Separation and Detection Method of Sclareol, Sclareolide and Sclareodiol

Abstract

The present invention discloses a method for separating and detecting sclareol, sclareolide and sclareodiol, which relates to the technical field of substance separation and detection. The separation and detection method includes: preparing a mixed standard solution of sclareol, sclareolide and sclareodiol; detecting the mixed standard solution by high performance liquid chromatography to obtain the standard curves and linear regression equations corresponding to sclareol, sclareolide and sclareodiol; separating and detecting sclareol, sclareolide and sclareodiol in the to-be-detected catalytic solution by high performance liquid chromatography. The present invention uses high performance liquid chromatography to realize the qualitative analysis, quantitative detection and component separation of sclareol, sclareolide and sclareodiol in the to-be-detected catalytic solution catalyzed by fungus ATCC 20624, and the precision of its quantitative detection approaches the detection result of gas chromatography.

Description

Technical Field

[0001] The invention relates to the technical field of substance separation and detection, and in particular to a method for separating and detecting sclareol, sclareolide and sclarediol. Background Art

[0002] Sclareol has a faint amber aroma, and the aroma is delicate, highly diffusible and long-lasting. It is mainly used to manufacture natural ambergris substitutes such as sclareolide and ambrox. That is, the semi-synthetic route using sclareol as the raw material, the sclareol extracted from the plant sclareol has a carbon atom skeleton similar to ambrox, the sclareol side chain is oxidized by an oxidant to generate sclareolide, and then reduced to obtain sclarediol, and finally under acidic conditions, dehydration cyclization is performed to generate ambrox. However, this method of obtaining sclareol using sclareol as the raw material requires extraction, purification and other steps, and the growth cycle of sclareol is long and greatly affected by environmental factors.

[0003] In the prior art, there is a technical solution for synthesizing sclareol by fermentation with microorganisms, that is, Cryptococcus albidus can ferment sclareol as a substrate and convert it into sclareolide, while yeast fungi (Hyphozyma roseonigra ATCC) can degrade sclareol into sclareoldiol and produce a small amount of sclareolide. However, due to the relatively low hydrophobicity of sclareol, sclareolide and sclareolide, similar structures, polarity and retention time, liquid chromatography cannot effectively separate and detect the mixed solution of sclareol, sclareolide and sclareolide, and gas chromatography is usually used to separate and detect them. Summary of the invention

[0004] An object of the present invention is to provide a method for separating and detecting sclareol, sclareolide and sclarediol, so as to solve the technical problem that the high performance liquid chromatography in the prior art cannot effectively separate the mixture of sclareol, sclareolide and sclarediol.

[0005] Another object of the present invention is to improve the detection accuracy of qualitative analysis and quantitative detection of sclareol, sclareolide and sclarediol by high performance liquid chromatography.

[0006] According to the purpose of the present invention, the present invention provides a method for separating and detecting sclareol, sclareolide and sclarediol, comprising:

[0007] preparing a mixed standard solution of sclareol, sclareolide and sclarediol;

[0008] The mixed standard solution was detected by high performance liquid chromatography (HPLC) to obtain the standard curves and linear regression equations corresponding to sclareol, sclareolide and sclareodiol;

[0009] The sclareol, sclareolide and sclareodiol in the catalytic solution to be measured were separated and detected by the above-mentioned HPLC;

[0010] Among them, the catalytic solution to be measured was obtained by catalyzing sclareol with the fungus with the preservation number of ATCC 20624 to generate sclareolide and sclareodiol. The chromatographic column of the HPLC was a C18 column, and the detection wavelength of the ultraviolet detector was any value in the range of 190 nm - 200 nm. Acetonitrile was used as mobile phase A and ultrapure water was used as mobile phase B for gradient elution. The gradient elution program was as follows: at 0 - 5 min, the volume fraction of mobile phase A was 60% and the volume fraction of mobile phase B was 40%; at 5 - 10 min, the volume fraction of mobile phase A increased from 60% to 90% and the volume fraction of mobile phase B decreased from 40% to 10%; at 10 - 20 min, the volume fraction of mobile phase A was 90% and the volume fraction of mobile phase B was 10%; at 20 - 30 min, the volume fraction of mobile phase A decreased from 90% to 80% and the volume fraction of mobile phase B increased from 10% to 20%; at 30 - 45 min, the volume fraction of mobile phase A decreased from 80% to 60% and the volume fraction of mobile phase B increased from 20% to 40%.

