CN115989812A - Preparation method of enhanced cumin volatile oil antibacterial active agent - Google Patents
Preparation method of enhanced cumin volatile oil antibacterial active agent Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention belongs to the technical field of biological pesticides, and discloses a preparation method of an enhanced cumin volatile oil antibacterial active agent, which comprises the steps of selecting a dried cumin seed, and preparing cumin volatile oil by adopting a steam distillation method; screening the surfactant, the cosurfactant and the Km value respectively, and determining km=2 as the optimal mass ratio of the surfactant to the cosurfactant; preparing a cumin essential oil nanoemulsion by using a surfactant and a cosurfactant; and finally, performing GC-MS analysis and detection on the emulsion and the cumin volatile oil nanoemulsion prepared by the traditional cumin volatile oil process, and testing the stability of the cumin volatile oil nanoemulsion. The cumin volatile oil nanoemulsion provided by the invention is safe and nontoxic to human and animals, is environment-friendly, has a good prevention and treatment effect on root rot caused by fusarium, solves the serious problems of agricultural product quality and industrial sustainable development influence caused by using a large amount of chemical pesticides in current agricultural planting, and saves cost.
Description
Technical Field
The invention belongs to the technical field of biological pesticides, and particularly relates to a preparation method of an enhanced cumin volatile oil antibacterial active agent.
Background
At present, the pathogen of the root rot is complex, fungi are considered as the most main causative factors of the root rot, wherein Fusarium spp is the highest in proportion, and the Fusarium spp is also strong in parasitism and pathogenicity and is the most harmful pathogenic bacterium in the root rot. Wherein Fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani) and Fusarium layering (Fusarium proliferatum) can all cause root rot of multiple crops.
The primary step of pathogen infection is to adsorb on the surface of the host, the fusarium pathogenic fungi do not differentiate into obvious infection structure, but rather rely on releasing certain enzymes and generating substances and toxins for degrading plant defense reaction to achieve the infection purpose, wherein spore germination is the first step of pathogen infection. At present, the prevention and treatment of root rot mainly depends on the application of chemical pesticides. Although the chemical bacteriostatic agent has good bacteriostatic effect, long-term repeated use of the chemical bacteriostatic agent leads pathogenic bacteria to generate drug resistance. In addition, chemical bacteriostats are destructive to the ecological environment and threaten the biological life safety. Therefore, it is urgent to find a safe and environment-friendly bacteriostatic agent. Many researches show that the plant essential oil has the potential of inhibiting the growth of pathogenic bacteria, and a certain result is achieved in the research of a bacteriostasis mechanism. Plant essential oil is a secondary metabolic substance with aromatic smell widely existing in plant bodies, and the complexity of the composition components is closely related to the types, growth conditions, collection positions, collection seasons and the like of plants. The complex diversity of essential oil compositions makes it biologically active with multiple targets and broad spectrum antimicrobial. The main components of the essential oil are low boiling point compounds such as monoterpenes, sesquiterpenes, aromatics, and aliphatics. The volatile oil can inhibit the generation, germination and hypha growth of spores, thereby reducing the harm of pathogenic bacteria. Essential oils contain a variety of components, and thus the antibacterial effect cannot be fully generalized by the action of a single compound. Compared with chemical pesticides, the volatile oil is derived from natural plants, and has the characteristics of safe use, drug residue compensation, no threat to human safety, wide sources and simple extraction process.
Cuminum cyminum L is also called as Cuminum celery, is a one-year or two-year old herb plant of Cuminum (Umbelliferae), is 20-40 cm high, is smooth and hairless, is green or pink and green and white, and has a yellow brown, flat arc shape, rich in essential oil and strong aromatic smell. Cumin is a very important seasoning, and essential Cumin oil is an extract prepared by taking Cumin spice as a raw material and extracting. The essential oils are extremely limited in practical use because they have strong fragrance, low water solubility and instability in variable environments, and some essential oils exert bacteriostatic activity only at high concentrations. Nanoemulsions are a dosage form which has been studied more recently, and are composed of components such as water phase, oil phase, surfactant, cosurfactant and the like, and are a uniform emulsion system. The volatile oil nanoemulsion is a kind of emulsion product obtained by nanocrystallizing volatile oil by utilizing a nanotechnology, and can be called nanoemulsion generally below 100 nm. Nanoemulsions generally appear clear and transparent because they have particle sizes smaller than the wavelength range of visible light. The nanometer emulsification technology is one new kind of medicine preparation technology, and has nanometer size to make the medicine particle penetrate the cell membrane or cell gap of pathogenic bacteria to produce bacteriostasis. The small-size effect is an important point of the nanoemulsion formulation different from other formulations, and the specific surface area of the drug is greatly increased after the particle size is reduced, the contact surface of the target protein is also increased, so that the bioavailability of the drug is finally improved. This small size effect directly solves many of the challenges that conventional techniques cannot address.
Through the above analysis, the problems and defects existing in the prior art are as follows: at present, the root rot is prevented and treated mainly by applying chemical pesticides, and the chemical bacteriostat is reused for a long time to cause pathogenic bacteria to generate drug resistance, and meanwhile, the chemical bacteriostat has destructive effect on ecological environment and threatens biological life safety.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a preparation method of an enhanced cumin volatile oil antibacterial active agent, and particularly relates to a novel process for preparing a nano emulsion of cumin volatile oil.
