Extraction method of fresh rhizoma nardostachyos essential oil

Abstract

The invention relates to the field of other living property management, in particular to a method for extracting fresh rhizoma nardostachyos essential oil, which comprises the following raw materials: mannosylerythritol lipid, spiraea glycoside, xylanase-chitinase-beta-glucanase compound enzyme, rhizoma nardostachyos grandiflorus, diatomite loaded nano zinc oxide, sodium chloride, distilled water and polysorbate-80. The mannosylerythritol lipid, the xylanase-chitinase-beta-glucanase compound enzyme and the spiramerin play a role through a synergistic effect, and the xylanase-chitinase-beta-glucanase compound enzyme effectively degrades a complex polysaccharide structure in cell walls of the rhizoma nardostachyos, so that the cell walls become looser; the mannosylerythritol lipid reduces the surface tension of a cell membrane lipid layer and enhances the permeation of hydrophilic components, the spiramerin forms an interface active layer between the essential oil and a water phase and promotes the emulsification and dispersion of the essential oil, and the extraction efficiency and quality of the rhizoma nardostachyos essential oil are remarkably improved through the combined action of the mannosylerythritol lipid, the spiramerin and the essential oil.

Description

Technical Field

[0001] The present invention relates to the field of other biological industries, and specifically to a method for extracting fresh Nardostachys jatamansi essential oil. Background Art

[0002] Currently, the extraction methods of Nardostachys jatamansi essential oil mainly include steam distillation, organic solvent extraction, and supercritical CO2 extraction. However, these methods all have significant limitations. Steam distillation is prone to destroying heat-sensitive components due to high temperatures (95 - 100°C). The oxidation or degradation loss rate of active substances such as α-nardostachone and acoradiene can reach more than 30%. Moreover, due to the dense cell walls of the rhizomes of Nardostachys jatamansi, the penetration of steam is difficult, resulting in low extraction efficiency. Long-time distillation is required, and the yield is only 1.5 - 2.0%. Organic solvent extraction has problems of solvent residue and pollution, high recovery cost, poor selectivity, easy extraction of impurities, and requires additional purification steps. Supercritical CO2 extraction has high equipment costs and low extraction efficiency for polar components, and entrainers need to be added. In addition, the limitations of the singularity of existing auxiliary technologies are also relatively obvious. The salting-out method can reduce the solubility of essential oil, but has no effect on cell wall destruction, and the yield improvement is limited. Single enzymatic hydrolysis can only degrade some cell wall polysaccharides and cannot completely break down the lignin-cellulose composite structure. Although ultrasonic or microwave pretreatment can accelerate cell rupture, it is easy to cause isomerization of essential oil components and change the aroma characteristics. There are obvious shortcomings in the existing processes in terms of component stability and resource utilization. There is a lack of a protection mechanism for heat-sensitive components during the extraction process, resulting in the retention rate of α-nardostachone generally being lower than 70%. At the same time, most of the residues after extraction are discarded, and the high-value utilization of resources has not been realized. Summary of the Invention

[0003] (1) Technical Problems to be Solved

[0004] The purpose of the present invention is to provide a method for extracting fresh Nardostachys jatamansi essential oil. This method selects Nardostachys jatamansi DC. var. grandiflora as the main extract, and through the synergistic effect of three components: mannosylerythritol lipid, spiraeoside (CAS No. 618 - 65 - 5), and a composite enzyme of xylanase - chitinase - β - glucanase, significantly improves the oil yield and quality of Nardostachys jatamansi essential oil, and solves the core problems such as low efficiency, easy destruction of active ingredients, and insufficient resource utilization rate in traditional processes.

[0005] (2) Technical Solution

[0006] To achieve the above object, on the one hand, the present invention provides a fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 10 - 20 parts of mannosylerythritol lipid, 10 - 30 parts of spiraeoside, 50 - 70 parts of Nardostachys jatamansi DC. var. grandiflora, 20 - 40 parts of diatomite-supported nano-zinc oxide, 5 - 15 parts of sodium chloride, 100 - 200 parts of distilled water, and 5 - 10 parts of polysorbate - 80;

[0007] The described fresh Nardostachys jatamansi essential oil further includes:

[0008] Xylanase-chitinase-β-glucanase complex enzyme;

[0009] The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora is (10 - 15):1;

[0010] Furthermore, the extraction method of the xylanase-chitinase-β-glucanase complex enzyme includes:

[0011] S11. Select alkalotolerant xylanase, Aspergillus, and Aspergillus niger as the production sources of xylanase, chitinase, and β-glucanase;

[0012] S12. Ferment the above three strains in a culture medium, where the fermentation temperature of alkalotolerant xylanase and Aspergillus niger is 37 - 39°C or 25 - 28°C, and the fermentation temperature of Aspergillus is 20 - 25°C, and culture for 48 - 52 h;

[0013] S13. Inoculate the activated strains into the fermentation medium, control the inoculation amount to be 5 - 8%, and shake culture at 28 - 30°C and 180 rpm for 72 - 80 h. Among them, 1 - 2% Triton X-100 needs to be added to Aspergillus to obtain three fermentation broths;

[0014] S14. Mix the three fermentation broths together, centrifuge at 7500 - 8000 rpm for 15 - 20 min, and separately collect the supernatants after centrifugation of the three fermentation broths to obtain the supernatant;