[0011] Optionally, the specification of the chromatographic column was 4.6 mm × 250 mm, 5 μm.

[0012] Optionally, the material of the packing particles of the chromatographic column was octadecylsilane-bonded silica gel.

[0013] Optionally, the column temperature of the chromatographic column was any value in the range of 25°C - 35°C.

[0014] Optionally, the volume flow rate of the gradient elution was any value in the range of 0.3 mL / min - 0.6 mL / min.

[0015] Optionally, the steps of using the fungus with the preservation number of ATCC 20624 to catalyze sclareol to generate sclareolide and sclareodiol for the catalytic solution to be measured further included:

[0016] Preparing a cell suspension of the fungus ATCC 20624;

[0017] Adding the sclareol solution to the basic inorganic salt maintenance solution to prepare a mixed solution;

[0018] Add the cell suspension to the mixed solution to prepare a reaction solution, and the cell suspension is set to have an OD when inoculated into the reaction solution 600 which is any value from 28 to 32;

[0019] Control the reaction solution to react at a first preset temperature for a first preset time at a preset rotation speed;

[0020] Perform extraction, centrifugation and filtration on the reaction solution in sequence to prepare the catalytic solution to be measured.

[0021] Optionally, the first preset temperature is any value from 20°C to 28°C, and the first preset time is any value from 60 h to 80 h.

[0022] Optionally, the step of preparing the cell suspension of fungus ATCC 20624 further includes:

[0023] Add a solution of sclareol with a preset concentration to a yeast malt medium containing the fungus ATCC 20624 to prepare a culture solution, and culture it for a second preset time under the condition of a second preset temperature;

[0024] Centrifuge and wash the culture solution in sequence;

[0025] Resuspend the culture solution in the basic inorganic salt maintenance solution to prepare the cell suspension.

[0026] Optionally, the second preset temperature is any value from 20°C to 28°C, and the second preset time is any value from 36 h to 60 h.

[0027] The present invention realizes qualitative analysis, quantitative analysis and component separation of sclareol, sclareolide and sclareodiol by using high performance liquid chromatography. At the same time, in multiple high performance liquid chromatography analyses, the above gradient elution program is used for mobile phase elution, so that sclareol, sclareolide and sclareodiol have good retention and separation in high performance liquid chromatography. This gradient program optimizes the separation effect of sclareol, sclareolide and sclareodiol by first rising, then stabilizing and then falling, and at the same time improves the repeatability, stability and flux adaptability of the analysis method.

[0028] Furthermore, the present invention selects a reversed-phase C18 chromatographic column with a particle size of 5 μm, an inner diameter of 4.6 mm and a column length of 250 mm. During the separation of sclareol, sclareolide and sclareodiol, it can provide good resolution and peak shape stability, and realize high-resolution analysis of structurally similar compounds on the premise of maintaining moderate column pressure and flow rate, improving the detection accuracy of qualitative analysis and quantitative detection.

[0029] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and be able to implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and describes them in detail in conjunction with the drawings as follows. Description of the Drawings

[0030] Some specific embodiments of the present invention will be described in detail hereinafter with reference to the drawings in an exemplary but not restrictive manner. The same reference numerals in the drawings denote the same or similar components or parts. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings:

[0031] Figure 1 is a schematic flowchart of a separation detection method according to an embodiment of the present invention;

[0032] Figure 2 is a schematic flowchart of the preparation of a catalytic solution to be measured according to an embodiment of the present invention;

[0033] Figure 3 is a schematic flowchart of the preparation of a cell suspension according to an embodiment of the present invention;

[0034] Figure 4 is a liquid chromatogram of a sclareol standard solution according to an embodiment of the present invention;

[0035] Figure 5 is a liquid chromatogram of a sclareodiol standard solution according to an embodiment of the present invention;

[0036] Figure 6 is a liquid chromatogram of a sclareolide standard solution according to an embodiment of the present invention;

