CN116076491A - Preparation method of enhanced lemon grass volatile oil antibacterial active agent - Google Patents
Preparation method of enhanced lemon grass volatile oil antibacterial active agent Download PDFInfo
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- CN116076491A CN116076491A CN202211592404.5A CN202211592404A CN116076491A CN 116076491 A CN116076491 A CN 116076491A CN 202211592404 A CN202211592404 A CN 202211592404A CN 116076491 A CN116076491 A CN 116076491A
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- volatile oil
- lemon grass
- oil
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the technical field of biological pesticides, and discloses a preparation method of a lemon grass volatile oil antibacterial active agent, which comprises the steps of selecting a lemon grass plant dried in the shade, and extracting volatile oil by adopting a steam distillation method; screening a surfactant, a cosurfactant and a Km value respectively to determine that the optimal essential oil microemulsion system is km=2, and mixing the surfactants, wherein the essential oil microemulsion system comprises volatile oil=8:2; preparing a mixed surfactant with km=2, and preparing a volatile oil nanoemulsion; and (3) respectively carrying out GC-MS analysis and detection on the emulsion prepared by the traditional process of the volatile oil and the volatile oil nanoemulsion, and measuring the antibacterial activity of the volatile oil nanoemulsion. The lemon grass volatile oil nanoemulsion prepared by the invention can achieve the purposes of synergism, improvement of the bacteriostatic activity of the volatile oil, reduction of the consumption of the volatile oil and cost saving. The preparation method of the lemon grass volatile oil provided by the invention 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.
Description
Technical Field
The invention belongs to the technical field of biological pesticides, and particularly relates to a preparation method of a lemon grass volatile oil enhanced antibacterial active agent.
Background
At present, plant diseases are a major threat in agricultural production, and especially fungal infestation causes great economic losses to agricultural production. Continuous cropping obstacles are common in agriculture and are difficult to control. Notoginseng radix is a perennial medicinal plant of Panax of Araliaceae, has effects of removing blood stasis, stopping bleeding, promoting blood circulation, and relieving pain, and is mainly used for hematemesis, hemoptysis, and traumatic hemorrhage. However, serious continuous cropping obstacles exist in the process of planting pseudo-ginseng. One of the reasons for continuous cropping obstacles is that increased soil borne pathogens cause a change in the rhizosphere soil microflora. Currently, the main pathogenic bacteria of pseudo-ginseng mainly include Fusarium oxysporum (Fusarium oxysporum, f.oxysporum), fusarium solani (f.solani), and the like. In order to prevent and treat the soil-borne root rot of pseudo-ginseng, the conventional measure is to continuously apply chemical pesticides, so that a series of potential safety hazards are brought. Therefore, searching for green and healthy antifungal drugs becomes a hotspot for the study of scholars at home and abroad.
Plant essential oils are products of secondary metabolism of plants that have aromatic odor and volatility. They are biologically active and are widely used as fragrances and perfumes in the food industry. Many studies have shown that essential oils have a strong antibacterial effect. For example, the chrysanthemum volatile oil has good antibacterial activity on several gram-negative bacilli. The volatile oil extracted from the origanum vulgare and the fennel can obviously inhibit the growth of soil fungi and improve the survival rate of tomato seedlings. The antifungal properties of plant volatile oils have been widely accepted, meaning that plant volatile oils may be a good source of antifungal agents.
In the screening process of the inhibition effect of the volatile oil of the spice on the pseudo-ginseng related pathogenic bacteria, the volatile oil of the lemon grass (Cymbopogom citratus) is found to have stronger antifungal activity. It is a perennial dense-cluster herb of citronella of Gramineae, has aromatic smell, and has antibacterial and antiviral effects. Previous researches on lemon grass show that the lemon grass volatile oil has the characteristics of resisting bacteria, fungi, killing insects and the like. The lemon grass essential oil is an extract prepared by steam distillation with lemon grass as raw material. 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 an emulsion product obtained by nanocrystallizing volatile oil by a nanotechnology, and the particle size of the emulsion product is generally smaller than 100nm and can be called as nanoemulsion. 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:
(1) The measure of plant disease control is carried out by continuously increasing chemical pesticides, so that the safety problems that the quality of agricultural products is reduced, the toxicity of liver and kidney of human bodies is caused and the sustainable development of agriculture is threatened are existed in chemical pesticide residues.