The invention is realized in this way, a preparation method of the essential oil antibacterial active agent of the reinforced cumin, the preparation method of the essential oil antibacterial active agent of the reinforced cumin comprises the following steps: selecting a cumin seed dried in the shade, and preparing cumin volatile oil by adopting a steam distillation method; screening the surfactant, the cosurfactant and the Km value respectively, and determining km=2 as the optimal mass ratio of the surfactant to the cosurfactant; preparing a cumin essential oil nanoemulsion by using a surfactant and a cosurfactant; and finally, performing GC-MS analysis and detection on the emulsion and the cumin volatile oil nanoemulsion prepared by the traditional cumin volatile oil process, and testing the stability of the cumin volatile oil nanoemulsion.
Further, the preparation method of the enhanced cumin volatile oil antibacterial active agent comprises the following steps:
step one, preparing cumin volatile oil;
step two, screening the mass ratio of the surfactant to the auxiliary surfactant;
step three, preparing a cumin essential oil nanoemulsion;
and step four, GC-MS analysis and detection of the cumin essential oil nanoemulsion.
Further, the preparation of the cumin volatile oil in the first step comprises the following steps:
(1) Respectively placing the dried cumin seeds and distilled water into a 10L round-bottom flask in a ratio of 1:8, and distilling for 8h by adopting a steam distillation method;
(2) Removing excessive water from the collected essential oil by using anhydrous sodium sulfate, dehydrating until the anhydrous sodium sulfate has crystals and powder, and precisely weighing to obtain the cumin volatile oil;
(3) The cumin volatile oil is stored in a brown bottle, sealed by a sealing film and placed in a refrigerator at the temperature of minus 20 ℃ for standby.
Further, the screening of the mass ratio of the surfactant to the auxiliary surfactant in the second step comprises the following steps:
(1) Weighing surfactant Tween 80 and cosurfactant absolute ethyl alcohol, and placing the mixture in a beaker according to the mass ratio of Km of 1.0, 2.0, 2.5, 3.0 and 4.0 respectively;
(2) Under the working power of 300W, carrying out ice bath ultrasonic treatment for 20min; mixing the mixed surfactant and cumin volatile oil phase in a mass ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 6:4, 3:7, 2:8 and 1:9, wherein the total mass is 5 g;
(3) Standing the mixture at room temperature for 15min, gradually dropwise adding distilled water with a 5mL pipette while stirring, repeating the operation until the solution becomes turbid from clarification, recording the adding amount of distilled water at a critical point when the state is unchanged, repeating the process for two times to obtain the average value, and calculating the mass fraction of the components at the transition point;
(4) The mixed surfactant, essential oil and distilled water are used as three phases to manufacture a three-phase diagram by using origin, and the area of the emulsion is calculated to determine the optimal Km of the essential oil microemulsion system.
Further, the preparation of the cumin essential oil nanoemulsion in the third step comprises the following steps:
(1) Mixing Tween 80 and absolute ethyl alcohol according to a mass ratio of 2:1 with a magnetic stirrer for 10min; after being uniformly mixed, the mixture is subjected to ice bath ultrasonic treatment for 20min for standby;
(2) Mixing the oil and the surfactant according to the mass ratio of 2:8, and magnetically stirring for 10min; carrying out ice bath ultrasonic treatment for 30min, and standing for 10min;
(3) Distilled water is added dropwise and stirred continuously to make the water content reach 80%, and finally ice bath ultrasound is carried out for 30min.
Further, the GC-MS analysis and detection of the cumin essential oil nanoemulsion in the fourth step comprises the following steps:
and (3) carrying out GC-MS analysis on the emulsion prepared by the traditional process and the cumin essential oil nanoemulsion prepared in the step (III), and detecting by adopting a meteorological chromatograph. Wherein, the chromatographic column: HP-5MS 30 m.times.250 μm.times.0.25. Mu.m. EI source ionization source temperature: 230 ℃; four-stage bar temperature: 150 ℃, scanning range: 30-500 m/z; sample inlet temperature: 285 ℃, the split ratio is 10:1; sample injection amount: 1 μl; electron energy: 70eV; column incubator temperature program: keeping at 50 ℃ for 4min, heating to 120 ℃ at 5 ℃/min, heating to 180 ℃ at 1 ℃/min, heating to 280 ℃ at 10 ℃/min, and keeping for 16min; RI values were calculated from the retention times of normal paraffins at C9-C25, and the compound retention times and mass spectra were compared to the NIST 17.L database to determine the final compound.
The invention further aims at providing the cumin essential oil nanoemulsion prepared by implementing the preparation method of the enhanced cumin essential oil antibacterial active agent.
The invention further aims to provide an application of the cumin essential oil nanoemulsion in preparing a medicament for preventing and treating root rot.
Further, the root rot is any one or three of fusarium oxysporum, fusarium putrescens and fusarium layering.
The invention further aims to provide an application of the cumin essential oil nanoemulsion in preparing fertilizer additives or chemical pesticide additives.
