Pomegranate peel-clove essential oil nano microemulsion as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food, and discloses pomegranate peel-clove essential oil nano microemulsion as well as a preparation method and application thereof. A conversion method is adopted for preparation, clove essential oil is used as an oil phase, a pomegranate peel aqueous solution containing a mixed surfactant is used as a water phase, and filling or high-pressure homogenization is performed to prepare the clove essential oil-containing pomegranate peel essence. The prepared microemulsion is uniform in structure, free of layering and precipitation and good in fluidity, is in an O / W type, has the particle size between 93.76 + / -2.08 nm and 257.62 + / -19.49 nm, does not produce layering after being centrifuged for 15 minutes, and keeps stable after being placed at normal temperature for 30 days. The pomegranate peel essential oil has a bacteriostatic effect on food spoilage bacteria (shewanella putrefaciens, pseudomonas fluorescens and pseudomonas aeruginosa), it is determined that the pomegranate peel and the clove essential oil have a synergistic effect, and the FIC of the pomegranate peel and the FIC of the clove essential oil are 0.25, 0.5 and 0.5 respectively. The microemulsion can provide reference for solubilizing application and bacteriostatic ability improvement of plant extracts researched and developed by production enterprises and scientific research institutions.

Description

Technical Field

[0001] The invention belongs to the technical field of food, and relates to a pomegranate peel-clove essential oil nano-microemulsion and a preparation method and application thereof. Background Art

[0002] As consumers' demands for healthy, green and environmentally friendly food increase, the use of natural preservatives or the absence of chemical preservatives has become a hot topic in the current food industry. Studies have found that some natural plant products such as pomegranate peel, licorice, turmeric, cloves, etc., due to the presence of acids, polyphenols, natural essential oils and other active ingredients, can show a certain antibacterial effect and can be used as an alternative to chemical preservatives. However, a single natural preservative is often difficult to achieve a good preservative effect and the cost is high. Therefore, the industry has been looking for ways to enhance the antiseptic and antibacterial effects of natural extracts. Combined antibacterial uses the synergistic effect of different antibacterial substances, which can reduce the amount of use while expanding the antibacterial spectrum and improving the antibacterial effect. It has become a hot topic in the current natural antibacterial field.

[0003] Clove essential oil has attracted attention with its abundant sources and strong antibacterial and antiviral activity. It is a light yellow or colorless transparent oily substance with a special aroma of cloves, insoluble in water, easily soluble in alcohol, ether or glacial acetic acid, and its main bactericidal substance is eugenol (2-methoxy-4-allylphenol). Although clove essential oil has good biological activity, due to its volatility and insoluble in water, its application in the field of food is greatly limited. Therefore, how to improve its water solubility and reduce the dosage used and reduce its impact on the food flavor system is the main factor restricting clove essential oil as a natural preservative. At present, the application of clove essential oil mainly adopts microcapsule embedding technology, which reduces the volatility of clove essential oil and alleviates its impact on flavor by wall material screening, but the physical form of solid powder restricts its application in liquid food. Pomegranate peel, as a kind of medicine and food homologous raw material, is rich in polyphenols, flavonoids, alkaloids, and has good antioxidant and broad-spectrum antibacterial properties. However, pomegranate peel extract has a strong astringent taste and dark color, and is easy to precipitate in the food system, affecting the sensory quality of the food. If a substance with a synergistic effect with pomegranate peel can be found and its synergistic effect can be utilized, its dosage can be reduced, and its broad-spectrum antibacterial effect can be exerted without affecting the color and flavor of the food. CN200910113503.9 discloses a production method of pomegranate peel extract with antiviral effect and determines the use of macroporous resin separation technology to enrich flavonoid components; CN202110451310.5 provides a combined antibacterial substance containing pomegranate peel water extract and pepper seed water extract, which can effectively inhibit Staphylococcus aureus; most of these technologies are synergistic enhancement of aqueous phase systems.

[0004] Microemulsion is a system formed by water phase, oil phase, emulsifier and co-emulsifier in appropriate proportions. Studies have found that emulsions with an average droplet diameter of less than 500nm can show good stability and can therefore be used as carriers for poorly soluble volatile essential oils. By selecting surfactants with suitable hydrophilic-lipophilic balance (HBL) values ​​and co-surfactants with high safety, a microemulsion system with good water solubility and high stability is prepared. Microemulsions have the advantages of solubilization and stability, and are highly safe and suitable for use in food systems. If pomegranate peel and clove essential oil can be prepared into microemulsions, while exerting their synergistic effects, the product stability is increased and the antibacterial effect is ultimately improved. Summary of the invention

[0005] In view of the problems existing in the prior art, the primary purpose of the present invention is to provide a method for preparing a pomegranate peel-clove essential oil nano-microemulsion.

