline, and finally preparing a bacterial suspension with the concentration of 1 multiplied by 10 by using the sterile physiological saline, wherein the bacterial suspension has the concentration of 1 multiplied by 10⁷CFU/mL。

(2) Determination of minimum inhibitory concentration

And (3) determining the minimum bacteriostatic concentration of the nano emulsion by a double gradient dilution method. The indicator bacteria in logarithmic phase are diluted to 10 degrees by LB culture medium gradient⁵CFU/mL, 100. mu.L of the bacterial liquid is respectively taken and put in a 96-well plate, and 100. mu.L of the nano-emulsion gradient diluent is added into each well. Six replicates were prepared with 200. mu.L LB medium as negative blank, and 100. mu.L of diluted broth and 100. mu.L of LB broth as positive blank. After mixing, the 96-well plate is placed in an incubator for culturing for 18h at 37 ℃, and after taking out, the OD600 is measured by an enzyme-labeling instrument. Taking the concentration of the nano emulsion as an abscissa and the OD600 value as an ordinate to make a curve, and determining the inflection point as the minimum bacteriostatic concentration.

(3) Bacteriostatic effect of nano emulsion

Treating the bacterial suspension by using the nano emulsion with the minimum bacteriostatic concentration, placing the bacterial suspension in an incubator for 24 hours at 37 ℃, taking out the bacterial suspension, and measuring OD600 by using an enzyme-labeling instrument. Six replicates of each sample were run against a blank supplemented with 100. mu.L of broth and 100. mu.L of LB broth. The bacteriostatic rate is calculated according to the following steps: the bacteriostatic ratio = (control group bacterial quantity-test group bacterial quantity)/control group bacterial quantity · 100%.

And evaluating the bacteriostasis aging by adopting a flat plate coating method. Specifically, 100. mu.L of the control or nanoemulsion-treated bacterial liquid was applied to a plate containing a broth medium, cultured at 37 ℃ for 72 hours, and sampled at 1 hour, 2 hours, 24 hours, and 72 hours to observe the growth of the bacteria. The inhibition result after 24h of culture is shown in fig. 2, and according to the graph, 0.5g/L eugenol-sophorolipid nano-emulsion is added into the bacterial liquid according to the minimum inhibitory concentration, and according to the OD600 results after 24h of culture of bacillus cereus, staphylococcus aureus and shigella shown in fig. 2(A, B, C), respectively, the bacterial liquid OD treated by the nano-emulsion is obviously reduced compared with that of a control group (CK), which indicates that the prepared eugenol-sophorolipid nano-emulsion has obvious inhibition effect on bacillus cereus, staphylococcus aureus and shigella. Wherein the content of the first and second substances,

the eugenol-sophorolipid nanoemulsion prepared in example 1 has an inhibition rate of 73.5% for bacillus cereus, 80.3% for staphylococcus aureus and 84.2% for shigella;

the eugenol-sophorolipid nanoemulsion prepared in example 2 has an inhibition rate of 82.9% for bacillus cereus, 67.9% for staphylococcus aureus and 76.0% for shigella;

the eugenol-sophorolipid nanoemulsion prepared in example 3 has an inhibition rate of 57.6% for bacillus cereus, 55.0% for staphylococcus aureus and 77.9% for shigella;

the eugenol-sophorolipid nanoemulsion prepared in example 4 has an inhibitory rate against bacillus cereus of 85.3%, an inhibitory rate against staphylococcus aureus of 79.4% and an inhibitory rate against shigella of 82.1%;

the eugenol-sophorolipid nanoemulsion prepared in example 5 had an inhibitory rate against bacillus cereus of 60.4% and an inhibitory rate against staphylococcus aureus of 64.3% and an inhibitory rate against shigella of 70.6%.

The growth condition after 72h of culture is shown in fig. 3, and according to the graph, the bacteria density is obviously reduced after 1h, 2h, 24h or 72h of culture of the bacteria in the eugenol-sophorolipid nanoemulsion treatment group compared with the control group, so that the onset time of the eugenol-sophorolipid nanoemulsion to the bacillus cereus is 1h, and the bacteriostatic time is 72 h.

And determining the influence of the nano emulsion on the bacterium morphology by adopting a scanning electron microscope. After the cultured bacterial suspension is centrifuged, 2.5% (v/v) glutaraldehyde solution is added into the bacterial sediment and fixed for 24h at 4 ℃. After the immobilization, the cells were collected by centrifugation and fixed again for 1.5h using a 1% osmate solution of pH 7.2. The fixed mycelia were rinsed 3 times with PBS buffer to remove excess fixative. The samples were sequentially placed in a series of 30%, 50%, 70%, 80%, 90% and 100% ethanol solutions for 15min each time for dehydration displacement. The displaced sample was rinsed twice with 100% isoamyl acetate to displace the ethanol, 15min each; spraying the thallus onto the critical point of copper net, drying, and spraying gold. The prepared slices are observed on a scanning electron microscope and images are collected. As shown in fig. 4 and 5, it is clear that, compared with the thallus phase state (fig. 4) of the bacillus cereus in the control group, the bacillus cereus treated with the eugenol-sophorolipid nanoemulsion shown in fig. 5 for 2 hours is morphologically destroyed and aggregated, which indicates that the eugenol-sophorolipid nanoemulsion has a significant inhibitory effect on bacillus cereus.

Comparative example 1 growth inhibition of Bacillus cereus by sophorolipid alone

Sophorolipid emulsion was prepared at the same concentration (0.05g/L) as used in the examples, and the results obtained by following the procedure of example 6 are shown in FIG. 6. Compared with the results in fig. 4 and 5, the single sophorolipid has a significantly poorer bacteriostatic effect on bacillus cereus for 2 hours than the eugenol-sophorolipid nanoemulsion.

Comparative example 2 growth inhibition of Bacillus cereus by eugenol alone

Eugenol emulsion (0.45g/L) with the same concentration as used in the example was prepared, and the results obtained by following the procedure of example 6 are shown in FIG. 7. Compared with the figures 4 and 5, the single eugenol has the obviously poorer bacteriostatic effect on the bacillus cereus for 2 hours than the eugenol-sophorolipid nanoemulsion.

Comparative example 3 growth inhibition of Bacillus cereus by Nisin

The natural bacteriostatic nisin commonly used in food is selected as a positive control. A nisin solution was prepared at a concentration of 2.0g/L at the minimum inhibitory concentration, according to the procedure of example 6, and the results are shown in FIG. 8. Compared with the figures 4 and 5, the bacteriostatic effect of nisin on bacillus cereus for 2 hours is obviously inferior to that of eugenol-sophorolipid nanoemulsion.

As can be seen from the above, the sophorolipid and eugenol of the nano emulsion provided by the invention have a synergistic antibacterial effect, have a remarkable antibacterial effect on common food-borne pathogens, are suitable for the fields of preservatives, antibacterial coating films, antibacterial packaging films and the like, and have a wide application prospect.