In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:
first, aiming at the technical problems in the prior art and the difficulty of solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:
the invention provides a preparation process of a nano emulsion for enhancing a cumin volatile oil antibacterial active agent and a method for reducing pathogenic fungus spore germination, hypha growth and root rot occurrence. The process comprises the steps of extracting the cumin volatile oil, establishing an essential oil microemulsion system, configuring the nanoemulsion, and root irrigation of plant roots. The invention provides a cumin volatile oil nanoemulsion for preventing and treating root rot, belongs to a botanical fungicide, is easy to volatilize and decompose, does not pollute the environment, and is safe to people and livestock. The cumin volatile oil provided by the invention is a mixture of various active ingredients, has a multi-target effect special effect on pathogenic bacteria, is not easy to generate drug resistance, and can be used for a long time; compared with the traditional process, the nano emulsion has smaller particle size, greatly reduces the application amount and effectively reduces the use cost. The cumin volatile oil nanoemulsion has wide raw material sources, low price and simple and easy preparation method, can be used as a fertilizer additive or a plant source pesticide development precursor, and is widely used for preventing and treating root rot caused by fusarium.
The invention provides a preparation method of a cumin volatile oil nanoemulsion, which greatly enhances the biological activity of the volatile oil and improves the efficacy period of the volatile oil; wherein, the combination of the surfactant (Tween 80) and the cosurfactant (absolute ethyl alcohol) can dissolve the cumin volatile oil, thereby increasing the stability of the emulsifier and being more mutually soluble with the aqueous solution; the particle size of the nano emulsion is smaller than 100nm, and the volatile oil is easier to penetrate through pathogenic bacteria cell membranes due to the small particle size, so that the antibacterial effect is exerted, and the use of chemical pesticides can be greatly reduced; the cumin volatile oil nanoemulsion is derived from natural plants, belongs to biological pesticides, and is environment-friendly; the volatile oil has multiple components, is not easy to generate resistance to pathogenic bacteria, and overcomes the phenomenon that chemical pesticides are used for multiple times to generate resistance to pathogenic bacteria.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
the invention provides a preparation process of a cumin volatile oil nanoemulsion, which is safe and nontoxic to human and animals, is environment-friendly, has a good prevention and treatment effect on root rot caused by fusarium, and solves the important problems that the quality of agricultural products is improved and the sustainable development of industry is influenced due to the fact that a large amount of chemical pesticides are used in the current agricultural planting. The volatile oil nanoemulsion prepared by the invention can achieve the purposes of synergism, improvement of the antibacterial activity of the volatile oil, reduction of the consumption of the volatile oil and cost saving. The preparation method of the cumin volatile oil is simple, the cost of the required reagent is low, mass production is facilitated, and the prevention and the treatment of root rot caused by fusarium pathogenic bacteria are facilitated.
Thirdly, as inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:
(1) The expected benefits and commercial values after the technical scheme of the invention is converted are as follows: the preparation can be used as a plant source additive to be added into foliar fertilizer for spraying, thereby promoting the plant growth and improving the disease prevention and disease resistance of the plants. The preparation can also be directly used for developing and utilizing plant-source pesticides, and compared with chemical synthesized pesticides, the plant-source pesticides have the characteristics of low toxicity, easy degradation, various action modes, difficult generation of drug resistance by pathogenic bacteria and the like. The raw materials have wide sources, low cost, simple preparation process and good popularization and application prospects.
(2) The technical scheme of the invention fills the technical blank in the domestic and foreign industries: because the volatile oil is of opposite polarity to water, the oil and water do not naturally form a homogeneous mixture and tend to separate. Due to the low toxicity, low residue and natural antibacterial properties of essential oils, development and utilization have been the subjects of continuous search. However, the loss of biological activity of volatile oils due to environmental fluctuations is a major factor limiting their exploitation. The nanoemulsion improves the water solubility of the volatile oil by enlarging the dispersibility of the volatile oil, so that the application of the volatile oil in agriculture is realized.
(3) Whether the technical scheme of the invention solves the technical problems that people want to solve all the time but fail to obtain success all the time is solved: volatile oils are a very complex natural mixture that appears to have no specific cellular targets due to the co-action of multiple components. Since volatile oils are a complex mixture of many molecules, in the sense that studying volatile oils provides more information than studying certain of its components, the concept of synergy appears to be more meaningful. In order to make the volatile oil a new generation of plant-derived pesticide, and make field application possible, the preparation of nanoemulsion is considered to be more effective and a formulation with wider application range. Nanoemulsions have many advantages such as improved water solubility, stability of active ingredients, larger specific surface area, etc., thereby improving bioavailability.