(2) The essential oils are greatly limited in practical use because they have strong fragrance, low water solubility and instability in variable environments, and some essential oils only exert bacteriostatic activity at high concentrations.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a preparation method of a lemon grass volatile oil antibacterial active agent, and particularly relates to a novel process for improving antibacterial activity by preparing lemon grass volatile oil nanoemulsion.
The invention is realized in this way, a preparation method of the lemon grass essential oil enhancement antibacterial active agent comprises the following steps: selecting a lemon grass plant dried in the shade, and extracting volatile oil by adopting a steam distillation method; screening a surfactant, a cosurfactant and a Km value respectively to determine that the optimal essential oil microemulsion system is km=2, and mixing the surfactants, wherein the essential oil microemulsion system comprises volatile oil=8:2; preparing a mixed surfactant with km=2, and preparing a volatile oil nanoemulsion; and (3) respectively carrying out GC-MS analysis and detection on the emulsion prepared by the traditional process of the volatile oil and the volatile oil nanoemulsion, and measuring the antibacterial activity of the volatile oil nanoemulsion.
Further, the preparation method of the enhanced lemon grass volatile oil antibacterial active agent comprises the following steps:
step one, preparing volatile oil: extracting volatile oil of the dried lemon grass plants by adopting a steam distillation method;
step two, an essential oil microemulsion system is established: screening a surfactant, a cosurfactant and a Km value;
step three, preparing volatile oil nanoemulsion: preparing a mixed surfactant with km=2 and preparing an emulsion;
step four, antibacterial activity determination: and respectively carrying out GC-MS analysis and detection on the traditional emulsion and the nano emulsion.
Further, the preparing of the volatile oil in the first step includes:
placing the dried lemon grass product in the shade and distilled water in a 10L round-bottom flask according to the proportion of 1:8, and soaking for 3-6 h; distilling for 8 hr with steam distillation method, removing excessive water from the volatile oil with anhydrous sodium sulfate, and precisely weighing; preserving in brown bottle, sealing with sealing film, and placing in-20deg.C refrigerator.
Further, the establishment of the essential oil microemulsion system in the second step comprises the following steps:
(1) Weighing surfactant Tween80 and cosurfactant absolute ethyl alcohol, and placing the mixture in a beaker according to the mass ratio of Km of 1:1, 2:1 and 3:1 respectively;
(2) Under the working power of 300W, carrying out ice bath ultrasonic treatment for 20min; mixing the mixed surfactant and the oil phase in a mass ratio of 8:2, 6:4, 4:6 and 2:8, wherein the total mass is 4 g;
(3) Standing the mixture at room temperature for 30min, 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 component at the transition point;
(4) And respectively taking the mixed surfactant, the oil phase and distilled water as 3 vertexes of the phase diagram, manufacturing a three-phase diagram by using origin, calculating the area, and screening to determine the optimal essential oil microemulsion system.
Further, the configuration of the volatile oil nanoemulsion in the third step includes:
(1) Mixing Tween80 and absolute ethyl alcohol according to a mass ratio of 2:1, and mixing for 5min by a magnetic stirrer, wherein the mixing is uniform; placing for 5min at room temperature after ice bath ultrasonic treatment for 20min for standby;
(2) Mixing according to the mass ratio of the oil to the mixed surfactant of 2:8, magnetically stirring for 5min, carrying out ice bath ultrasonic treatment for 20min, and standing for 15min;
(3) Stirring with a multi-head magnetic heating stirrer at 25deg.C, dropwise adding 8g distilled water, stirring, and performing ice bath ultrasonic treatment for 20min to obtain volatile oil nanoemulsion.