[0015] S15. Slowly add ammonium sulfate to the supernatant to 30 - 35% saturation, let it stand for 2 - 3 h, then centrifuge at 7500 - 8000 rpm for 15 - 20 min. Subsequently, continue to add ammonium sulfate to the supernatant to 70 - 75% saturation, let it stand at 4°C for 10 - 12 h, centrifuge at 7500 - 8000 rpm for 15 - 20 min, and dissolve the precipitate with 0.1M phosphate buffer to obtain the first mixed solution;

[0016] S16. Add 0.1M phosphate buffer to the first mixed solution, adjust the pH to 6.0 - 6.5 for dialysis, change the buffer every 6 - 8 h, and change it at least three times to obtain the second mixed solution;

[0017] S17. Load the second mixture onto a DEAE-cellulose chromatography column and elute it with a linear gradient of 0.1 - 0.5 M NaCl, gradually increasing the NaCl concentration to elute different components in sequence. Subsequently, load the active components after DEAE-cellulose chromatography onto a Sephadex G-75 column and elute it with a phosphate buffer solution at pH = 6.0 - 6.5. Collect the eluate, measure the enzyme activity of the eluate, combine the active components of xylanase, chitinase, and β-glucanase, and then perform lyophilization to obtain the xylanase-chitinase-β-glucanase complex enzyme.

[0018] Further, the loading amount of nano-zinc oxide in the diatomite-supported nano-zinc oxide is 5 - 9%.

[0019] Further, the mass ratio of mannitol erythritol lipid to spiraeoside is 1:(2 - 4).

[0020] Further, the weight ratio of distilled water, sodium chloride, and polysorbate-80 is (100 - 120):(3 - 6):(8 - 10).

[0021] Further, the mass ratio of Nardostachys jatamansi DC., diatomite-supported nano-zinc oxide, and sodium chloride is 1:(20 - 40):(1 - 3).

[0022] On the other hand, based on the same inventive concept, the present invention also provides a method for extracting fresh Nardostachys jatamansi DC. essential oil, which is applied to the above-mentioned fresh Nardostachys jatamansi DC. essential oil and includes the following steps:

[0023] S21. Take 100 g of fresh Nardostachys jatamansi DC. rhizomes (with a moisture content of 60 - 65%), quickly freeze them in crystalline liquid nitrogen at -196 - -190 °C for 10 - 15 min, and then immediately place them in a freeze grinder and grind them to 60 - 65 mesh to obtain Nardostachys jatamansi DC. particles;

[0024] S22. Weigh 2 - 3 g of citric acid and 12 - 15 g of disodium hydrogen phosphate, dissolve them in 1 - 1.5 L of ultrapure water, adjust the pH to 5.5 - 5.8, sterilize them at 121 °C for 20 min under this condition to obtain a buffer solution. Subsequently, weigh 50 - 65 g of the xylanase-chitinase-β-glucanase complex enzyme and dissolve it in the buffer solution to obtain a complex enzyme solution;

[0025] S23. Add the Nardostachys jatamansi DC. particles and the complex enzyme solution to a first container according to a mass ratio of 1:(10 - 12), place them in a constant temperature oscillator at 45 °C and react at 150 - 160 rpm for 2 - 3 h. Subsequently, continue to shake at 80 °C for 5 - 10 min, cool to room temperature and then centrifuge at 3500 - 4000 rpm for 5 - 10 min to obtain a first solution;

[0026] S24. Mix mannosylerythritol lipid and lactic acid at a mass ratio of 1:2, place them in a constant temperature oscillator at 60 °C and react at 150 - 180 rpm for 2 - 3 h to obtain a second mixture. Mix the second mixture and spiraeoside at a mass ratio of 1:(2 - 4), and then perform ultrasonic treatment under the conditions of 40 - 50 kHz for 10 - 15 min to obtain a third solution;

[0027] S25. Mix the first solution and the third solution at a mass ratio of 1:10, then sequentially add sodium chloride and polysorbate - 80 at a mass ratio of (2 - 6):1, spread them evenly on the surface of the porous plate inside the distillation kettle, and the filling height ≤ 2 / 3 of the kettle body height. Turn on the steam generator, introduce saturated steam, control the steam pressure at 0.1 - 0.12 MPa, control the temperature inside the kettle at 95 - 98 °C, and the distillation time is 4 - 4.5 h;

[0028] S26. The steam is sequentially passed through the primary condenser to trap high - boiling components and the secondary condenser to efficiently recover volatile essential oils to obtain a condensate. Then the condensate is stratified by an oil - water separator. The essential oil phase is filled with diatomaceous earth - supported nano - zinc oxide in the adsorption column, and the packing density is 0.45 g / cm 3 , and then pass through the aqueous phase to obtain crude essential oil;

[0029] S27. Elute the adsorption column with n - hexane in a counter - current manner, collect the eluate, and then concentrate the eluate by rotary evaporation at 40 - 45 °C until there is no solvent residue to obtain light yellow transparent Nardostachys jatamansi essential oil.