[0006] Another object of the present invention is to provide a pomegranate peel-clove essential oil nano-microemulsion.

[0007] Another object of the present invention is to provide an application of the pomegranate peel-clove essential oil nano-microemulsion.

[0008] To solve the above problems, the present invention adopts the following technical solutions:

[0009] A method for preparing a pomegranate peel-clove essential oil nano-microemulsion comprises the following steps: preparing the pomegranate peel-clove essential oil nano-microemulsion by a phase inversion method, taking clove essential oil as an oil phase and a pomegranate peel aqueous solution containing a mixed surfactant as a water phase, and preparing the nano-microemulsion by filling or high-pressure homogenization.

[0010] Preferably, the preparation method specifically comprises the following steps:

[0011] (1) Adding a mixed surfactant to the pomegranate peel aqueous solution, stirring to obtain a stable and uniform pomegranate peel aqueous phase system S SL ;

[0012] (2) Using clove essential oil as the oil phase, adding pomegranate peel water phase system S to clove essential oil SL , after stirring and balancing for a period of time, an oil-water mixture is obtained, and the pomegranate peel-clove essential oil nano-microemulsion is obtained by filling or high-pressure homogenization;

[0013] Wherein, the mixed surfactant is composed of a surfactant and a co-surfactant, and the Km value (surfactant / co-surfactant) = 2:1 to 6:1;

[0014] The volume ratio of the mixed surfactant to the clove essential oil is 9:1 to 7:3.

[0015] Preferably, it is characterized in that Km=2:1.

[0016] Preferably, the HLB value of the surfactant is 13-15.

[0017] Preferably, the surfactant is at least one of Tween60 (HLB=14.5), Tween80 (HLB=15), and polyoxyethylene castor oil EL (HLB=13.3);

[0018] The co-surfactant is anhydrous ethanol.

[0019] Preferably, the pomegranate peel aqueous phase system S SL The mass concentration of Tween60 is 1.5% to 2.5%, and the mass concentration of polyoxyethylene castor oil EL is 4% to 8%;

[0020] The amount of the co-surfactant added is formulated according to the Km value.

[0021] Preferably, the mass concentration of the pomegranate peel aqueous solution is 5% to 10%, and it is prepared from pomegranate peel concentrate;

[0022] The equilibrium temperature is 20-40° C. and the equilibrium time is 15-60 min.

[0023] Preferably, homogenization is performed at 40 to 80 MPa for 2 to 6 minutes, and the homogenization is circulated for 3 to 6 times.

[0024] Preferably, the stirring speed in step (1) is 100 to 300 r / min, and the stirring time is 30 to 60 min;

[0025] The stirring speed in step (2) is 100 to 300 r / min, and the stirring time is 5 to 30 min.

[0026] Preferably, the pomegranate peel concentrate is prepared by the following method:

[0027] The pomegranate peel is crushed, and ultrasonic extraction is performed with a 45%-75% ethanol solution at a solid-liquid ratio of 1:20 to 1:30, the ultrasonic extraction power is 100 to 300w, the extraction temperature is 40°C to 60°C, and the ultrasonic time is 30 to 90min; the ultrasonically treated solution is refluxed at 60°C to 100°C for 60 to 90min, and the extract 1 is collected after centrifugation at 4000r / min for 15min; the residue is refluxed twice with a 45%-75% ethanol solution at a solid-liquid ratio of 1:20 to 1:30, and the extract 2 is collected after centrifugation;

[0028] The extract 1 and the extract 2 were combined, and the total extract was heated and concentrated to a concentration of 1 g (crude drug) / mL to obtain a pomegranate peel concentrate, which was sterilized and refrigerated for later use.

[0029] Preferably, the eugenol content in the clove essential oil is ≥ 85%.

[0030] A pomegranate peel-clove essential oil nano-microemulsion is prepared by the above method.

[0031] Preferably, the pomegranate peel-clove essential oil nanoemulsion is of O / W type, with a particle size of <500 nm, more preferably 90 to 300 nm.

[0032] Application of the pomegranate peel-clove essential oil nano-microemulsion in inhibiting food spoilage bacteria.

