CN112075564A - Eucalyptus citriodora essential oil/nisin nano-emulsion and preparation method thereof - Google Patents
Eucalyptus citriodora essential oil/nisin nano-emulsion and preparation method thereof Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3544—Organic compounds containing hetero rings
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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- A23L29/035—Organic compounds containing oxygen as heteroatom
- A23L29/04—Fatty acids or derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/10—Foods or foodstuffs containing additives; Preparation or treatment thereof containing emulsifiers
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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- A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention belongs to the field of food antibacterial and fresh-keeping materials, and particularly relates to eucalyptus citriodora essential oil/nisin nano emulsion and a preparation method thereof. The preparation method comprises the steps of dissolving nisin with a certain mass in deionized water, adding a composite emulsifier consisting of tween 80 and soybean lecithin to form a water phase, dropwise adding corn oil which is used as an oil phase and is loaded with eucalyptus citriodora essential oil after magnetic stirring, and forming the oil-in-water type nano emulsion under the action of stirring homogenization and ultrasonic crushing. The nano emulsion prepared by the invention can effectively improve the water solubility and the biocompatibility of the eucalyptus citriodora essential oil, and can achieve the purposes of efficient utilization and long-acting antibiosis by virtue of the synergistic effect of the nano emulsion and the nisin, thereby expanding the application of the eucalyptus citriodora essential oil and the nisin in the field of food antibacterial and fresh-keeping materials.
Description
Technical Field
The invention belongs to the field of food antibacterial and fresh-keeping materials, and particularly relates to eucalyptus citriodora essential oil/nisin nano emulsion and a preparation method thereof.
Background
The eucalyptus citriodora essential oil is plant essential oil extracted from the leaves of the eucalyptus citriodora trees, emits pungent aromatic odor, has strong bacteriostatic ability and broad-spectrum bacteriostatic activity, is mainly applied to the fields of essence and spice, medicine, pest control and the like, and is a natural and nontoxic additive. However, the eucalyptus citriodora essential oil is easy to volatilize, is sensitive to light, heat, oxygen and the like, and has pungent flavor when in use, so the utilization rate is low. Few domestic patent applications are made for improving the stability and utilization rate of the eucalyptus citriodora essential oil. At present, only Chinese patent CN105030677A application discloses a high-stability eucalyptus citriodora essential oil nano liposome antibacterial agent and a preparation method thereof, but chloroform is adopted as an organic solvent in the preparation process, so that solvent residue is inevitable, and certain toxic action is brought to human bodies; in addition, the antibacterial agent is not easy to disperse uniformly, and the antibacterial effect of the eucalyptus citriodora essential oil cannot be effectively exerted.
Nisin is a natural biological preservative synthesized by some strains of lactococcus lactis subsp lactis in a metabolic process, has good bacteriostatic activity, can effectively inhibit most gram-positive bacteria, and has no direct inhibition effect on gram-negative bacteria, mold and yeast. The preservative is approved by the world health organization as a food preservative as early as the last century, and is widely applied to food preservation industries of fruits and vegetables, dairy products, meat products, wine products, instant products, bean products and the like by a plurality of countries and regions.
The nano emulsion is an emulsification transportation system with the particle size of 10-200nm generally, and generally consists of an oil phase, a water phase and an emulsifier, so that the water solubility of embedded fat-soluble components can be obviously improved, and the problems of low bioavailability, easy volatilization, easy oxidation, light and heat sensitivity and the like of the plant essential oil can be solved to a great extent. The emulsion is mainly prepared by three high-energy emulsification methods of high-pressure homogenization, micro-jet homogenization, ultrasonic homogenization and the like and two low-energy emulsification methods of spontaneous emulsification, phase transition temperature and the like. Compared with the conventional micron-sized coarse emulsion, the nano emulsion basically has no flocculation, aggregation, precipitation and Australian curing phenomena in the storage process, and has excellent dispersibility and stability.