[0030] The mechanism of action of the above raw material components is as follows:

[0031] The xylanase - chitinase - β - glucanase complex enzyme is formed by xylanase - chitinase - β - glucanase. Xylan is one of the main components of plant cell walls. Xylanase can degrade xylan and destroy the structure of the cell wall. By hydrolyzing the β - 1,4 - glycosidic bond of the xylan molecule, xylanase decomposes xylan into small molecules such as xylo - oligosaccharides and xylose, thereby weakening the integrity of the cell wall. Chitin is an important component in plant cell walls, and chitinase can decompose chitin, further destroying the structure of the cell wall. β - Glucan is another important component in plant cell walls, and β - glucanase can hydrolyze β - glucan, completely disintegrating the lignin - hemicellulose composite structure of the cell wall. Xylanase first degrades xylan, and chitinase and β - glucanase further decompose other polysaccharide components, thus completely destroying the integrity of the cell wall and making it easier to release essential oil components.

[0032] Mannosvlerythritol Lipids (MELs) are a kind of biosurfactants produced by yeasts. They have a unique chemical structure and multifunctionality, are non-toxic and biodegradable. They are composed of mannose, erythritol and fatty acid chains, and are divided into four types, namely MEL-A, MEL-B, MEL-C and MEL-D according to the degree of hydroxylation. Their hydrophilic head (glycosyl group) and hydrophobic tail (fatty acid) enable them to reduce the surface tension of liquids. Mannosvlerythritol lipids improve the contact efficiency between water vapor and cell membranes by reducing the surface tension of the aqueous phase, thereby enhancing the extraction efficiency of Nardostachys jatamansi essential oil.

[0033] The molecular formula of spiraeoside is C 21 H 20 O 12 , which is a flavonoid glycoside compound and widely exists in plants such as Spiraea, Ginkgo biloba, and buckwheat. It is formed by glycosylation with quercetin as the aglycone and has significant biological activities. It is soluble in hot water, methanol, and ethanol, slightly soluble in acetone, and insoluble in petroleum ether. Spiraeoside has an amphiphilic structure, that is, it contains both hydrophilic (glycoside part) and hydrophobic (triterpene or steroid skeleton) groups. When saponins are present at the oil-water interface, the hydrophobic part tends to combine with the essential oil, while the hydrophilic part combines with water. This effect reduces the interfacial tension between oil and water, making it easier for oil droplets to disperse into smaller sizes.

[0034] Nardostachys grandiflora is the original species of the genus Nardostachys. The main components in its essential oil include nardol, β-elemene (2.9%), and 1,2,9,10-tetradehydroaristolene (2.9%). The essential oil of Nardostachys grandiflora has a rich and lasting woody aroma. Nardostachys jatamansi esters and nardostachone are its main aroma components, which are particularly suitable for formulating fragrances such as sandalwood type, rose sandalwood type, and fougere type, and have a good fixative effect.

[0035] Sodium chloride is a colorless and transparent cubic crystal or white crystalline powder, which is easily soluble in water, slightly soluble in ethanol, insoluble in concentrated sulfuric acid, has stable chemical properties and is not easily decomposed, but can be melted at high temperatures. Sodium chloride mainly promotes the release of essential oil through salting-out effect and osmotic pressure drive in the extraction of Nardostachys jatamansi essential oil. Sodium chloride increases the ionic strength of the aqueous phase, reduces the solubility of essential oil components (mainly hydrophobic terpene compounds) in water, and promotes the precipitation of essential oil from the aqueous phase, thereby improving the extraction rate and separation efficiency. In steam distillation, the addition of sodium chloride can increase the boiling point of the system, enhance the penetration of steam into plant tissues, and reduce the volatilization loss of essential oil components at the same time.

[0036] Polysorbate 80 (also known as Tween 80) is a non-ionic surfactant that is readily soluble in polar solvents such as water, ethanol, and glycerol and forms a stable emulsion with the oil phase. It is polymerized from sorbitan monooleate and ethylene oxide. Polysorbate 80 consists of a hydrophilic polyoxyethylene chain (PEO) and a lipophilic sorbitan-oleate group, forming an amphiphilic molecule (HLB value of about 15), which has emulsifying, solubilizing, and dispersing functions. By reducing the oil-water interfacial tension, it forms micelles or microemulsions (particle size <100 nm), encapsulating hydrophobic essential oil components (such as terpenes and sesquiterpenes) in the micelle core, enhancing their dispersibility and solubility in the aqueous phase.

[0037] In the extraction of Nardostachys jatamansi essential oil, mannosylerythritol lipid, xylanase-chitinase-β-glucanase complex enzyme, and spiraeoside play a role through synergistic effects. During the extraction process of Nardostachys jatamansi essential oil, the xylanase-chitinase-β-glucanase complex enzyme can destroy the structure of the cell wall of Nardostachys jatamansi rhizome by synergistically degrading xylan, chitin, and β-glucan in the cell wall, selectively softening the cell wall and activating the active sites of polysaccharidase, making it easier for essential oil components to be released. Mannosylerythritol lipid can significantly reduce the liquid surface tension, enhance the permeability of water vapor or other solvents, reduce the solvent viscosity and improve solubility, thereby promoting the release of essential oil components. At the same time, under steam heating conditions, it can promote the transformation of the membrane structure from the gel state to the liquid crystal state, protecting essential oil molecules and creating a better working environment for the complex enzyme, increasing the contact opportunity between the complex enzyme and Nardostachys jatamansi cells. Spiraeoside forms a stable interfacial active layer, reducing the surface tension between the essential oil and the aqueous phase, promoting emulsification and dispersion, preventing the coalescence of essential oils, which may lead to a decrease in extraction efficiency and unstable quality of Nardostachys jatamansi essential oil. It improves the penetration ability of the solvent and the dispersibility of essential oil components, while stabilizing the extraction process and protecting essential oil components from oxidation and degradation. The synergistic effect of the three greatly destroys the integrity of the cell wall, enabling the essential oil components inside the cell to be released more smoothly, greatly improving the extraction efficiency and essential oil yield.