(4) The technical scheme of the invention overcomes the technical bias: nanoemulsions are a dosage form which has been studied more recently, and are composed of components such as water phase, oil phase, surfactant, cosurfactant and the like, and are a uniform emulsion system. The nanometer emulsification technology is one new kind of medicine preparation technology, and has nanometer size to make the medicine particle penetrate the cell membrane or cell gap of pathogenic bacteria to produce bacteriostasis. The small-size effect is an important point of the nanoemulsion formulation different from other formulations, and the specific surface area of the drug is greatly increased after the particle size is reduced, the contact surface of the target protein is also increased, so that the bioavailability of the drug is finally improved. This small size effect directly solves many of the challenges that conventional techniques cannot address. Compared with the traditional process, the nano emulsion has smaller particle size, greatly reduces the application amount and effectively reduces the use cost.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a preparation method of the enhanced cumin volatile oil antibacterial active agent provided by the embodiment of the invention;
FIG. 2 is a graph showing the effect of different Km values on the formation of cumin nanoemulsions provided by an example of the present invention;
FIG. 3 is a graph of the principal components versus heat and Wen for the conventional cumin emulsions, nanoemulsions and crude oils provided by the present invention;
FIG. 4 is a schematic diagram of the stability test results of the cumin nanoemulsion provided by the example of the present invention;
FIG. 5 is an electron microscope photograph of the cumin essential oil prepared by the traditional process and the new process provided by the embodiment of the invention; wherein, the figure (a) is a cumin essential oil electron microscope photograph prepared by the traditional process, and the figure (b) is a cumin essential oil electron microscope photograph prepared by the new process;
FIG. 6 is a graph showing the particle size of the cumin essential oil solvent prepared by the conventional process and the novel process according to the embodiment of the invention; wherein, the graph (a) is a cumin essential oil solvent particle size graph prepared by the traditional process, and the graph (b) is a cumin essential oil solvent particle size graph prepared by the new process.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides a preparation method of an enhanced cumin volatile oil antibacterial active agent, and the invention is described in detail below with reference to the accompanying drawings.
In order to fully understand how the invention may be embodied by those skilled in the art, this section is an illustrative embodiment in which the claims are presented for purposes of illustration.
As shown in fig. 1, the preparation method of the enhanced cumin volatile oil antibacterial active agent provided by the embodiment of the invention comprises the following steps:
s101, selecting a cumin seed dried in the shade, and extracting cumin volatile oil by adopting a steam distillation method;
s102, screening a surfactant, a cosurfactant and Km values;
s103, preparation of the cumin essential oil nanoemulsion and GC-MS analysis and detection.
The cumin volatile oil provided by the embodiment of the invention is prepared by taking cumin seeds as raw materials and adopting a steam distillation method.
The method for extracting and preparing the cumin volatile oil provided by the embodiment of the invention comprises the following steps:
(1) Respectively placing the dried cumin seeds and distilled water into a 10L round-bottom flask in a ratio of 1:8, and distilling for 8h by adopting a steam distillation method;
(2) Removing excessive water from the collected essential oil by using anhydrous sodium sulfate, dehydrating until the anhydrous sodium sulfate has crystals and powder, and precisely weighing to obtain the cumin volatile oil; preserving in brown bottle, sealing with sealing film, and placing in-20deg.C refrigerator.
The method for screening Km values by using the cumin volatile oil provided by the embodiment of the invention with a surfactant (Tween 80) and a cosurfactant (absolute ethyl alcohol) as a solvent and a dispersing agent comprises the following steps:
(1) Accurately weighing a surfactant (Tween 80) and a cosurfactant (absolute ethyl alcohol) in a certain mass ratio (Km is selected to be 1.0, 2.0, 2.5, 3.0 and 4.0 respectively) in a beaker;
(2) Under the working power of 300W, carrying out ice bath ultrasonic treatment for 20min, and then mixing a mixed surfactant and an oil phase (cumin volatile oil) according to the mass ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 6:4, 3:7, 2:8 and 1:9, wherein the total mass is 5 g;
(3) Standing the mixture at room temperature for 15min, gradually dropwise adding distilled water with a 5mL pipette while stirring, repeatedly operating until the solution becomes turbid from clarification, recording the adding amount of distilled water at a critical point (taking average value by repeating twice) when the state is unchanged, and calculating the mass fraction of the component at the transition point;
(4) The mixed surfactant, essential oil and distilled water are used as three phases to manufacture a three-phase diagram by using origin, and the area of the emulsion is calculated to determine the optimal Km of the essential oil microemulsion system.
The preparation method of the cumin essential oil nanoemulsion provided by the embodiment of the invention comprises the following steps:
(1) Mixing Tween 80 and absolute ethyl alcohol according to a mass ratio of 2:1 by a magnetic stirrer for 10min, and uniformly mixing (no separation and no flocculence are observed by naked eyes);
(2) Ultrasonic treatment in ice bath for 20min, and keeping (if the mixed surfactant is not used immediately, sealing with fresh-keeping film, and stirring properly before use);
(3) Mixing according to the mass ratio of oil to surfactant of 2:8, magnetically stirring for 10min, performing ice bath ultrasound for 30min, and standing for 10min;
(4) Distilled water is added dropwise and stirred continuously to make the water content reach 80%, and finally ice bath ultrasound is carried out for 30min.
The embodiment of the invention provides a cumin essential oil nanoemulsion prepared by a preparation method of an enhanced cumin essential oil antibacterial active agent.
The embodiment of the invention provides application of a cumin essential oil nanoemulsion in preparing medicines for preventing and treating root rot, wherein the root rot is any one or three of fusarium oxysporum, fusarium putrescens and fusarium layering.
The embodiment of the invention provides an application of a cumin essential oil nanoemulsion as a fertilizer additive or used with chemical pesticides to reduce the application amount of the chemical pesticides.