[0037] Figure 7 is a liquid chromatogram of a single-concentration mixed standard solution according to an embodiment of the present invention;

[0038] Figure 8 is a liquid chromatogram of a gradient-concentration mixed standard solution according to an embodiment of the present invention;

[0039] Figure 9 is a standard curve of sclareodiol according to an embodiment of the present invention;

[0040] Figure 10 is a standard curve of sclareolide according to an embodiment of the present invention;

[0041] Figure 11 is a standard curve of sclareol according to an embodiment of the present invention;

[0042] Figure 12 is a liquid chromatogram of the catalytic solution to be measured detected according to Embodiment 1 of the present invention;

[0043] Figure 13 It is the gas chromatogram of the catalytic solution to be detected in Comparative Example 3 according to the present invention. Detailed implementation manners

[0044] The following will further describe in detail the specific implementation manners of the present invention in conjunction with the drawings and embodiments. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0045] To make the above objects, features, and advantages of the present application more obvious and understandable, the following will describe in detail the specific implementation manners of the present application in conjunction with the drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, rather than limiting the present application. Additionally, it should be noted that for the sake of convenience of description, only the parts related to the present application rather than all the structures are shown in the drawings. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present application.

[0046] The terms "comprise" and "have" and any variations thereof in the present application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products, or devices.

[0047] Referring to "embodiments" herein means that the specific features, structures, or characteristics described in conjunction with the embodiments can be included in at least one embodiment of the present application. The phrase appears in various positions in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0048] As Figure 1 shown, the present invention provides a method for separating and detecting sclareol, sclareolide, and sclaridiol. The fungus used in the present invention is a yeast-like fungus (Hyphozyma roseonigra ATCC), and its preservation number is ATCC20624, which can be purchased from commercial channels. The fungus ATCC 20624 used in the present invention is a freeze-dried powder purchased from Beijing Biovector Science Lab Co., Ltd., and can be cultured by dissolving it in yeast malt medium. The yeast malt medium includes 3 g / L yeast extract, 3 g / L malt extract, 10 g / L glucose, and 5 g / L tryptophan. The separation and detection method includes:

[0049] Step S100: Prepare a mixed standard solution of sclareol, sclareolide, and sclareodiol;

[0050] Step S200: Detect the mixed standard solution by high performance liquid chromatography to obtain the standard curves and linear regression equations corresponding to sclareol, sclareolide, and sclareodiol;

[0051] Step S300: Separate and detect sclareol, sclareolide, and sclareodiol in the catalytic solution to be tested by high performance liquid chromatography;

[0052] Among them, the catalytic solution to be tested is obtained by using the fungus with the preservation number of ATCC 20624 to catalyze sclareol to generate sclareolide and sclareodiol. The chromatographic column of the high performance liquid chromatography is a C18 column, and the detection wavelength of the ultraviolet detector is any value between 190 nm and 200 nm. Acetonitrile is used as mobile phase A and ultrapure water is used as mobile phase B for gradient elution. The gradient elution program is as follows: at 0 - 5 min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%; at 5 - 10 min, the volume fraction of mobile phase A increases from 60% to 90%, and the volume fraction of mobile phase B decreases from 40% to 10%; at 10 - 20 min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%; at 20 - 30 min, the volume fraction of mobile phase A decreases from 90% to 80%, and the volume fraction of mobile phase B increases from 10% to 20%; at 30 - 45 min, the volume fraction of mobile phase A decreases from 80% to 60%, and the volume fraction of mobile phase B increases from 20% to 40%. Here, the linear regression equation corresponding to each component reflects the relationship between the concentration and the peak area in the corresponding standard curve.

[0053] It should be noted that sclareol, sclareolide, and sclareodiol have similar structures, similar polarities, and weak ultraviolet absorption abilities, and the molecular structures of sclareol, sclareolide, and sclareodiol are small and have high volatility. Using traditional high performance liquid chromatography usually results in close retention times of the three components and serious peak tailing, and it is impossible to separate sclareol, sclareolide, and sclareodiol in a timely and effective manner. Moreover, since the elution time in the gradient elution stage, the proportion of the mobile phase components eluted in each stage, the change rate of each mobile phase, and the mobile phase composition all have a great impact on the retention time and peak shape of sclareol, sclareolide, and sclareodiol, thus affecting the peak areas and separation effects of sclareol, sclareolide, and sclareodiol, that is, it is necessary to select the corresponding mobile phase composition and simultaneously optimize the parameters related to the elution time, the proportion of the mobile phase components eluted in each stage, and the change rate of each mobile phase according to the selection of the corresponding mobile phase.