[0038] (3) Beneficial effects

[0039] Compared with the prior art, the beneficial effects of the present invention are:

[0040] 1. Mannosylerythritol lipid, xylanase-chitinase-β-glucanase complex enzyme, and spiraeoside, through their synergistic effects, jointly constitute an efficient and refined method for extracting Nardostachys jatamansi essential oil, greatly optimizing the extraction process, improving the quality of essential oil, and providing new ideas for the production and application of Nardostachys jatamansi essential oil;

[0041] 2. The xylanase-chitinase-β-glucanase complex enzyme has a higher degradation rate on the cell wall, is more efficient than single enzymes, makes the release channel of Nardostachys jatamansi essential oil smoother, reduces the pretreatment time at the same time, and the synergistic effect accelerates the decomposition of the cell wall, thus shortening the time of the whole extraction process;

[0042] 3. Diatomite-supported nano-zinc oxide can selectively capture essential oil molecules, reduce thermal degradation at high temperatures, and extend the shelf life of essential oils through the antibacterial property of nano-zinc oxide. Moreover, the adsorbent can be regenerated by ethanol washing and drying after elution, saving costs. Description of the Drawings

[0043] Figure 1 SEM image of untreated Nardostachys jatamansi cells in Example 1 of the present invention;

[0044] Figure 2 SEM image of Nardostachys jatamansi cells treated with xylanase-chitinase-β-glucanase complex enzyme in Example 1 of the present invention. Detailed Embodiments

[0045] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0046] The test equipment and preparations in the following embodiments are as follows: electronic balance (Sartorius, Germany), electrothermal constant temperature water bath (Kedao, Jiangsu), magnetic stirrer (Meiyingpu, Shanghai), ultrasonic instrument (Yixin, Shanghai), high-speed centrifuge (Jidi, Guangzhou), vacuum drying oven (Jiecheng, Shanghai), gas chromatograph (Shimadzu), rotary evaporator (Yaote, Shanghai), scanning electron microscope (Zeiss, Germany), freeze dryer (Pudong Freeze Drying, Shanghai), pH meter (Yidian, Shanghai), constant temperature incubator (Hetian, Shanghai), ultrasonic vibrator (ELMA-E5K), distillation kettle (GEA, Germany); chemical drugs and reagents are purchased from Sigma-Aldrich.

[0047] Example 1: This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 10 parts of mannosylerythritol lipid, 10 parts of spiraeoside, 50 parts of Nardostachys jatamansi DC., 150 parts of distilled water, 10 parts of sodium chloride, 20 parts of diatomite-supported nano-zinc oxide, and 5 parts of polysorbate-80. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys jatamansi DC. is 10:1.

[0048] The core of the extraction of essential oil of Nardostachys raphanus lies in the multi-dimensional synergistic effect of the three-level system of mannosyl erythritol lipids, xylanase-chitinase-β-glucanase complex enzymes and meadowfoam glycosides. Its mechanism is progressive and forms a chain synergistic network of "penetration deconstruction-enzymatic targeting-dynamic encapsulation". First, the xylanase-chitinase-β-glucanase complex enzyme begins to work. Xylanase preferentially cuts the β-1,4-glycosidic bonds of the hemicellulose main chain to release xylo-oligosaccharide fragments; β-glucanase targets the decomposition of β-1,3 / 1,6-glucan in the cross-linking area of ​​cellulose and hemicellulose to release the interlocking of the polysaccharide network; chitinase specifically hydrolyzes the N-acetylglucosamine chain in the fungal symbiotic structure to eliminate exogenous biofilm interference. Figure 1 and Figure 2 The SEM comparison of nard before and after adding xylanase-chitinase-β-glucanase complex enzymes shows that the structure of nard without adding xylanase-chitinase-β-glucanase complex enzymes is network-like. After being treated with mannosyl erythritol lipids, the network-like structure disappears and begins to disperse into flakes, with a tendency to merge into one, indicating that the structure has changed after treatment and the cell wall has been destroyed. The hydrophobic end (alkyl chain) of mannosyl erythritol lipids can bind to the lipid layer of the cell membrane, while the hydrophilic end (sugar group) binds to water molecules to form a transmembrane channel, thereby reducing the interfacial tension of water vapor passing through the cell membrane and accelerating the diffusion of water molecules into the cell. In addition, the insertion of mannosyl erythritol lipids can change the fluidity of the lipid bilayer of the cell membrane, promote the local phase change of the cell membrane structure under steam heating conditions, enhance the permeability of the cell membrane, and facilitate the escape of small molecules of essential oils. At the same time, the critical micelle concentration (CMC) of mannosyl erythritol lipid is low, and it can form micro-micelles in the aqueous phase, encapsulating the released essential oil molecules, preventing them from being re-adsorbed on the surface of cell fragments, and reducing the loss of essential oils. Spiraea glycosides are arranged in a directional manner at the water-oil interface, with the hydrophobic groups anchoring the naringa essential oil molecules, while the hydrophilic groups form a dense interfacial film outward, effectively preventing the droplets from coalescing, thereby maintaining the stability of the solution. In addition, spiraea glycosides carry a negative charge, which disperses the droplets evenly through electrostatic repulsion, avoiding stratification due to gravity, thereby extending the shelf life of the emulsified system. At the same time, in the synergistic effect with mannosyl erythritol lipids, the long-chain micelles of spiraea glycosides and the short-chain micelles of mannosyl erythritol lipids form a mixed micelle network, which enhances the mechanical strength of the interfacial film and enables it to be compatible with essential oil components of different polarities. The cell wall fragments released by the directional cutting of the complex enzyme, the joint action of mannoerythritol lipids and meadowfoam glycosides stabilize the essential oil in the solution after it escapes, further forming the "enzyme-solvent-carrier" three-phase interface, and promoting the directional enrichment of essential oil molecules. The synergistic mechanism of these three substances regulates the interface behavior and energy transfer path at the molecular scale, transforming the passive diffusion of the traditional process into an active synergistic mode of directional deconstruction-dynamic capture, providing an innovative paradigm for the green manufacturing of plant essential oils.