The volatile oil nanoemulsion prepared by the invention can achieve the purposes of synergism, improvement of the antibacterial activity of the volatile oil, reduction of the consumption of the volatile oil and cost saving. The preparation method of the cumin volatile oil provided by the invention is simple, the cost of the required reagent is low, mass production is facilitated, and the prevention and treatment of root rot caused by fusarium pathogenic bacteria are facilitated.
The technical principle of the invention is as follows:
1. cumin is a very important spice, and has the advantages of fragrant smell, fragrance channeling and good resistance to dirt. The volatile oil is composed of small molecular substances, so that the volatile oil is easy to diffuse in soil.
2. The nanoemulsion system consists of an oil phase, a water phase, a surfactant, a cosurfactant and the like, wherein the surfactant has a molecular structure with one hydrophilic end and one oleophilic end, and the special structure can enable the system to dissolve the oil phase into water or dissolve the water phase into the oil under the action of the surfactant.
3. The hydrophilic-lipophilic balance value (HLB value) of the surfactant is the same as or similar to the hydrophilic-lipophilic balance value required by oil phase emulsification, and the prepared system is relatively stable, so that the oil phase and the water phase are non-uniformly integrated together, and the layering phenomenon is caused when the oil phase floats on the water phase.
4. After the volatile oil is prepared into the nano emulsion, the solubility of the volatile oil in water is improved.
5. The nano emulsion prepared from the volatile oil is easier to penetrate through pathogenic bacteria cell membranes or cell gaps after the particle size is reduced, meanwhile, the specific surface area is greatly increased, the contact surface of target protein is also increased, and the antibacterial activity of the volatile oil is enhanced.
6. The volatile oil is a plant source extract, and the cumin is a traditional Chinese medicine with homology of medicine and food and a common spice, is harmless to human bodies, and solves the threat of the current common chemical pesticide on the toxicity of the liver and kidney of the human bodies.
7. The volatile oil is easy to volatilize, so the invention solves the troublesome problems that the chemical pesticide residue reduces the quality of agricultural products and threatens the sustainable development of agriculture.
In order to prove the inventive and technical value of the technical solution of the present invention, this section is an application example on specific products or related technologies of the claim technical solution.
The volatile oil nanoemulsion is applied to food preservation, for example, in bread preservation, and is mostly used as an additive, so that the influence on the flavor of bread can be reduced, and meanwhile, the volatile oil nanoemulsion can be used as a natural preservative to prolong the shelf life of the food. Fruits and vegetables provide many nutrients for humans and are a necessity for human life, but can cause significant economic loss during post-harvest storage due to infection by pathogenic fungi. The garlic oil nanoemulsion can be used as a preservative for vegetables, melons and fruits after harvesting due to the antibacterial effect on penicillium.
The embodiment of the invention has a great advantage in the research and development or use process, and has the following description in combination with data, charts and the like of the test process.
Example 1: preparation method of cumin volatile oil
And (3) respectively placing the dried cumin seeds and distilled water into a 10L round-bottom flask in a ratio of 1:8, distilling for 8 hours by adopting a steam distillation method, removing excessive moisture from the collected essential oil by using anhydrous sodium sulfate, dehydrating until the anhydrous sodium sulfate has crystals and powder, and precisely weighing to obtain the cumin volatile oil. Preserving in brown bottle, sealing with sealing film, and placing in-20deg.C refrigerator.
Example 2: screening of surfactant to Co-surfactant and Mass ratio of active Agents (Km value)
Step one: accurately weighing a surfactant (Tween 80) and a cosurfactant (absolute ethyl alcohol) in a certain mass ratio (Km is selected to be 1.0, 2.0, 2.5, 3.0 and 4.0 respectively) in a beaker;
step two: under the working power of 300W, carrying out ice bath ultrasonic treatment for 20min, and then mixing a mixed surfactant and an oil phase (cumin volatile oil) according to the mass ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 6:4, 3:7, 2:8 and 1:9, wherein the total mass is 5 g;
step three: standing the mixture at room temperature for 15min, gradually dropwise adding distilled water with a 5mL pipette while stirring, repeatedly operating until the solution becomes turbid from clarification, recording the adding amount of distilled water at a critical point (taking average value by repeating twice) when the state is unchanged, and calculating the mass fraction of the component at the transition point;
step four: preparing a three-phase diagram by taking a mixed surfactant, essential oil and distilled water as three phases by using origin, calculating the area of the emulsion, and determining the optimal Km of an essential oil microemulsion system;
step five: calculating the shadow area of the three-phase diagram by using software AutoCAD, and obtaining that the area ratio is maximum when Km=2, wherein the area ratio is 37.0683 (see FIG. 2); so the mass ratio of the surfactant with Km=2 to the cosurfactant is selected to prepare the cumin volatile oil nanoemulsion.
Example 3: preparation of cumin essential oil nanoemulsion
Step one: mixing Tween 80 and absolute ethyl alcohol according to a mass ratio of 2:1 by using a magnetic stirrer for 10min, and uniformly mixing (layering and flocculence are not observed by naked eyes);
step two: ultrasonic treatment in ice bath for 20min, and keeping (if the mixed surfactant is not used immediately, sealing with fresh-keeping film, and stirring properly before use);
step three: mixing according to the mass ratio of oil to surfactant of 2:8, magnetically stirring for 10min, performing ice bath ultrasound for 30min, and standing for 10min;
step four: distilled water is added dropwise and stirred continuously to make the water content reach 80%, and finally ice bath ultrasound is carried out for 30min.