[0054] In this embodiment, first, a mixed standard solution of sclareol, sclareolide, and sclareodiol is prepared. Then, the mixed standard solution is detected by high-performance liquid chromatography to obtain the standard curves and linear regression equations corresponding to sclareol, sclareolide, and sclareodiol. After that, high-performance liquid chromatography is used to separate and detect sclareol, sclareolide, and sclareodiol in the catalytic solution to be measured. That is, according to the peak areas of sclareol, sclareolide, and sclareodiol detected in the catalytic solution to be measured, they are substituted into the linear regression equations of the standard curves of the corresponding components to calculate the contents of sclareol, sclareolide, and sclareodiol in the catalytic solution to be measured. When using liquid chromatography to test the mixed standard solution and the catalytic solution to be measured, gradient elution procedure is used for elution, and the detection instrument is an ultraviolet detector. The detection wavelength of the ultraviolet detector can be 190 nm, 195 nm, or 200 nm, or any value in the range of 190 nm - 200 nm.

[0055] In this embodiment, before detecting the mixed standard solution by liquid chromatography, liquid chromatography is first used to qualitatively analyze the sclareol standard solution, sclareolide standard solution, and sclareodiol standard solution respectively, and the retention times corresponding to sclareol, sclareolide, and sclareodiol are recorded. Here, the concentration of the sclareol standard solution is 3 g / L, the concentration of the sclareolide standard solution is 2 g / L, and the concentration of the sclareodiol standard solution is 2 g / L. The solvents of the sclareol standard solution, sclareolide standard solution, and sclareodiol standard solution are chromatographic grade methanol solutions. As Figures 4 to 6 shown, the retention times of sclareodiol, sclareolide, and sclareol are 18.874 min, 23.556 min, and 24.579 min respectively.

[0056] In this embodiment, chromatographic analysis is performed on the standard solutions and mixed standard solution corresponding to sclareol, sclareolide, and sclareodiol in sequence by using high performance liquid chromatography (HPLC) to achieve qualitative analysis of sclareol, sclareolide, and sclareodiol. At the same time, the standard curves and linear regression equations corresponding to sclareol, sclareolide, and sclareodiol are obtained. Then, chromatographic analysis is performed on the catalytic solution to be measured by using HPLC. The concentration of each component is calculated according to the peak area corresponding to each component and the corresponding linear regression equation, so as to achieve quantitative analysis and component separation of sclareol, sclareolide, and sclareodiol in the catalytic solution to be measured. At the same time, in multiple HPLC analyses, the above gradient elution program is used for mobile phase elution, so that sclareol, sclareolide, and sclareodiol have good retention and separation in HPLC. This gradient program optimizes the separation effect of sclareol, sclareolide, and sclareodiol by first rising, then stabilizing, and then falling, while improving the repeatability, stability, and throughput adaptability of the analysis method.

[0057] In this embodiment, in the gradient elution program, during the elution process from 0 to 5 minutes, the volume fraction of mobile phase A is set to 60%, and the volume fraction of mobile phase B is set to 40%. This is beneficial to eluting impurities or precursor components with stronger polarity in advance, reducing their interference with subsequent analysis, and preparing for the full retention of the target substances. During the elution process from 5 to 10 minutes, by gradually increasing the proportion of mobile phase A in the mobile phase, that is, increasing the volume fraction of mobile phase A from 60% to 90% and decreasing the volume fraction of mobile phase B from 40% to 10%, a smooth transition to strong elution force conditions is achieved, avoiding peak crowding or overly strong elution, which is beneficial to separating target substances with medium polarity such as sclareol. During the isocratic elution process from 10 to 20 minutes, high acetonitrile strong elution improves the elution efficiency of substances with long retention time and low polarity, that is, improves the elution efficiency of sclareodiol or sclareolide, ensuring their elution from the column within a reasonable time and avoiding tailing peaks or non-elution phenomena. During the process from 20 to 30 minutes, the volume fraction of mobile phase A is decreased from 90% to 80%, and the volume fraction of mobile phase B is increased from 10% to 20%, that is, slowly restored to moderate conditions, balancing the retention behavior of the previous and subsequent stages, further optimizing the peak shape and facilitating system callback. During the elution process from 30 to 45 minutes, the volume fractions of mobile phase A and mobile phase B gradually return to the initial conditions, which can keep the system clean, avoid residues, be beneficial to the repeatability of subsequent injections, and at the same time complete system rebalancing, improving the continuity and stability of the method.