[0033] Preferably, the food spoilage bacteria are Shewanella putrefciens, Pseudomonas fluorescens and Pseudomonas aeruginosa.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] (1) Using the Oxford cup method, it was determined that pomegranate peel and clove essential oils had a synergistic effect against Shewanella putrefaciens, Pseudomonas fluorescens, and Pseudomonas aeruginosa, with FICs of 0.25, 0.5, and 0.5, respectively.

[0036] (2) The product is bright yellow-brown in color, has a uniform texture, no stratification, no precipitation, and good fluidity. The microemulsion configuration is determined by the conductivity method, and its water content is determined to be >62.5%, which is an O / W type. The sample is tested for particle size using a Summary particle size analyzer, and it is determined that the particle size of the prepared pomegranate peel-clove essential oil nanoemulsion is 93.76±2.08nm to 257.62±19.49nm, all <500nm. No stratification occurs after centrifugation for 15 minutes, and it remains stable at room temperature for 30 days.

[0037] (3) The prepared pomegranate peel-clove essential oil nanoemulsion showed a strong antibacterial effect against Shewanella putrefaciens, Pseudomonas fluorescens and Pseudomonas aeruginosa. At the same time, high-pressure homogenization had little effect on the antibacterial ability of the microemulsion system. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 For WR SL-DX Inhibitory plot against Shewanella putrefaciens.

[0039] Figure 2 For WR SL-DX Antibacterial plot against Pseudomonas aeruginosa. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0041] The eugenol content in clove essential oil is ≥85%.

[0042] The pomegranate peel concentrate can be extracted by a common method in the art, or prepared by the following method:

[0043] The pomegranate peel is crushed, and ultrasonic extraction is performed with a 45%-75% ethanol solution at a solid-liquid ratio of 1:20 to 1:30, the ultrasonic extraction power is 100 to 300w, the extraction temperature is 40°C to 60°C, and the ultrasonic time is 30 to 90min; the ultrasonically treated solution is refluxed at 60°C to 100°C for 60 to 90min, and the extract 1 is collected after centrifugation at 4000r / min for 15min; the residue is refluxed twice with a 45%-75% ethanol solution at a solid-liquid ratio of 1:20 to 1:30, and the extract 2 is collected after centrifugation;

[0044] The extract 1 and the extract 2 were combined, and the total extract was heated and concentrated to a concentration of 1 g (crude drug) / mL to obtain a pomegranate peel concentrate, which was sterilized and refrigerated for later use.

[0045] The test methods involved in the following embodiments are:

[0046] Oxford cup method: Pour 10 mL of 1.5% agar into the plate, and after solidification, gently place the autoclaved Oxford cup on the agar; wait for the nutrient agar medium to cool to 45°C, add 100 μL of spoilage bacteria (1×10 7 CFU / mL), mix well and pour 15mL broth medium around the Oxford cup to make the surface of the plate flat. Place the plate on the clean bench and wait for the second layer of agar to solidify. Use tweezers to carefully pull out the Oxford cup (be careful not to damage the surrounding agar). Add 100μl of antibacterial substance to each well, transfer it to a 4℃ refrigerator for diffusion and penetration for 24h, take it out and place it in a 37℃ biochemical incubator for 24h. Determine the antibacterial ability of the antibacterial substance according to the size of the antibacterial circle.

[0047] Conductivity method: Place the pomegranate peel-clove essential oil microemulsion in a beaker and stir it magnetically at room temperature for 15 minutes (150r / min). Operate the conductivity meter according to the instruction manual and record the readings.

[0048] Example 1

[0049] (1) The pomegranate peel concentrate was prepared into a solution with a mass fraction of 10% and used as the solvent of a mixed surfactant, wherein the mixed surfactant was composed of Tween 60 and anhydrous ethanol in a certain proportion, wherein Tween 60 was a surfactant and anhydrous ethanol was a co-surfactant (Km=2:1), and the mass concentration of Tween 60 in the solution was 2%. After magnetic stirring for 45 min (150 r / min), a stable and uniform pomegranate peel aqueous phase system (S SL );

[0050] (2) Add pomegranate peel aqueous phase system to clove essential oil in a ratio of mixed surfactant: clove essential oil = 8:2 (volume ratio), stir magnetically for 15 min (150 r / min), place in a 30° C. water bath for equilibration for 60 min to obtain an oil-water mixture, and directly fill it to obtain Example 1 (WR SL-DX ).