[0049] The preparation method of the xylanase-chitinase-β-glucanase complex enzyme includes:

[0050] S11. Select alkali-tolerant xylanase, Aspergillus, and Aspergillus niger as the production sources of xylanase, chitinase, and β-glucanase;

[0051] S12. Ferment the above three strains in a culture medium, where the fermentation temperature of alkali-tolerant xylanase and Aspergillus niger is 37-39°C or 25-28°C, and the fermentation temperature of Aspergillus is 20-25°C, and culture for 48-52 h;

[0052] S13. Inoculate the activated strains into the fermentation medium, control the inoculation amount to be 5-8%, and shake culture at 28-30°C and 180 rpm for 72-80 h. Among them, 1-2% Triton X-100 needs to be added to Aspergillus to obtain three fermentation broths;

[0053] S14. Mix the three fermentation broths together, centrifuge at 7500-8000 rpm for 15-20 min, and separately collect the supernatants after centrifugation of the three fermentation broths to obtain the supernatants;

[0054] S15. Slowly add ammonium sulfate to the supernatant to a saturation of 30-35%, let it stand for 2-3 h, then centrifuge at 7500-8000 rpm for 15-20 min. Subsequently, continue to add ammonium sulfate to the supernatant to a saturation of 70-75%, let it stand at 4°C for 10-12 h, centrifuge at 7500-8000 rpm for 15-20 min, and dissolve the precipitate with 0.1 M phosphate buffer to obtain the first mixed solution;

[0055] S16. Add 0.1 M phosphate buffer to the first mixed solution, adjust the pH to 6.0-6.5 for dialysis, change the buffer every 6-8 h, and change it at least three times to obtain the second mixed solution;

[0056] S17. Load the second mixed solution onto a DEAE-cellulose chromatography column, elute with a linear gradient of 0.1-0.5 M NaCl, gradually increase the NaCl concentration to elute different components in sequence. Subsequently, load the active components after DEAE-cellulose chromatography onto a Sephadex G-75 column, elute with a phosphate buffer at pH = 6.0-6.5, collect the eluate, measure the enzyme activity of the eluate, combine the xylanase, chitinase, and β-glucanase active components, and then perform lyophilization to obtain the xylanase-chitinase-β-glucanase complex enzyme.

[0057] The loading amount of nano-zinc oxide in the diatomite-supported nano-zinc oxide is 5-9%.

[0058] The mass ratio of the mannosylerythritol lipid to the spiraeoside is 1:2.

[0059] The weight ratio of the distilled water, sodium chloride, and polysorbate-80 is 100:3:8.

[0060] The mass ratio of the Nardostachys jatamansi DC., diatomite-supported nano-zinc oxide, and sodium chloride is 1:20:1.

[0061] A method for extracting fresh Nardostachys jatamansi DC. essential oil, which is applied to the fresh Nardostachys jatamansi DC. essential oil described above, comprises the following steps:

[0062] S21. Take 100 g of fresh Nardostachys jatamansi DC. rhizomes (with a moisture content of 60-65%), quickly freeze them in crystalline liquid nitrogen at -196 to -190 °C for 10-15 min, and then immediately place them in a cryogenic grinder and grind them to 60-65 mesh to obtain Nardostachys jatamansi DC. granules;

[0063] S22. Weigh 2-3 g of citric acid and 12-15 g of disodium hydrogen phosphate, dissolve them in 1-1.5 L of ultrapure water, adjust the pH to 5.5-5.8, sterilize them at 121 °C for 20 min under this condition to obtain a buffer solution. Subsequently, weigh 50-65 g of a xylanase-chitinase-β-glucanase complex enzyme and dissolve it in the buffer solution to obtain a complex enzyme solution;

[0064] S23. Add the Nardostachys jatamansi DC. granules and the complex enzyme solution to a first container according to a mass ratio of 1:(10-12), place them in a constant temperature shaker at 45 °C, and react at 150-160 rpm for 2-3 h. Subsequently, continue shaking at 80 °C for 5-10 min, cool to room temperature, and then centrifuge at 3500-4000 rpm for 5-10 min to obtain a first solution;

[0065] S24. Mix the mannosylerythritol lipid and lactic acid according to a mass ratio of 1:2, place them in a constant temperature shaker at 60 °C, and react at 150-180 rpm for 2-3 h to obtain a second mixture. Mix the second mixture and the spiraeoside according to a mass ratio of 1:(2-4), and then perform ultrasonic treatment under the conditions of 40-50 kHz for 10-15 min to obtain a third solution;