[0058] In a preferred embodiment, the detection wavelength of the ultraviolet detector is 195 nm. The peak shape of sclareol is good and the intensity is high, and the resolution of sclareodiol and sclareolide is effectively improved.

[0059] In a further embodiment, the specifications of the chromatographic column are 4.6 mm × 250 mm, 5 μm, that is, the column length of the chromatographic column is 250 mm, the inner diameter of the chromatographic column is 4.6 mm, and the particle size of the packed particles in the chromatographic column is 5 μm. In this embodiment, a reversed-phase C18 chromatographic column with a particle size of 5 μm, an inner diameter of 4.6 mm, and a column length of 250 mm is selected. During the separation of sclareol, sclareolide, and sclareodiol, it can provide good resolution and peak shape stability. On the premise of maintaining a moderate column pressure and flow rate, high-resolution analysis of structurally similar compounds can be achieved, and the detection accuracy of qualitative analysis and quantitative detection can be improved. Here, the specifications of the chromatographic column are 4.6 mm × 250 mm, 5 μm, and the model of the chromatographic column is SunFire.

[0060] In a further embodiment, the material of the packed particles of the chromatographic column is octadecylsilyl-bonded silica gel. In this embodiment, the chromatographic column uses octadecylsilyl-bonded silica gel as the stationary phase filler. This material has good hydrophobicity and chemical stability, and can have a significant retention effect on target compounds such as sclareol, sclareodiol, and sclareolide, providing high separation selectivity and resolution, which is beneficial to the effective separation and accurate quantification of structurally similar compounds.

[0061] In a further embodiment, the column temperature of the chromatographic column is any value between 25°C and 35°C, that is, the column temperature of the chromatographic column can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C, or it can also be any value between 25°C and 35°C. The molecular thermal motion slows down, the interaction with the stationary phase increases, and the retention is longer. By setting the column temperature of the chromatographic column within the above range, it helps to improve the stability and reproducibility of the analytical method. At the same time, the separation behavior of compounds in the stationary phase can be adjusted, the peak shape and retention time can be optimized, and the separation efficiency and detection accuracy of target compounds can be improved. Here, when the column temperature of the chromatographic column increases, the movement of compounds in the mobile phase will be accelerated, and the retention time will be slightly advanced. At the same time, it is beneficial to reduce chromatographic tailing and improve the peak shape. When the column temperature of the chromatographic column decreases, the molecular thermal motion slows down, the interaction with the stationary phase increases, and the retention time is extended. Regarding the column temperature of the chromatographic column, it can be flexibly selected according to actual detection needs.

[0062] In a further embodiment, the volumetric flow rate of the gradient elution is any value in the range of 0.3 mL / min to 0.6 mL / min, that is, the volumetric flow rate of the gradient elution can be 0.3 mL / min, 0.4 mL / min, 0.5 mL / min or 0.6 mL / min, or any value in the range of 0.3 mL / min to 0.6 mL / min. In this embodiment, when the flow rate is decreased, that is, when the volumetric flow rate of the gradient elution approaches 0.3 mL / min, the analysis time can be prolonged and the resolution can be improved. When the volumetric flow rate of the gradient elution program is increased and approaches 0.6 mL / min, the analysis time is shortened, which is helpful for improving the sample throughput. Since the polarities and structures of the three salviol substances are slightly different, the retention time difference between them can be optimized by adjusting the flow rate, and the separation clarity can be improved. Moreover, by setting the flow rate range of the high-performance liquid chromatography in the gradient elution program, it is possible to adapt to changes in different column lengths, column inner diameters or solvent viscosities, avoid abnormal high pressure, and improve the operation stability of the high-performance liquid chromatography instrument.