Further, the determination of bacteriostatic activity in step four comprises:
GC-MS analysis is carried out on the crude oil, the emulsion prepared by the traditional process and the lemon grass volatile oil nanoemulsion, and a meteorological chromatograph is adopted for detection. 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 also aims to provide the lemon grass volatile oil prepared by implementing the preparation method of the lemon grass volatile oil-enhancing antibacterial active agent.
The invention also aims to provide an application of the lemon grass volatile oil 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 also aims to provide an application of the lemon grass volatile oil 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 nanoemulsion for enhancing bacteriostatic activity of volatile oil, and a method for reducing spore germination, hypha growth and root rot occurrence of pathogenic fungi. The invention provides a 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 volatile oil is a mixture of various active ingredients, has the characteristic of multi-target action on pathogenic bacteria, is not easy to generate drug resistance on pathogenic bacteria, can be used for a long time, but has low bioavailability due to hydrophobicity and volatility of the volatile oil, and cannot fully exert biological activity. Therefore, it is necessary to construct a delivery system that improves the hydrophobicity and reduces the volatility of the volatile oils. The nano emulsion has the advantages of fine and uniform liquid drops, good physical stability, strong permeability, high bioavailability and the like, has smaller particle size than the emulsion prepared by the traditional process, can fully exert the biological activity, greatly reduces the application amount and can effectively reduce the use cost. The raw materials of the composition are wide in sources and low in price, the preparation method is simple and easy to implement, and the composition can be used as a fertilizer additive or a plant source pesticide development precursor and can be widely applied to preventing and treating the occurrence of root rot caused by fusarium.
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 preparation process of the lemon grass volatile oil nanoemulsion provided by the invention is safe and nontoxic to human and animals, is environment-friendly, has a good control 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 affected due to the fact that a large amount of chemical pesticides are used in the current agricultural planting. The lemon grass volatile oil nanoemulsion prepared by the invention can achieve the purposes of synergism, improvement of the bacteriostatic activity of the volatile oil, reduction of the consumption of the volatile oil and cost saving. The preparation method of the lemon grass 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.
The invention provides a preparation method of lemon grass volatile oil nanoemulsion, which greatly enhances the biological activity of 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 lemon grass volatile oil, which can increase the stability of the emulsifier and make the emulsifier and the aqueous solution more mutually soluble; 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 lemon grass 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 the pathogenic bacteria generate resistance due to the repeated use of chemical pesticides.
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 lemon grass essential oil antibacterial active agent provided by the embodiment of the invention;
FIG. 2 is a graph showing the effect of different Km values on lemon grass nanoemulsion formation provided by the examples of the present invention;
FIG. 3 is a graph showing the main components of the conventional lemon grass emulsion, the nano emulsion and the crude oil according to the embodiment of the invention;
FIG. 4 is a schematic diagram showing a stability test of a lemon grass nanoemulsion provided by an embodiment of the present invention;
FIG. 5 is an electron microscope photograph of lemon grass essential oil prepared by the conventional process and the novel process provided by the embodiment of the invention; wherein, the figure (a) is a lemon grass essential oil electron microscope photograph prepared by the traditional process, and the figure (b) is a lemon grass essential oil electron microscope photograph prepared by the new process;
FIG. 6 is a graph showing the particle size of the lemon grass essential oil solvent prepared by the novel process and the conventional process provided by the embodiment of the invention; wherein, the figure (a) is a particle size diagram of the lemon grass essential oil solvent prepared by the traditional process, and the figure (b) is a particle size diagram of the lemon grass essential oil solvent 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 a lemon grass volatile oil enhanced 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 lemon grass volatile oil antibacterial active agent provided by the embodiment of the invention comprises the following steps:
s101, selecting a lemon grass plant dried in the shade, and extracting volatile oil by adopting a steam distillation method;
s102, screening a surfactant, a cosurfactant and a Km value respectively, determining that an optimal essential oil microemulsion system is Km=2, and mixing the surfactants, wherein the volatile oil=8:2;
s103, preparing volatile oil nanoemulsion by preparing a mixed surfactant with Km=2;
s104, performing GC-MS analysis and detection on the emulsion prepared by the traditional process of the volatile oil and the volatile oil nanoemulsion respectively, and determining the antibacterial activity of the volatile oil nanoemulsion.