Example 4: GC-MS analysis and detection of emulsion and nanoemulsion prepared by cumin volatile oil traditional process
The traditional process comprises the following steps: the medicament comprises a solvent and a dispersing agent, wherein the solvent is DMSO, and the dosage of the solvent is 2% of the total mass of the volatile oil; the dispersing agent is Tween 80, and the total weight of the volatile oil is 0.1%. The volatile oil was dissolved in 2% DMSO and 0.1% Tween 80 (2-DMSO-T) suspension.
GC-MS analysis is carried out on the emulsion prepared by the traditional process and the nano emulsion of the cumin volatile oil of example 3, and detection is carried out by adopting a weather chromatograph model Agilengt Technologies 7890B-5977B.
Chromatographic column: HP-5MS 30 m.times.250 μm.times.0.25. Mu.m. EI source ionization source temperature: 230 ℃; four-stage bar temperature: 150 ℃, scanning range: 30-500 m/z; sample inlet temperature: 285 ℃, the split ratio is 10:1; sample injection amount: 1 μl; electron energy: 70eV; column incubator temperature program: keeping at 50 ℃ for 4min, heating to 120 ℃ at 5 ℃/min, heating to 180 ℃ at 1 ℃/min, heating to 280 ℃ at 10 ℃/min, and keeping for 16min; wherein RI is calculated from the retention time of normal alkane continuous carbon (C9-C25), the retention time and mass spectrum of the compound are compared with NIST 17.L database, and the final compound is determined by combining the relevant literature, and the identification result is shown in figure 3.
As can be seen from fig. 3, crude oil identified 41 major components, 2-methyl-3-phenylpropionaldehyde (37.637%), 2-caren-10-ol (24.346%) being more abundant; 21 main components were identified in the traditional emulsion, 2-methyl-3-phenylpropionaldehyde (32.53%), 2-caren-10-ol (8.38%) being more abundant; 24 main components were identified in the nanoemulsion, wherein 2-methyl-3-phenylpropionaldehyde (46.032%), 7-dimethyl bicyclo <4.1.0> hept-3-ene-3-formaldehyde (16.685%) accounted for more, and crude oil and both emulsions contained 2-methyl-3-phenylpropionaldehyde in high amounts, indicating that it may be the main component for bacteriostasis. The essential components of crude oil are still remained and the content is similar after the cumin volatile oil is prepared into nano emulsion.
Example 5: nanoemulsion stability test
1. Centrifugal test
Centrifuging the prepared cumin essential oil microemulsion at 2000r/min, 5000r/min and 8000r/min for 15min, and observing whether layering occurs. Absorbance at 595nm before and after centrifugation was measured, and light transmittance was calculated.
(T＝10 -A T: light transmittance; a: absorbance of light
2. Effect of salt on microemulsion stability
And 5mL of the essential oil microemulsion is placed in 3 clean test tubes, sodium chloride is respectively added into the test tubes, the mass fractions of the sodium chloride are respectively 1%, 3% and 5%, the test tubes are oscillated to dissolve the essential oil microemulsion, and the change condition of the microemulsion is observed.
Absorbance was measured at a wavelength of 595nm, and light transmittance was calculated.
3. Effect of acid base on microemulsion stability
Respectively placing 5mL of the microemulsion into four small beakers, and gradually dropwise adding sodium hydroxide solution with the concentration of 0.1mol/L into the three beakers to ensure that the pH is 5,6 and 7; to the other small beaker, a hydrochloric acid solution having a concentration of 0.1mol/L was gradually added dropwise to give a pH of 8. The light transmittance was calculated by measuring with a precision pH meter and observing the change of the microemulsion, and measuring absorbance at a wavelength of 595 nm.
4. Impact of high and low temperatures on microemulsion stability
Low temperature: packaging the prepared essential oil microemulsion in a glass bottle, sealing, placing in an environment of-20deg.C and 4deg.C, standing for 12 hr for sampling, recovering room temperature, observing its properties, measuring absorbance of the microemulsion before and after heating, and calculating light transmittance.
High temperature: heating the prepared essential oil microemulsion at 25deg.C, 30deg.C and 35deg.C for 30min, observing whether the microemulsion is layered, and measuring absorbance of the microemulsion before and after heating if no layering exists, and calculating light transmittance.
As can be seen from fig. 4, the light transmittance of the cumin nanoemulsion at different salt concentrations is not significantly different from that of the control group, and the cumin nanoemulsion has no turbidity and layering phenomenon and shows good stability; the light transmittance at the temperature of-20 ℃,4 ℃,30 ℃ and 40 ℃ is not remarkably different from the light transmittance at the temperature of 25 ℃ of the control group, and the turbidity layering phenomenon does not occur at each temperature, so that the light transmittance with good stability under different rotation speeds is not remarkably different from the light transmittance of the control group, is not layered and is always stable; the cumin nanoemulsion showed turbidity at pH 5,6 compared to control ph=7.6, the light transmittance was significantly reduced and the nanoemulsion tended to stabilize at pH 7, 8.