[0063] As Figure 2 shown, in a further embodiment, for the catalytic solution to be measured, the steps of using the fungus with the preservation number of ATCC 20624 to catalyze the formation of sclareolide and sclareodiol from sclareol further include:

[0064] Step S310: Prepare a cell suspension of the fungus ATCC 20624;

[0065] Step S320: Add the sclareol solution to the basic inorganic salt maintenance solution to prepare a mixed solution;

[0066] Step S330: Add the cell suspension to the mixed solution to prepare a reaction solution, and the cell suspension is set to have an OD 600 value of any value between 28 and 32 when inoculated into the reaction solution;

[0067] Step S340: Control the reaction solution to react at a first preset temperature for a first preset time at a preset rotation speed;

[0068] Step S350: Extract, centrifuge and filter the reaction solution in sequence to prepare the catalytic solution to be measured.

[0069] In this embodiment, the catalytic solution to be measured includes sclareol raw material and fungus ATCC 20624. During the preparation of the catalytic solution to be measured, first, a cell suspension of fungus ATCC 20624 is prepared, and the sclareol solution is added to the basic inorganic salt maintenance solution to prepare a mixed solution. Then, the cell suspension is added to the mixed solution to prepare a reaction solution, and the reaction solution is controlled to react at a preset rotation speed at a first preset temperature for a first preset time. Finally, the reaction solution is successively extracted, centrifuged, and filtered to obtain the catalytic solution to be measured. Here, when adding the cell suspension to the mixed solution, it is necessary to make the inoculation of the cell suspension to the reaction solution have an OD 600 value of any one of 28 - 32, that is, the addition of the cell suspension needs to make the OD 600 of the reaction solution be 28, 29, 30, 31, or 32, or it can also be any value in 28 - 32. The preset rotation speed is any value in 150 rpm / min - 200 rpm / min, that is, the preset rotation speed can be 150 rpm / min, 160 rpm / min, 170 rpm / min, 180 rpm / min, 190 rpm / min, or 200 rpm / min, or it can also be any value in 150 rpm / min - 200 rpm / min.

[0070] In this embodiment, since a higher cell density can provide more catalytic sites, shorten the reaction time, and increase the product yield, by inoculating the cell suspension of fungus ATCC 20624 into the mixed solution and controlling the addition amount of the cell suspension to adjust the OD 600 of the reaction solution to be any value in 28 - 32, it helps to improve the conversion efficiency of the catalytic system for the substrate sclareol while ensuring sufficient catalytic activity.

[0071] In a further embodiment, the first preset temperature is any value in 20°C - 28°C, and the first preset time is any value in 60 h - 80 h. That is, the reaction time for the cell suspension to catalyze the conversion of sclareol to sclareodiol and sclareolide can be 60 h, 64 h, 68 h, 70 h, 72 h, 76 h, or 80 h, or it can also be any value in 60 h - 80 h. The reaction temperature can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, or 28°C, or it can also be any value in 20°C - 28°C. By setting the reaction temperature within the above range, it can ensure that the microbial catalytic system operates efficiently under mild conditions, avoid inactivation at high temperature or the generation of by-products, and is conducive to the stable expression of the catalytic performance of fungus ATCC 20624 cells and the maintenance of enzyme activity. Setting the reaction time to any value in 60 h - 80 h can provide sufficient reaction time, balance the conversion rate and reaction efficiency, adapt to different substrate concentrations or cell states, and improve the applicability and flexibility of the catalytic system.

[0072] As shown Figure 3 in a further embodiment, step S310 further includes:

[0073] Step S311: Add a sclareol solution with a preset concentration to a yeast malt medium containing fungus ATCC 20624 to prepare a culture solution, and culture it for a second preset time under a second preset temperature condition;

[0074] Step S312: Centrifuge and wash the culture solution in sequence;

[0075] Step S313: Resuspend the culture solution in a basic inorganic salt maintenance solution to prepare a cell suspension.