[0066] S25. Mix the first solution and the third solution according to a mass ratio of 1:10, and then sequentially add sodium chloride and polysorbate-80 according to a mass ratio of (2-6):1, spread them evenly on the surface of the porous plate inside the distillation kettle, and the filling height ≤ 2 / 3 of the kettle body height. Turn on the steam generator, introduce saturated steam, control the steam pressure at 0.1-0.12 MPa, control the temperature inside the kettle at 95-98 °C, and the distillation time at 4-4.5 h;

[0067] S26. The steam is sequentially passed into a primary condenser to trap high-boiling components, and a secondary condenser to efficiently recover volatile essential oils, obtaining condensate. Subsequently, the condensate is separated into layers by an oil-water separator. The essential oil phase is filled with diatomite-supported nano-zinc oxide in an adsorption column, with a packing density of 0.45 g / cm 3 , and then passes through the aqueous phase to obtain crude essential oil;

[0068] S27. The adsorption column is eluted countercurrently with n-hexane, and the eluate is collected. Subsequently, the eluate is concentrated by rotary evaporation at 40 - 45 °C until there is no solvent residue, obtaining light yellow transparent Nardostachys jatamansi essential oil.

[0069] Example 2: This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 15 parts of mannosylerythritol lipid, 10 parts of spiraeoside, 50 parts of Nardostachys grandiflora, 150 parts of distilled water, 5 parts of sodium chloride, 20 parts of diatomite-supported nano-zinc oxide, and 5 parts of polysorbate-80. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora is 10:1. The extraction method of the xylanase-chitinase-β-glucanase complex enzyme and Nardostachys grandiflora in this example is the same as that in Example 1. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0070] Example 3: This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 20 parts of mannosylerythritol lipid, 10 parts of spiraeoside, 50 parts of Nardostachys grandiflora, 150 parts of distilled water, 5 parts of sodium chloride, 20 parts of diatomite-supported nano-zinc oxide, and 5 parts of polysorbate-80. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora is 10:1. The extraction method of the xylanase-chitinase-β-glucanase complex enzyme and Nardostachys grandiflora in this example is the same as that in Example 1. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0071] Example 4: This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 10 parts of mannosylerythritol lipid, 10 parts of spiraeoside, 50 parts of Nardostachys grandiflora, 150 parts of distilled water, 5 parts of sodium chloride, 20 parts of diatomite-supported nano-zinc oxide, and 5 parts of polysorbate-80. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora is 12:1. The extraction method of the xylanase-chitinase-β-glucanase complex enzyme and Nardostachys grandiflora in this example is the same as that in Example 1. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0072] Example 5: This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 10 parts of mannosylerythritol lipid, 10 parts of spiraeoside, 50 parts of Nardostachys grandiflora DC., 150 parts of distilled water, 5 parts of sodium chloride, 20 parts of diatomite-supported nano-zinc oxide, and 5 parts of polysorbate-80. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora DC. is 15:1. The extraction method of the xylanase-chitinase-β-glucanase complex enzyme and Nardostachys grandiflora DC. in this example is the same as that in Example 1. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0073] Example 6: This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 10 parts of mannosylerythritol lipid, 20 parts of spiraeoside, 50 parts of Nardostachys grandiflora DC., 150 parts of distilled water, 5 parts of sodium chloride, 20 parts of diatomite-supported nano-zinc oxide, and 5 parts of polysorbate-80. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora DC. is 10:1. The extraction method of the xylanase-chitinase-β-glucanase complex enzyme and Nardostachys grandiflora DC. in this example is the same as that in Example 1. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0074] Example 7: This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 10 parts of mannosylerythritol lipid, 30 parts of spiraeoside, 50 parts of Nardostachys grandiflora DC., 150 parts of distilled water, 5 parts of sodium chloride, 20 parts of diatomite-supported nano-zinc oxide, and 5 parts of polysorbate-80. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora DC. is 10:1. The extraction method of the xylanase-chitinase-β-glucanase complex enzyme and Nardostachys grandiflora DC. in this example is the same as that in Example 1. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0075] Control Group 1: The difference between this example and Example 1 is that it does not contain the xylanase-chitinase-β-glucanase complex enzyme. This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, including the following raw materials in parts by weight: 10 parts of mannosylerythritol lipid, 10 parts of spiraeoside, 50 parts of Nardostachys grandiflora DC., 150 parts of distilled water, 5 parts of sodium chloride, 20 parts of diatomite-supported nano-zinc oxide, and 5 parts of polysorbate-80. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0076] Control Group 2: The difference between this example and Example 1 is that it does not contain spiraeoside. This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, which includes the following raw materials in parts by weight: mannosylerythritol lipid 10 parts, Nardostachys grandiflora 50 parts, distilled water 150 parts, sodium chloride 5 parts, diatomite-supported nano-zinc oxide 20 parts, polysorbate-80 5 parts. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora is 10:1. The extraction method of the xylanase-chitinase-β-glucanase complex enzyme in this example is the same as that in Example 1. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0077] Control Group 3: The difference between this example and Example 1 is that it does not contain mannosylerythritol lipid. This example discloses a method for extracting fresh Nardostachys jatamansi essential oil, which includes the following raw materials in parts by weight: spiraeoside 10 parts, Nardostachys grandiflora 50 parts, distilled water 150 parts, sodium chloride 5 parts, diatomite-supported nano-zinc oxide 20 parts, polysorbate-80 5 parts. The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora is 10:1. The extraction method of the xylanase-chitinase-β-glucanase complex enzyme in this example is the same as that in Example 1. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0078] Control Group 4: The difference between this example and Example 1 is that it does not contain mannosylerythritol lipid, xylanase-chitinase-β-glucanase complex enzyme, and spiraeoside. This example discloses a fresh Nardostachys jatamansi essential oil, which includes the following raw materials in parts by weight: Nardostachys grandiflora 50 parts, distilled water 150 parts, sodium chloride 5 parts, diatomite-supported nano-zinc oxide 20 parts, polysorbate-80 5 parts. The method for extracting fresh Nardostachys jatamansi essential oil in this example is the same as that in Example 1.