Example 6: emulsion Transmission Electron Microscopy (TEM) observation of particle size
The emulsion particle size was observed under a transmission electron microscope (JEM-1011, JEOL, co.Ltd., tokyo, japan).
The emulsion prepared according to the screening formulations of examples 2 and 3 was a clear, transparent, pale yellow liquid, which was still clear in appearance after centrifugation, free of flocculent precipitate and delamination, and was infinitely dilutable with water, indicating that the prepared sample was a nanoemulsion. The traditional cumin essential oil solvent is a turbid milky white liquid.
Under the transmission electron microscope, the cumin essential oil nanoemulsion is in a regular spherical shape, the nano emulsion liquid drops are distributed uniformly, adhesion and agglomeration are avoided, and compared with the traditional cumin essential oil solvent, the cumin essential oil nanoemulsion has smaller particles (see figure 5).
The results of the laser particle size analyzer show that the average particle size of the traditional cumin essential oil solvent is 782.287 +/-139.594 nm, and the average particle size of the cumin essential oil nanoemulsion is 17.327 +/-0.049 nm (see figure 6).
Example 7: determination of Minimum Inhibitory Concentration (MIC) of emulsion and nanoemulsion prepared by traditional process on fusarium
Step 1: washing colony with 1/4PDB liquid medium, filtering mycelium with eight layers of gauze, and washing with blood cell counting plateCounting under microscope, and finally configuring concentration to be 1×10 4 Individual/ml spore suspension.
Step 2: the emulsion and the nano-emulsion prepared by the traditional process are respectively filtered by an organic filter head of 0.22 mu m to obtain sterile filtrate.
Step 3: the emulsion and the nano emulsion prepared by the traditional process are respectively diluted by a double dilution method, the initial concentration is 15mg/mL, and a series of concentrations of 15-0.0146 mg/mL are obtained by dilution by the double dilution method. In a 96-well plate, filtered volatile oil solution (50. Mu.L) and fungal suspension (150. Mu.L) were added to each well. Wherein 200. Mu.L of 1/4PDB solution was used as a blank and 200. Mu.L of fungal suspension was used as a positive control. The 96-well plate was incubated at constant temperature in a 28℃microbial incubator for 36 hours. The absorbance of each well was measured at 595nm absorbance using a microplate reader (thermo. Model: 1510), and when the difference between the absorbance of the sample well and the absorbance of the blank well was 0, it was considered that fungal growth did not occur, and the corresponding volatile oil concentration value was MIC value. 8 duplicate wells were provided for each treatment.
TABLE 1 determination of Minimum Inhibitory Concentration (MIC) of emulsions and nanoemulsions prepared by conventional Process on Fusarium oxysporum
Note that: MIC is the minimum inhibitory concentration. The data were repeated 8 times and each value represents 8 data analyzed with mean ± standard deviation. Differences were checked for significance using ANOVA (analysis of variance) with a significance level of p <0.05. Different alphabetic letters in the same column represent significant differences.
The results in Table 1 show that after the cumin volatile oil is prepared into the nano emulsion, the antibacterial effect is obviously improved.
Example 8: determination of prevention and control effect of nano emulsion on root rot of potted pseudo-ginseng
Step 1: selecting annual Notoginseng radix seedling (plant height about 10 cm) with consistent growth vigor, injecting 10mL of spore with concentration of 2×10 into soil around plant stem base by syringe 6 Mixed bacterial suspension of individual/mLFusarium oxysporum, fusarium putrescens and Fusarium layering at a volume ratio of 1:1:1), 50 plants per treatment.
Step 2: preparation of a Cumin nanoemulsion, mixing the nanoemulsion with a Fusarium layering bacterial suspension to a final concentration of 0.45mg/mL, the Fusarium layering spore concentration of 2X 10 6 10mL of sterile water is irrigated at the base of each plant stem, and 10mL of sterile water is irrigated at the blank control; it was poured every 7 d.
Step 3: pseudo-ginseng plants were cultivated in a greenhouse at 24℃for 12h light/12 h dark cultivation, and the disease state was observed and data recorded.
Morbidity (DI) =number of sick plants/total number of plants×100%;
the disease classification was as follows:
level 0: the plants are healthy;
stage 1: plant leaves are provided with disease spots;
2 stages: wilting the plants;
3 stages: the plants die.
Disease index (Di) = Σ [ (dn×dg)/(tn×mg) ]x100;
wherein Dn represents the number of plants with the same disease grade, dg represents the corresponding disease grade, tn represents the total number of plants, and Mg represents the highest disease grade.
Chlorophyll content was measured with SPAD instrument.
TABLE 2 prevention and control effects of volatile oil on Fusarium inoculation of Notoginseng radix
Note that: each value represents 20 data analyzed with mean ± standard deviation. Differences were checked for significance using ANOVA (analysis of variance) with a significance level of p <0.05. Different alphabetic letters in the same column represent significant differences.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (10)
1. The preparation method of the enhanced cumin volatile oil antibacterial active agent is characterized by comprising the following steps of: selecting a cumin seed dried in the shade, and preparing cumin volatile oil by adopting a steam distillation method; screening the surfactant, the cosurfactant and the Km value respectively, and determining km=2 as the optimal mass ratio of the surfactant to the cosurfactant; preparing a cumin essential oil nanoemulsion by using a surfactant and a cosurfactant; and finally, performing GC-MS analysis and detection on the emulsion and the cumin volatile oil nanoemulsion prepared by the traditional cumin volatile oil process, and testing the stability of the cumin volatile oil nanoemulsion.