The volatile oil provided by the embodiment of the invention takes various plants as raw materials and is prepared by adopting a steam distillation method.
The volatile oil extraction preparation method provided by the embodiment of the invention comprises the following steps:
(1) Respectively putting the shade-dried seeds or plants and distilled water into a 10L round-bottom flask according to the proportion of 1:8, soaking for 3-6 h, and distilling for 8h by adopting a steam distillation method;
(2) And 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 volatile oil. Preserving in brown bottle, sealing with sealing film, and placing in-20deg.C refrigerator.
The method for establishing the essential oil microemulsion system by using the surfactant (Tween 80) and the cosurfactant (absolute ethyl alcohol) as the solvent and the dispersing agent comprises the following steps of:
(1) Accurately weighing a surfactant (Tween 80) and a cosurfactant (absolute ethyl alcohol) according to a certain mass ratio (Km is selected to be 1:1, 2:1 and 3:1 respectively) in a beaker;
(2) Under the working power of 300W, carrying out ice bath ultrasonic treatment for 20min, and then mixing the mixed surfactant and oil phase (volatile oil) according to the mass ratio of 8:2, 6:4, 4:6 and 2:8, wherein the total mass is 4 g;
(3) Standing the mixture at room temperature for 30min, 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) And drawing a ternary phase diagram by taking the mixed surfactant, the oil phase and the water phase as 3 vertexes of the phase diagram, and determining the optimal Km value according to the size of the emulsion region.
The preparation method of the essential oil nanoemulsion provided by the embodiment of the invention comprises the following steps:
(1) Mixing Tween80 and absolute ethyl alcohol according to a mass ratio of 2:1, firstly mixing for 5min by a magnetic stirrer, and uniformly mixing (layering and flocculence are not observed by naked eyes);
(2) Placing at room temperature for 5min after ice bath ultrasonic treatment for 20min for standby (if the mixed surfactant is not used immediately, sealing by using a preservative film, and stirring properly before use);
(3) Mixing according to the mass ratio of the oil to the mixed surfactant of 2:8, magnetically stirring for 5min, carrying out ice bath ultrasonic treatment for 20min, and standing for 15min;
(4) Stirring on a multi-head magnetic heating stirrer with the temperature of 25 ℃, dropwise adding 8g of distilled water, continuously stirring and uniformly mixing, and finally carrying out ice bath ultrasonic treatment for 20min.
The embodiment of the invention provides an application of lemon grass volatile oil as a fertilizer additive or used together with chemical pesticides to reduce the application amount of the chemical pesticides.
The embodiment of the invention provides an application of lemon grass volatile oil in preparing a medicine for preventing and treating root rot, wherein the root rot is any one or three of fusarium oxysporum, fusarium putrescens or fusarium layering.
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 volatile oil nanoemulsion has the advantages of simple preparation method, low cost of required reagents, contribution to mass production and convenience for preventing and treating root rot caused by fusarium pathogenic bacteria.
The technical principle of the invention is as follows:
1. lemon grass is a very important spice, has fragrant smell and is good at avoiding 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 lemon grass is a medicinal and edible traditional Chinese medicine 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 volatile oil
Putting the dried lemon grass and distilled water in a ratio of 1:8 into a 10L round bottom flask, soaking for 3-6 h, distilling for 8h by adopting a steam distillation method, removing excessive water from the collected volatile oil by using anhydrous sodium sulfate, and precisely weighing. Preserving in brown bottle, sealing with sealing film, and placing in-20deg.C refrigerator.
Example 2: essential oil microemulsion system establishment
Step one: km is the mass ratio of surfactant to cosurfactant. The Km value was determined by pseudo ternary phase diagram method with the selection of surfactant (Tween 80) and cosurfactant (absolute ethanol).