[0051] Example 2

[0052] (1) The pomegranate peel concentrate was prepared into a 7.5% solution and used as a solvent for a mixed surfactant, wherein the mixed surfactant was composed of Tween 60 and anhydrous ethanol in a certain proportion, wherein Tween 60 was selected as a surfactant and anhydrous ethanol was selected as a co-surfactant (Km=2:1), the mass concentration of Tween 60 in the solution was 2%, and a stable and uniform pomegranate peel aqueous phase system (S) was obtained after magnetic stirring for 45 min (150 r / min). SL );

[0053] (2) Add pomegranate peel aqueous phase system to clove essential oil in a ratio of mixed surfactant: clove essential oil = 7:3 (volume ratio), stir magnetically for 15 min (150 r / min), place in a 30° C. water bath for equilibration for 30 min to obtain an oil-water mixture, and homogenize the prepared oil-water mixture 5 times at 60 MPa for 4 minutes to obtain Example 2 (WR SL-DX ).

[0054] Example 3

[0055] (1) A 7.5% solution of pomegranate peel concentrate was prepared as a solvent for a mixed surfactant, wherein the mixed surfactant was composed of polyoxyethylene castor oil EL and anhydrous ethanol in a certain proportion, wherein the mass concentration of polyoxyethylene castor oil EL in the solution was 6%, and anhydrous ethanol was used as a co-surfactant (Km=2:1). After magnetic stirring for 45 min (150 r / min), a stable and uniform pomegranate peel aqueous phase system (S SL );

[0056] (2) Adding a mixed surfactant to clove essential oil in a ratio of 8:2 (volume ratio) to the pomegranate peel aqueous phase system, stirring the mixture magnetically for 15 min (150 r / min) and placing the mixture in a 30° C. water bath for 30 min to obtain an oil-water mixture. The prepared oil-water mixture was homogenized 5 times at 60 MPa for 4 minutes to obtain Example 3 (WR SL-DX ).

[0057] Example 4

[0058] (1) A 7.5% solution of pomegranate peel concentrate was prepared as a solvent for a mixed surfactant, wherein the mixed surfactant was composed of polyoxyethylene castor oil EL and anhydrous ethanol in a certain proportion, wherein the mass concentration of polyoxyethylene castor oil EL in the solution was 6%, and anhydrous ethanol was used as a co-surfactant (Km=2:1). After magnetic stirring for 45 min (150 r / min), a stable and uniform pomegranate peel aqueous phase system (S SL );

[0059] (2) Adding a mixed surfactant to clove essential oil in a ratio of 7:3 (volume ratio) to the pomegranate peel aqueous phase system, stirring the mixture magnetically for 15 min (150 r / min) and placing the mixture in a 30° C. water bath for 30 min to obtain an oil-water mixture. The prepared oil-water mixture was homogenized 5 times at 60 MPa for 4 minutes to obtain Example 4 (WR SL-DX ).

[0060] Test Example 1

[0061] The conductivity method was used to measure the microemulsion configuration, and it was determined that its water content was >62.5%, which was an O / W type.

[0062] Test Example 2

[0063] The particle size of the sample was measured using the Summary particle size analyzer, and it was determined that the particle size of the prepared microemulsion was nanometer-scale (see Table 1). As shown in Table 1, the particle size of clove essential oil is relatively large (3.50×10 3 nm), and using microemulsion preparation technology, the particle size of pomegranate peel-clove essential oil microemulsion ranged from 93.76±8.08nm to 257.62±19.49nm, all <500nm.

[0064] Table 1 Particle size of several samples (nm)

[0065]

[0066] Test Example 3

[0067] The product is bright yellow-brown in color, with uniform texture, no stratification, no precipitation, and good fluidity; no stratification occurs after centrifugation at 4000r / min for 15 minutes; after the emulsion is placed at room temperature for 30 days, except for slight stratification in Implementation Sample 1, Implementation Samples 2 and 3 are relatively stable.

[0068] Test Example 4

[0069] The prepared clove essential oil-pomegranate peel microemulsion was diluted 20 times by the Oxford cup method and then tested to determine its antibacterial ability against spoilage bacteria (the results are shown in Table 2). As can be seen from the table, the microemulsion has different antibacterial abilities against different spoilage bacteria, and high-pressure homogenization treatment and different emulsifiers have different effects on the antibacterial ability of the microemulsion system. High-pressure homogenization has little effect on its antibacterial ability, but improves the stability of the microemulsion.

[0070] Table 2 Size of inhibition zone of several samples (diameter of inhibition zone, mm)

[0071]

[0072] When the diameter of the inhibition zone is greater than 8 mm, there is an antibacterial effect, and the larger the diameter of the inhibition zone, the better the antibacterial performance; when the diameter of the inhibition zone is less than or equal to 8 mm, there is no antibacterial effect.