The domestic patent application of the nano emulsion for encapsulating the plant essential oil is less. Chinese patent CN104490661A application discloses an O/W type nano emulsion containing lemon essential oil and a preparation method thereof; chinese patent CN107981143A application discloses a preparation method and application of a nanoemulsion containing sweet orange essential oil. Chinese patent CN107714516A application discloses a preparation method of an embedded mint essential oil nano emulsion and application thereof in mouthwash. However, at present, there are few patent applications about bacteriostatic nanoemulsion encapsulating plant essential oil, and only chinese patent CN108719457A at present discloses a nano-emulsion with bacteriostatic activity thymol, its preparation method and application, although the method can prepare nano-emulsion, its preparation efficiency is low, and the prepared nano-emulsion has not strong enough stability. The invention adopts an ultrasonic emulsification method to overcome the defect, can greatly improve the preparation efficiency compared with the traditional stirring homogenization, and has stronger stability of the prepared nano emulsion.
The oil-in-water type nano emulsion is prepared from the eucalyptus citriodora essential oil by an ultrasonic emulsification method, and the synergistic effect of the oil-in-water type nano emulsion and the nisin overcomes the application defects of the eucalyptus citriodora essential oil, widens the antibacterial range of the nisin, improves the stability and the antibacterial activity of the nano emulsion, and has good application prospect and market value.
Disclosure of Invention
The invention aims to provide a eucalyptus citriodora essential oil/nisin nano emulsion and a preparation method thereof, wherein a certain mass of nisin is dissolved in deionized water, a composite emulsifier consisting of tween 80 and soybean lecithin is added to form a water phase, corn oil which is used as an oil phase and is loaded with the eucalyptus citriodora essential oil is dropwise added after magnetic stirring, and the oil-in-water nano emulsion is formed under the actions of stirring homogenization and ultrasonic crushing. The structural schematic diagram of the nano emulsion is shown in figure 1.
The invention relates to a nano emulsion prepared from nisin, corn oil, eucalyptus citriodora essential oil, tween 80 and soybean lecithin, which comprises the following preparation steps:
firstly, dissolving nisin with a certain mass in deionized water, adding a composite emulsifier consisting of tween 80 and soybean lecithin with a certain proportion, and magnetically stirring for 2 hours to form a water phase; adding a certain volume of eucalyptus citriodora essential oil into corn oil serving as carrier oil, mixing and stirring for 5min to form an oil phase, then dropwise adding the oil phase into a water phase, and forming an oil-in-water emulsion under the action of stirring homogenization and ultrasonic crushing.
The concentration of the nisin in the aqueous phase was 5 mg/mL.
The mass ratio of the tween 80 to the soybean lecithin is 2:1, and the mass ratio of the composite emulsifier to the oil phase is 2: 7.
The volume ratio of the corn oil to the eucalyptus citriodora essential oil is 3: 2.
The volume ratio of the water phase to the oil phase is 9: 1.
The rate of dropwise addition of the oil phase to the aqueous phase was 1 drop/s.
The rotating speed of stirring and homogenizing is 1500r/min, and the time of stirring and homogenizing is 30 min.
The power of ultrasonic crushing is 350W, the ultrasonic crushing time is 15min, the work time is 1s, the intermission time is 1s, and the specification of an ultrasonic amplitude transformer is 6 mm.
According to the invention, the eucalyptus citriodora essential oil is prepared into the oil-in-water type nano emulsion by an ultrasonic emulsification method, and the oil-in-water type nano emulsion and the lactic streptococcal bacteria have synergistic effect, so that the application defects of the eucalyptus citriodora essential oil are overcome, the antibacterial range of the lactic streptococcal bacteria is widened, the stability and the antibacterial activity of the nano emulsion are improved, and the nano emulsion has good application prospect and market value.
Drawings
FIG. 1 is a schematic structural diagram of a eucalyptus citriodora essential oil/nisin nanoemulsion
FIG. 2 particle size distribution diagram of Eucalyptus citriodora essential oil/nisin nanoemulsion
FIG. 3 Zeta potential distribution diagram of Eucalyptus citriodora essential oil/nisin nanoemulsion
FIG. 4 viscosity profile of Eucalyptus citriodora essential oil/nisin nanoemulsion
FIG. 5 shows the bacteriostatic effect of Eucalyptus citriodora essential oil/nisin nanoemulsion on E.coli O157: H7
FIG. 6 shows the bacteriostatic effect of Eucalyptus citriodora essential oil/nisin nanoemulsion on Staphylococcus aureus
Detailed Description
The following examples illustrate specific embodiments of the present invention, but the scope of the present invention is not limited thereto.