[0079] Effect evaluation: Quality analysis test of Nardostachys jatamansi essential oil: Select 100 g of Nardostachys grandiflora for extraction in each group, weigh the mass of the finally obtained essential oil, which is the yield of Nardostachys jatamansi essential oil, and the calculation method is as follows. Subsequently, take 10 μL of the Nardostachys jatamansi essential oil sample, add 990 μL of chromatographically pure n-hexane, dilute to 1 mL (concentration 1%), and vortex for 1 - 2 min to fully dissolve it. Filter using a 0.22 μm organic syringe filter to remove particulate impurities, transfer to a 2 mL brown injection vial, and seal and store in the dark. The distribution coefficients of each component in the Nardostachys jatamansi essential oil between the stationary phase (column coating) and the mobile phase (helium) are different. Components with low boiling points and small polarities (such as monoterpenes) elute first, and components with high boiling points and large polarities (such as sesquiterpene ketones) elute later. Optimize the separation effect by gradient temperature programming. For example, raise the temperature to 250 °C to ensure the full elution of the heavy components (such as α-nardostachone). Calculate the contents of α-nardostachone and total terpenoids using the external standard method. The extraction time is counted from the start of heating to the stop of heating.

[0080] Yield of Nardostachys jatamansi essential oil = (mass of essential oil / dry weight of raw material) × 100%

[0081] Table 1 shows the measurement and statistical results of Nardostachys jatamansi essential oil for each experimental group.

[0082]

[0083] Table 1 shows the measurement and statistical results of the content of Nardostachys jatamansi essential oil for each experimental group. It can be seen from Table 1 that the Nardostachys jatamansi essential oil obtained by extraction in each experimental group has a relatively high oil yield. α-Nardostachone and total terpenoids are the main components of Nardostachys jatamansi essential oil and maintain relatively high contents. The control group shows a relatively low oil yield and the contents of α-nardostachone and total terpenoids. By comparing the oil yields of the essential oils in Examples 1-7 and Control Groups 1-4, it can be found that overall, the Nardostachys jatamansi essential oil obtained by extraction in Examples 1-7 has a higher oil yield, which is higher than that of other experimental groups and control groups. This indicates that the Nardostachys jatamansi essential oil obtained by extraction in Example 1 has the best effect and higher quality. The contents of α-nardostachone and total terpenoids are the highest among all experimental groups, reaching 26.1% and 87.3% respectively, and the extraction time is the shortest, which is 4.5 h. The oil yields of the Nardostachys jatamansi essential oils obtained by extraction in Control Groups 1-4 are all lower than those of the experimental groups. Among them, the control group has the longest extraction time and the lowest contents of α-nardostachone and total terpenoids. This shows that by adding mannosylerythritol lipid, xylanase-chitinase-β-glucanase complex enzyme, and spiraeoside simultaneously during the extraction process of Nardostachys jatamansi essential oil, Nardostachys jatamansi essential oil with high quality and high oil yield can be obtained. Through the above limited experiments, the application effect of the method for extracting fresh Nardostachys jatamansi essential oil in Example 1 of the present invention is remarkable. By adding mannosylerythritol lipid, xylanase-chitinase-β-glucanase complex enzyme, and spiraeoside simultaneously during the extraction process, the utilization rate of Nardostachys jatamansi can be significantly improved, and it has both environmental protection and economic benefits, realizing the efficient, high-quality, and low-consumption extraction of Nardostachys jatamansi essential oil, providing equipment guarantee for efficient and green extraction.

[0084] Finally, it should be noted that although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fresh spikenard essential oil, characterized in that The method comprises the following raw materials in parts by weight: 10 to 20 parts of mannose erythritol lipid, 10 to 30 parts of meadowfoam glycoside, 50 to 70 parts of naringa chinensis, 20 to 40 parts of diatomaceous earth loaded nano zinc oxide, 5 to 15 parts of sodium chloride, 100 to 200 parts of distilled water, and 5 to 10 parts of polysorbate-805; The fresh spikenard essential oil also includes: Xylanase-chitinase-β-glucanase complex; The weight ratio of the xylanase-chitinase-β-glucanase complex enzyme to Nardostachys grandiflora is (10-15):

1.