Step two: weighing a surfactant (Tween 80) and a cosurfactant (absolute ethyl alcohol) in a certain mass ratio (Km=1, 2, 3) in a beaker;
step three: placing the beaker on a magnetic stirrer, stirring for 5min, performing ice bath ultrasonic treatment for 20min (power: 300 w), and mixing the mixed surfactant and the oil phase (volatile oil) according to the mass ratio of 8:2, 6:4, 4:6 and 2:8, wherein the total mass is 4 g;
step four: and (3) standing the mixture at room temperature for 15min, gradually dropwise adding distilled water by using a syringe, stirring while dropwise adding, repeating the operation until the solution becomes turbid from clear, recording the adding amount of distilled water at a critical point (taking the average value by repeating twice) when the solution is kept standing for 30min, and calculating the mass fraction of the component at the transition point.
Step five: the three-phase diagram is made by using the mixed surfactant, essential oil and distilled water as three phases, the area is calculated, and the optimal essential oil microemulsion system is determined to be km=2 through screening, and the mixed surfactant is volatile oil=8:2.
As shown in fig. 2, when km=2, the three-phase diagram has a hatched area ratio of 45.41% at maximum. So selecting the mass ratio of the surfactant with Km=2 to the cosurfactant to prepare the lemon grass volatile oil nanoemulsion.
Example 3: preparing volatile oil nano emulsion
Step one: a mixed surfactant (for example, 120 g) with km=2 is prepared;
80g of Tween80 and 40g of absolute ethyl alcohol are weighed into a 250mL conical flask, placed on a magnetic stirrer and stirred for 5min, and then subjected to ice bath ultrasonic treatment for 20min.
Step two: preparation of emulsion (for example, 18g was prepared at a concentration of 111 mg/mL)
8g of the mixed surfactant with Km=2 is weighed into a 50mL conical flask, 2g of volatile oil is added, the flask is sealed by a preservative film, the flask is placed on a magnetic stirrer for stirring for 5min, then placed in an ultrasonic machine for ice bath ultrasonic treatment for 20min, and then placed for 15min, and 8g of water is added to the conical flask while adding water and stirring (slowly adding) the conical flask. Stirring for 5min, and placing in an ultrasonic machine for ice bath and ultrasonic treatment for 20min to obtain the nanometer emulsion.
Example 4: GC-MS analysis and detection for preparing emulsion and nano emulsion by using traditional volatile oil process
The traditional process comprises the following steps: the medicament comprises a solvent and a dispersing agent, wherein the solvent is DMSO, the dosage of the solvent is 2% of the total mass of the volatile oil, the dispersing agent is Tween80, and the dosage of the Tween is 0.1% of the total mass of the volatile oil. The volatile oil was dissolved in 2% DMSO and 0.1% Tween80 (2-DMSO-T) suspension.
The crude oil, emulsion prepared by conventional process and nanoemulsion of example 3 were subjected to GC-MS analysis and detected using 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 61 major components, with geranylgeraniol (14.626%) and citronellal (9.205%) being more abundant; 57 main components were identified in the traditional emulsion, wherein nerol (5.806%) and atractylol (2.454%) account for a relatively large proportion; in the nanoemulsion, 57 main components were identified, among which citronellal (34.279%) and geranylgeraniol (18.375%) were more abundant. The essential components of the crude oil remain and the relative ratio increases after the lemon grass essential oil is prepared into the nanoemulsion, which may be related to the enhancement of the antibacterial activity thereof. From the wien diagram, the crude oil, the traditional emulsion and the nano emulsion have 7 total components, and the crude oil and the nano emulsion have 13 components identical to each other and 11 components identical to the traditional emulsion; nanoemulsions have 12 components identical to traditional emulsions. The total of 7 components may be associated with the antimicrobial activity of the volatile oil, while the 10 components specific to the nanoemulsion may be associated with their higher antimicrobial activity.