Example 1 measurement of particle size, PDI (polydispersity index) and Zeta potential of Eucalyptus citriodora essential oil/Nisin nanoemulsion
1.1 Experimental materials:
tween 80: AR; chemical reagents of national drug group Co Ltd
Eucalyptus citriodora essential oil: france Florihana Co Ltd
Soybean lecithin: AR; chemical reagents of national drug group Co Ltd
Nisin: AR; aladdin Chemicals Ltd
Corn oil: yihaijiali golden dragon fish food and food products GmbH
1.2 Experimental methods:
(1) preparation of eucalyptus citriodora essential oil/nisin nano emulsion
Weighing 45mg of nisin, adding the nisin into 9mL of distilled water, and magnetically stirring the nisin for 30min at normal temperature to prepare a nisin solution of 5 mg/mL.
② adding 180mg of Tween 80 and 90mg of soybean lecithin into the solution respectively, and magnetically stirring for 30min at normal temperature to form a water phase.
Respectively sucking 600 mu L of corn oil and 400 mu L of eucalyptus citriodora essential oil (total about 945mg) into a 2mL centrifuge tube, mixing and stirring for 5min, dropwise adding the mixture into the aqueous phase solution at the speed of 1 drop per second while timing through a 200uL pipette gun, and stirring and homogenizing at the rotating speed of 1500r/min for 30min to form the oil-in-water type coarse emulsion.
And fourthly, placing the coarse emulsion in an ultrasonic cell disruptor, performing ultrasonic treatment for 15min under the power of 350W in ice water bath for 1s, and performing intermittent treatment for 1s to obtain the eucalyptus citriodora essential oil/nisin nano emulsion.
(2) Particle size, PDI and Zeta potential measurements
And (3) placing a proper amount of the prepared nano emulsion sample in a sample groove of a laser particle analyzer, and respectively measuring the particle size, PDI and Zeta potential.
1.3 Experimental results:
the results of FIG. 2 show that the average particle size of the prepared eucalyptus citriodora essential oil/nisin nanoemulsion is 166.66nm, and the particle sizes are uniformly distributed between 70nm and 340 nm; the polydispersity index (PDI) is less than 0.300, which indicates that the nano emulsion has good dispersion performance and no agglomeration or oil drop fusion phenomenon; in addition, the results in FIG. 3 show that the average Zeta potential of the prepared nanoemulsion is-1.75 mV, and the Zeta potential is substantially uniformly distributed between-75 mV and 75 mV. The prepared nano emulsion is more stable by combining the analysis of the results.
Example 2 measurement of viscosity of Eucalyptus citriodora essential oil/Nisin lactate nanoemulsion
2.1 Experimental materials:
eucalyptus citriodora essential oil/nisin nanoemulsion
2.2 Experimental methods:
1.3mL of the eucalyptus citriodora essential oil/nisin nanoemulsion prepared in the example 1 is placed on a clamp of a rotational rheometer, a conical plate with the diameter of 40mm is selected, and the viscosity change of the nanoemulsion with the shear rate within the range of 10-100l/s is measured at normal temperature.
2.3 Experimental results:
the results in fig. 4 reflect that the viscosity of the prepared nano emulsion is overall lower, and is not as low as 0.0025Pa · s when the viscosity is maximum, and the viscosity of the nano emulsion is gradually reduced along with the increase of the shear rate, which indicates that the fluidity of the nano emulsion is better.
Example 3 measurement of synergistic bacteriostatic Properties of Eucalyptus citriodora essential oil/Nisin lactate nanoemulsion
3.1 Experimental materials:
eucalyptus citriodora essential oil/nisin nanoemulsion
Sodium chloride: AR; chemical reagents of national drug group Co Ltd
Beef extract: BR; chemical reagents of national drug group Co Ltd
Fish meal peptone: BR; chemical reagents of national drug group Co Ltd
Agar powder: BR; chemical reagents of national drug group Co Ltd
Anhydrous disodium hydrogen phosphate: AR; chemical reagents of national drug group Co Ltd
Potassium dihydrogen phosphate: AR; chemical reagents of national drug group Co Ltd
Strain: coli O157: H7; staphylococcus aureus
3.2 Experimental methods:
and placing a proper amount of the eucalyptus citriodora essential oil/nisin nano emulsion prepared in the example 1 in an ultra-clean bench for ultraviolet sterilization treatment for 1 h.