2. The method for preparing the enhanced cumin essential oil antibacterial active agent according to claim 1, wherein the method for preparing the enhanced cumin essential oil antibacterial active agent comprises the following steps:
step one, preparing cumin volatile oil;
step two, screening the mass ratio of the surfactant to the auxiliary surfactant;
step three, preparing a cumin essential oil nanoemulsion;
and step four, GC-MS analysis and detection of the cumin essential oil nanoemulsion.
3. The method for preparing the enhanced cumin essential oil antibacterial active agent according to claim 2, wherein the preparation of the cumin essential oil in the step one comprises the following steps:
(1) Respectively placing the dried cumin seeds and distilled water into a 10L round-bottom flask in a ratio of 1:8, and distilling for 8h by adopting a steam distillation method;
(2) Removing excessive water from the collected essential oil by using anhydrous sodium sulfate, dehydrating until the anhydrous sodium sulfate has crystals and powder, and precisely weighing to obtain the cumin volatile oil;
(3) The cumin volatile oil is stored in a brown bottle, sealed by a sealing film and placed in a refrigerator at the temperature of minus 20 ℃ for standby.
4. The method for preparing the enhanced cumin volatile oil antibacterial active agent according to claim 2, wherein the screening of the mass ratio of the surfactant to the auxiliary surfactant in the second step comprises the following steps:
(1) Weighing surfactant Tween 80 and cosurfactant absolute ethyl alcohol, and placing the mixture in a beaker according to the mass ratio of Km of 1.0, 2.0, 2.5, 3.0 and 4.0 respectively;
(2) Under the working power of 300W, carrying out ice bath ultrasonic treatment for 20min; mixing the mixed surfactant and cumin volatile oil phase in a mass ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 6:4, 3:7, 2:8 and 1:9, wherein the total mass is 5 g;
(3) Standing the mixture at room temperature for 15min, gradually dropwise adding distilled water with a 5mL pipette while stirring, repeating the operation until the solution becomes turbid from clarification, recording the adding amount of distilled water at a critical point when the state is unchanged, repeating the process for two times to obtain the average value, and calculating the mass fraction of the components at the transition point;
(4) The mixed surfactant, essential oil and distilled water are used as three phases to manufacture a three-phase diagram by using origin, and the area of the emulsion is calculated to determine the optimal Km of the essential oil microemulsion system.
5. The method for preparing the enhanced cumin essential oil antibacterial active agent according to claim 2, wherein the preparation of the cumin essential oil nanoemulsion in the third step comprises the following steps:
(1) Mixing Tween 80 and absolute ethyl alcohol according to a mass ratio of 2:1 with a magnetic stirrer for 10min; after being uniformly mixed, the mixture is subjected to ice bath ultrasonic treatment for 20min for standby;
(2) Mixing the oil and the surfactant according to the mass ratio of 2:8, and magnetically stirring for 10min; carrying out ice bath ultrasonic treatment for 30min, and standing for 10min;
(3) Distilled water is added dropwise and stirred continuously to make the water content reach 80%, and finally ice bath ultrasound is carried out for 30min.
6. The method for preparing the enhanced cumin essential oil antibacterial active agent according to claim 2, wherein the GC-MS analysis and detection of the cumin essential oil nanoemulsion in the fourth step comprises:
performing GC-MS analysis on the emulsion prepared by the traditional process and the cumin essential oil nanoemulsion prepared in the step three, and detecting by adopting a meteorological chromatograph; wherein, the chromatographic column: HP-5MS 30 m.times.250 μm.times.0.25. Mu.m; EI source ionization source temperature: 230 ℃; four-stage bar temperature: 150 ℃, scanning range: 30-500 m/z; sample inlet temperature: 285 ℃, the split ratio is 10:1; sample injection amount: 1 μl; electron energy: 70eV; column incubator temperature program: keeping at 50 ℃ for 4min, heating to 120 ℃ at 5 ℃/min, heating to 180 ℃ at 1 ℃/min, heating to 280 ℃ at 10 ℃/min, and keeping for 16min; RI values were calculated from the retention times of normal paraffins at C9-C25, and the compound retention times and mass spectra were compared to the NIST 17.L database to determine the final compound.
7. A nano emulsion of essential oil of cumin prepared by carrying out the method for preparing the antibacterial active agent of essential oil of cumin as claimed in any one of claims 1 to 6.
8. Use of a cumin essential oil nanoemulsion of claim 7 for the preparation of a medicament for the prevention and treatment of root rot.
9. The use of the cumin essential oil nanoemulsion according to claim 8 for the preparation of a medicament for the prevention and treatment of root rot, wherein the root rot is any one or three of fusarium oxysporum, fusarium putrescens and fusarium layering.
10. Use of a cumin essential oil nanoemulsion of claim 7 for the preparation of fertilizer additives or chemical pesticide additives.
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