Example 5: nanoemulsion stability test
1. Centrifugal test
Centrifuging the prepared lemon grass 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 lemon grass volatile oil nanoemulsion has no significant difference between the light transmittance at the temperature of-20 ℃,4 ℃,30 ℃ and 40 ℃ and the light transmittance at the temperature of 25 ℃ of the control group, and has no turbidity and layering phenomena at each temperature, thus having good stability; the light transmittance at different salt concentrations is not obviously different from that of a control group, the turbidity layering phenomenon does not occur, and good stability is shown; the light transmittance under different rotation speed conditions is not significantly different from that of the control group, is not layered and is always stable; the lemon grass volatile oil nanoemulsion has turbidity when the pH is 5 and 6, compared with the pH=7.5 of a control group, the light transmittance is obviously reduced, and the lemon grass volatile oil nanoemulsion tends to be stable when the pH is 7 and 8.
Example 6: morphology observation and particle size analysis of lemon grass essential oil nanoemulsion and traditional lemon grass essential oil emulsion
The emulsion particle size morphology and the particle size were measured by a laser particle size analyzer 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 lemon grass essential oil solvent is a turbid milky white liquid.
Under the transmission electron microscope, the lemon grass 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 lemon grass essential oil solvent, the particles are small (see figure 5).
The results of the laser particle size analyzer showed that the conventional lemon grass essential oil solvent average particle size was 461.25nm and the lemon grass essential oil nanoemulsion average particle size was 15.86nm (see fig. 6).
Example 7: determination of Minimum Inhibitory Concentration (MIC) of emulsion and nanoemulsion prepared by traditional process on fusarium
Step 1: colonies were rinsed with 1/4PDB liquid medium, mycelia were filtered through eight layers of gauze, counted under a microscope using a blood cell counting plate, and finally prepared at a concentration of 1X 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 putrescens
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 lemon grass volatile oil is prepared into the nano emulsion, the antibacterial effect is obviously improved, and compared with the traditional emulsion, the antibacterial effect is improved by 6.25 times.
Example 8: determination of prevention and control effect of lemon grass nanoemulsion on root rot of 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 Each treatment was 50 strains per mL of fusarium solani bacterial suspension.
Step 2: preparing lemon grass nanoemulsion, mixing the lemon grass nanoemulsion with Fusarium solani suspension to obtain final concentration of 0.45mg/mL, and spore concentration of Fusarium solani of 2×10 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.
Step 4: collecting pseudo-ginseng leaves, cleaning surface dirty with distilled water, drying surface moisture, cutting into fragments, mixing, then randomly weighing 0.1g of the mixture into a test tube, adding 25mL of a mixed solution of 95% ethanol and acetone (V: V=1:1), sealing the dark place of the test tube by using a sealing film, placing the dark place for more than 24 hours, measuring the optical density (the optical density at the wavelengths 663 and 645 nm) of the test tube by using an enzyme-labeled instrument after the leaves are completely whitened, and then calculating the chlorophyll content of each sample.
Concentration of chlorophyll a: c (C) a ＝2.21D 663 -2.81D 646 ；
Concentration of chlorophyll b: c (C) b ＝20.13D 646 -5.03D 663 ；
Concentration of total chlorophyll: c (C) T ＝C a +C b ＝17.32D 646 -2.82D 663 。
TABLE 2 prevention and control effects of volatile oil on Fusarium inoculation of Notoginseng radix
Note that: each value represents 3 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.
Table 2 shows that the incidence and disease index of plants irrigated with the suspension of the lemon grass volatile oil nanoemulsion and the FS spore are significantly lower than those of plants irrigated with the suspension of the FS fungus only, and the chlorophyll content of pseudo-ginseng plants treated with the suspension of the lemon grass volatile oil nanoemulsion and the FS spore is significantly higher than those of pseudo-ginseng plants irrigated with the suspension of the FS fungus only.
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 lemon grass volatile oil antibacterial active agent is characterized by comprising the following steps of: selecting a lemon grass plant dried in the shade, and extracting volatile oil by adopting a steam distillation method; screening a surfactant, a cosurfactant and a Km value respectively to determine that the optimal essential oil microemulsion system is km=2, and mixing the surfactants, wherein the essential oil microemulsion system comprises volatile oil=8:2; preparing a mixed surfactant with km=2, and preparing a volatile oil nanoemulsion; and (3) respectively carrying out GC-MS analysis and detection on the emulsion prepared by the traditional process of the volatile oil and the volatile oil nanoemulsion, and measuring the antibacterial activity of the volatile oil nanoemulsion.