Inoculating E.coli O157: H7 and Staphylococcus aureus in Nutrient Broth (NB), shake culturing at 37 deg.C for 24H to obtain bacterial suspension in logarithmic growth phase, adding appropriate amount of E.coli O157: H7 and Staphylococcus aureus in logarithmic growth phase into phosphate buffer, and diluting with ten-fold stepwise gradient until the concentrations of E.coli O157: H7 and Staphylococcus aureus are 105-6CFU/mL。
Adding the sterilized eucalyptus citriodora essential oil/nisin nanoemulsion into the test tube containing E.coli O157: H7 and Staphylococcus aureus to enable the concentration of the nanoemulsion to be 25% (v: v), carrying out oscillation reaction at 37 ℃, taking a proper amount of culture solution to be diluted to a proper concentration in a ten-fold gradient manner at 0d, 1d, 2d, 3d and 4d respectively, then taking 100 mu L of diluted solution to drop on a solid culture medium, putting the solution into a constant-temperature incubator at 37 ℃ to carry out inversion culture after uniform coating, carrying out plate counting after 48H, repeating the experiment for three times, and taking the average value as a result. The control group replaced the nanoemulsion with an equal amount of deionized water.
3.3 results of the experiment
Fig. 5 and 6 show that as the action time of the nanoemulsion and the time of the E.coli O157, H7 and Staphylococcus aureus are respectively prolonged, the corresponding number of the residual bacteria gradually decreases, and the number of the residual bacteria of the control group without the nanoemulsion slowly increases to be stable, which indicates that the prepared nanoemulsion has a certain bacteriostatic effect.
Claims (9)
1. A preparation method of eucalyptus citriodora essential oil/nisin nano emulsion is characterized by comprising the following preparation steps: firstly, dissolving nisin with a certain mass in deionized water, adding a composite emulsifier consisting of tween 80 and soybean lecithin with a certain proportion, and magnetically stirring for 2 hours to form a water phase; adding a certain volume of eucalyptus citriodora essential oil into corn oil serving as carrier oil, mixing and stirring for 5min to form an oil phase, then dropwise adding the oil phase into a water phase, and forming an oil-in-water emulsion under the action of stirring homogenization and ultrasonic crushing.
2. The method for preparing the eucalyptus citriodora essential oil/nisin nanoemulsion according to claim 1, wherein the method comprises the following steps: the concentration of the nisin in the aqueous phase was 5 mg/mL.
3. The method for preparing the eucalyptus citriodora essential oil/nisin nanoemulsion according to claim 1, wherein the method comprises the following steps: the mass ratio of the tween 80 to the soybean lecithin is 2:1, and the mass ratio of the composite emulsifier to the oil phase is 2: 7.
4. The method for preparing the eucalyptus citriodora essential oil/nisin nanoemulsion according to claim 1, wherein the method comprises the following steps: the volume ratio of the corn oil to the eucalyptus citriodora essential oil is 3: 2.
5. The method for preparing the eucalyptus citriodora essential oil/nisin nanoemulsion according to claim 1, wherein the method comprises the following steps: the volume ratio of the water phase to the oil phase is 9: 1.
6. The method for preparing the eucalyptus citriodora essential oil/nisin nanoemulsion according to claim 1, wherein the method comprises the following steps: the rate of dropwise addition of the oil phase to the aqueous phase was 1 drop/s.
7. The method for preparing the eucalyptus citriodora essential oil/nisin nanoemulsion according to claim 1, wherein the method comprises the following steps: the rotating speed of stirring and homogenizing is 1500r/min, and the time of stirring and homogenizing is 30 min.
8. The method for preparing the eucalyptus citriodora essential oil/nisin nanoemulsion according to claim 1, wherein the method comprises the following steps: the power of ultrasonic crushing is 350W, the ultrasonic crushing time is 15min, the work time is 1s, the intermission time is 1s, and the specification of an ultrasonic amplitude transformer is 6 mm.
9. Use of the eucalyptus citriodora essential oil/nisin nanoemulsion prepared according to the process of any one of claims 1 to 8, for the antimicrobial preservation of food products.
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