2. A fresh spikenard essential oil according to claim 1, characterized in that, The method for preparing the xylanase-chitinase-β-glucanase complex enzyme comprises: S11. Selecting alkali-resistant xylanase, Aspergillus, and Aspergillus niger as the production source of xylanase, chitinase, and β-glucanase; S12. The three strains are fermented in a culture medium, wherein the fermentation temperature of alkali-resistant xylanase and Aspergillus niger is 37-39°C or 25-28°C, and the fermentation temperature of Aspergillus is 20-25°C, and the culture is carried out for 48-52 hours; S13. The activated strain was inoculated into the fermentation medium, the inoculation amount was controlled to be 5-8%, and the culture was shaken at 28-30°C and 180rpm for 72-80h, wherein Aspergillus needed to be added with 1-2% Triton X-100 to obtain three fermentation broths; S14. The three fermentation broths were mixed together, centrifuged at 7500-8000 rpm for 15-20 min, and the supernatants of the three fermentation broths after centrifugation were collected to obtain supernatants; S15. slowly adding ammonium sulfate to the supernatant to 30-35% saturation, standing for 2-3 hours, centrifuging at 7500-8000 rpm for 15-20 minutes, then continuing to add ammonium sulfate to the supernatant to 70-75% saturation, standing at 4°C for 10-12 hours, centrifuging at 7500-8000 rpm for 15-20 minutes, dissolving the precipitate with 0.1 M phosphate buffer to obtain a first mixed solution; S16. Add 0.1 M phosphate buffer to the first mixed solution, adjust the pH to 6.0-6.5 and perform dialysis, replace the buffer every 6-8 hours, and replace it at least three times to obtain a second mixed solution; S17. Load the second mixed solution onto a DEAE-cellulose chromatography column, and use a 0.1-0.5 M NaCl linear gradient elution, gradually increase the NaCl concentration, and elute different components in sequence; then load the active components after DEAE-cellulose chromatography onto a Sephadex G-75 column, and use a pH = 6.0-6.5 phosphate buffer for elution; collect the eluate, and perform enzyme activity assay on the eluate; combine the xylanase, chitinase, and β-glucanase active components, and then freeze-dry to obtain a xylanase-chitinase-β-glucanase complex enzyme.

3. A fresh spikenard essential oil according to claim 1, characterized in that, The loading amount of the nano zinc oxide in the diatomite-loaded nano zinc oxide is 5-9%.

4. A fresh spikenard essential oil according to claim 1, characterized in that The mass ratio of the mannose erythritol lipid to meadowfoam glycoside is 1:(2-4).

5. A fresh spikenard essential oil according to claim 1, characterized in that The distilled water, sodium chloride and polysorbate-80 are in a weight ratio of (100-120):(3-6):(8-10).

6. A fresh spikenard essential oil according to claim 1, characterized in that The mass ratio of the Nardostachys grandiflora and diatomaceous earth loaded nano zinc oxide and sodium chloride is 1:(20-40):(1-3).

7. A method for extracting fresh spikenard essential oil, used for preparing the fresh spikenard essential oil as claimed in any one of claims 1 to 6, characterized in that: The method comprises the following steps: S21. Take 100g of fresh rhizome of Nardostachys affine (water content 60-65%), freeze it in liquid nitrogen at -196-190°C for 10-15min, and then immediately place it in a freezer grinder and grind it to 60-65 mesh to obtain Nardostachys affine granules; S22. Weigh 2 to 3 g of citric acid and 12 to 15 g of disodium hydrogen phosphate, dissolve in 1 to 1.5 L of ultrapure water, adjust the pH to 5.5 to 5.8, sterilize at 121° C. for 20 min to obtain a buffer solution, then weigh 50 to 65 g of xylanase-chitinase-β-glucanase complex enzyme and dissolve in the buffer solution to obtain a complex enzyme solution; S23. Add the nard granules and the complex enzyme solution to the first container in a mass ratio of 1:(10-12), place in a constant temperature oscillator at 45°C, 150-160rpm for 2-3h, then continue to shake at 80°C for 5-10min, cool to room temperature and centrifuge, centrifuge at 3500-4000rpm for 5-10min to obtain a first solution; S24. Mannosyl erythritol lipid and lactic acid are mixed in a mass ratio of 1:2, placed in a constant temperature oscillator at 60° C., 150-180 rpm for reaction for 2-3 hours to obtain a second mixed solution, the second mixed solution is mixed with meadowfoam glycoside in a mass ratio of 1:(2-4), and then ultrasonically treated at 40-50 kHz for 10-15 minutes to obtain a third solution; S25. The first solution and the third solution are mixed in a mass ratio of 1:10, and then sodium chloride and polysorbate-80 are added in a mass ratio of (2-6):1, and spread on the surface of the porous plate in the distillation kettle, and the filling height is ≤ 2 / 3 of the height of the kettle body. The steam generator is turned on, saturated water vapor is introduced, and the steam pressure is controlled to 0.1-0.12MPa, the temperature in the kettle is controlled to 95-98°C, and the distillation time is 4-4.5h; S26. Steam is sequentially passed into the primary condenser to capture high-boiling point components, and the secondary condenser efficiently recovers volatile essential oils to obtain condensate. The condensate is then separated by an oil-water separator, and the essential oil phase is filled with diatomaceous earth-loaded nano-zinc oxide in an adsorption column with a filling density of 0.45 g / cm 3 , then through the aqueous phase, to obtain the crude essential oil; S27. Use n-hexane to countercurrently elute the adsorption column, collect the eluate, and then concentrate the eluate by rotary evaporation at 40-45°C until no solvent remains to obtain light yellow transparent nard essential oil.

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