[0073] Determination of Fractional Inhibitory Concentration (FIC) index:

[0074] The criteria for judging the FIC index are:

[0075] When FIC≤0.5, the two drugs act synergistically;

[0076] When 0.5<FIC≤1.0, the two drugs have an additive effect;

[0077] When 1.0<FIC≤2.0, there is no relevant effect between the two drugs;

[0078] When FIC>2.0, the two drugs have antagonistic effects.

[0079] Pomegranate peel and clove essential oils showed synergistic effects against Shewanella putrefaciens, Pseudomonas fluorescens, and Pseudomonas aeruginosa, with FICs of 0.25, 0.5, and 0.5, respectively.

[0080]

[0081] Table 3 Combined antibacterial effect of pomegranate peel and clove essential oil on spoilage bacteria

[0082] Corruption bacteria <![CDATA[MIC 丁香精油 (g / kg)]]> <![CDATA[MIC 石榴皮 (g / kg)]]> <![CDATA[MIC 联合 (g / kg)]]> FIC Functional relationship Shewanella putrefaciens 15.62 1.9525 1.953 / 0.244 0.25 Collaboration Pseudomonas fluorescens 3.905 7.81 0.976 / 1.953 0.5 Collaboration Pseudomonas aeruginosa 7.81 15.62 1.953 / 3.905 0.5 Collaboration

[0083] The above is only a preferred specific implementation of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved conception within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a pomegranate peel-clove essential oil nano-microemulsion, characterized in that: The method comprises the following steps: adopting a phase conversion method to prepare a pomegranate peel-clove essential oil nano-microemulsion, taking clove essential oil as an oil phase and a pomegranate peel aqueous solution containing a mixed surfactant as a water phase, and preparing the nano-microemulsion by filling or high-pressure homogenization.

2. The method for preparing the pomegranate peel-clove essential oil nano-microemulsion according to claim 1, characterized in that: The preparation method specifically comprises the following steps: (1) Adding a mixed surfactant to the pomegranate peel aqueous solution, stirring to obtain a stable and uniform pomegranate peel aqueous phase system S SL ; (2) Using clove essential oil as the oil phase, adding pomegranate peel water phase system S to clove essential oil SL , after stirring and balancing for a period of time, an oil-water mixture is obtained, and the pomegranate peel-clove essential oil nano-microemulsion is obtained by filling or high-pressure homogenization; Wherein, the mixed surfactant is composed of a surfactant and a co-surfactant, and Km=2:1-6:1; The volume ratio of the mixed surfactant to the clove essential oil is 9:1 to 7:

3.

3. The method for preparing the pomegranate peel-clove essential oil nano-microemulsion according to claim 2, characterized in that: The HLB value of the surfactant is 13 to 15; The Km=2:

1.

4. The method for preparing the pomegranate peel-clove essential oil nano-microemulsion according to claim 2, characterized in that: The surfactant is at least one of Tween60, Tween80, and polyoxyethylene castor oil EL; The co-surfactant is anhydrous ethanol.

5. The method for preparing the pomegranate peel-clove essential oil nano-microemulsion according to claim 4, characterized in that: The pomegranate peel aqueous phase system S SL The mass concentration of Tween60 is 1.5% to 2.5%, and the mass concentration of polyoxyethylene castor oil EL is 4% to 8%; The amount of the co-surfactant added is formulated according to the Km value.

6. The method for preparing the pomegranate peel-clove essential oil nano-microemulsion according to claim 2, characterized in that: The mass concentration of the pomegranate peel aqueous solution is 5% to 10%, and it is prepared from pomegranate peel concentrated solution; The equilibrium temperature is 20-40° C. and the equilibrium time is 15-60 min.

7. The method for preparing the pomegranate peel-clove essential oil nano-microemulsion according to claim 2, characterized in that: The high pressure homogenization conditions are: 40-80 MPa for homogenization for 2-6 minutes, and 3-6 cycles of homogenization.

8. A pomegranate peel-clove essential oil nano-microemulsion, characterized in that: The method is prepared by any one of claims 1 to 7.

9. The pomegranate peel-clove essential oil nano-microemulsion according to claim 8, characterized in that: The pomegranate peel-clove essential oil nanoemulsion is of O / W type, and the particle size is less than 500nm.

10. Use of the pomegranate peel-clove essential oil nano-microemulsion according to claim 8 or 9 in inhibiting food spoilage bacteria.

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