2. The method for preparing the enhanced lemon grass essential oil antibacterial active agent according to claim 1, wherein the method for preparing the enhanced lemon grass essential oil antibacterial active agent comprises the following steps:
step one, preparing volatile oil: extracting volatile oil of the dried lemon grass plants by adopting a steam distillation method;
step two, an essential oil microemulsion system is established: screening a surfactant, a cosurfactant and a Km value;
step three, preparing volatile oil nanoemulsion: preparing a mixed surfactant with km=2 and preparing an emulsion;
step four, antibacterial activity determination: and respectively carrying out GC-MS analysis and detection on the traditional emulsion and the nano emulsion.
3. The method for preparing the enhanced lemon grass essential oil antibacterial active agent according to claim 2, wherein the preparation of the essential oil in the first step comprises the following steps:
placing the dried lemon grass product in the shade and distilled water in a 10L round-bottom flask according to the proportion of 1:8, and soaking for 3-6 h; distilling for 8 hr with steam distillation method, removing excessive water from the volatile oil with anhydrous sodium sulfate, and precisely weighing; preserving in brown bottle, sealing with sealing film, and placing in-20deg.C refrigerator.
4. The method for preparing the enhanced lemon grass essential oil antibacterial active agent according to claim 2, wherein the establishment of the essential oil microemulsion system in the second step comprises the following steps:
(1) Weighing surfactant Tween80 and cosurfactant absolute ethyl alcohol, and placing the mixture in a beaker according to the mass ratio of Km of 1:1, 2:1 and 3:1 respectively;
(2) Under the working power of 300W, carrying out ice bath ultrasonic treatment for 20min; mixing the mixed surfactant and the oil phase in a mass ratio of 8:2, 6:4, 4:6 and 2:8, wherein the total mass is 4 g;
(3) Standing the mixture at room temperature for 30min, 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 component at the transition point;
(4) And respectively taking the mixed surfactant, the oil phase and distilled water as 3 vertexes of the phase diagram, manufacturing a three-phase diagram by using origin, calculating the area, and screening to determine the optimal essential oil microemulsion system.
5. The method for preparing the enhanced lemon grass essential oil antibacterial active agent according to claim 2, wherein the configuration of the essential oil nanoemulsion in the third step comprises the following steps:
(1) Mixing Tween80 and absolute ethyl alcohol according to a mass ratio of 2:1, and mixing for 5min by a magnetic stirrer, wherein the mixing is uniform; placing for 5min at room temperature after ice bath ultrasonic treatment for 20min for standby;
(2) Mixing according to the mass ratio of the oil to the mixed surfactant of 2:8, magnetically stirring for 5min, carrying out ice bath ultrasonic treatment for 20min, and standing for 15min;
(3) Stirring with a multi-head magnetic heating stirrer at 25deg.C, dropwise adding 8g distilled water, stirring, and performing ice bath ultrasonic treatment for 20min to obtain volatile oil nanoemulsion.
6. The method for preparing the enhanced lemon grass volatile oil antibacterial active agent according to claim 2, wherein the determination of the antibacterial activity in the fourth step comprises the following steps:
performing GC-MS analysis on crude oil, emulsion prepared by the traditional process and the lemon grass volatile oil nanoemulsion, 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 lemon grass essential oil prepared by carrying out the preparation method of the enhanced lemon grass essential oil antibacterial active agent according to any one of claims 1 to 6.
8. Use of the lemon grass essential oil of claim 7 in the preparation of a medicament for preventing and treating root rot.
9. The use of the lemon grass essential oil according to claim 8 for preparing a medicament for preventing and treating root rot, wherein the root rot is any one or three of fusarium oxysporum, fusarium putrescens and fusarium layering.
10. Use of the lemon grass volatile oil of claim 7 in the preparation of a fertilizer additive or a chemical pesticide additive.
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