[0076] In this embodiment, in the method for preparing a cell suspension of fungus ATCC 20624, first, a sclareol solution with a preset concentration is added to a yeast malt medium containing fungus ATCC 20624 to prepare a culture solution, and it is cultured for a second preset time under a second preset temperature condition. Then, the culture solution is centrifuged and washed in sequence. Finally, the culture solution is resuspended in a basic inorganic salt maintenance solution to prepare a cell suspension. Here, the yeast malt medium includes 3 g / L yeast extract, 3 g / L malt extract, 10 g / L glucose, and 5 g / L tryptophan, and the basic inorganic salt maintenance solution includes 2.44 g / L potassium dihydrogen phosphate, 14.04 g / L disodium hydrogen phosphate dodecahydrate, 2 g / L ammonium chloride, 0.2 g / L magnesium chloride hexahydrate, 1 mg / L calcium chloride dihydrate, 0.01 g / L yeast extract, 5 moL / L metal ion mixture, and 0.2 mL / L vitamin mixture.

[0077] In this embodiment, by adding a sclareol with a preset concentration as an inducing substrate in the cultivation stage of fungus ATCC 20624 in advance, it can not only effectively induce the expression of catalytic-related enzyme systems, but also enhance the tolerance and transformation ability of cells to substrates. At the same time, combined with subsequent centrifugation and washing to remove impurities, and resuspension in a basic inorganic salt maintenance solution to form a cell suspension, it is beneficial to obtain a cell catalytic system with stable enzyme activity, high catalytic efficiency, and low system background interference, thereby improving the conversion efficiency and specificity of catalytic reactions.

[0078] In a further embodiment, the second preset temperature is any value in the range of 20°C - 28°C, and the second preset time is any value in the range of 36h - 60h. That is, the reaction temperature for inducing the expression of the catalytic-related enzyme system is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C or 28°C, or any value in the range of 20°C - 28°C. The reaction time can be 36h, 40h, 44h, 48h, 52h, 56h or 60h, or any value in the range of 36h - 60h. By setting the second preset temperature and the second preset time within the above ranges, the effective induction expression of the catalytic-related enzyme system in the fungus ATCC 20624 can be achieved. The formed cell state is more suitable for the subsequent biotransformation reaction of sclareol, ensuring sufficient enzyme expression, high cell activity and low reaction background interference, thereby improving the conversion efficiency, specificity and repeatability of the catalytic reaction.

[0079] The present application will be further described in detail below with reference to specific embodiments.

[0080] Example 1

[0081] First, weigh 30 mg of the standards of sclareol, 20 mg of sclareodiol and 20 mg of sclareolide respectively, and dissolve them in 10 mL of chromatographic grade methanol solution to obtain a 3 g / L sclareol standard solution, a 2 g / L sclareodiol standard solution and a 2 g / L sclareolide standard solution respectively. Qualitative analysis of the sclareol standard solution, sclareodiol standard solution and sclareolide standard solution is carried out by high performance liquid chromatography respectively.

[0082] Weigh 125 mg of sclareol, 50 mg of sclareodiol and 50 mg of sclareolide, dissolve them in 5 mL of chromatographic grade methanol solution respectively, and prepare mixed standard solutions with different concentrations in Table 2 by adding the corresponding component volumes according to Table 1. Chromatographic analysis of the mixed standard solutions is carried out by high performance liquid chromatography to obtain the standard curves and linear regression equations corresponding to sclareol, sclareodiol and sclareolide.

[0083] Add the cell suspension of the fungus ATCC 20624 to the reaction mixture solution containing 4 g / L of sclareol raw material to prepare a reaction solution with an OD 600 of 30. React the reaction solution on a shaker at a speed of 180 rpm / min at 24°C for 72 h to prepare a catalytic solution to be measured. Chromatographic analysis of the catalytic solution to be measured is carried out by high performance liquid chromatography to obtain the high performance liquid chromatogram of the catalytic solution to be measured.

[0084] Chromatographic conditions for high performance liquid chromatography: The parameters of the chromatographic column are C18, 4.6 mm × 250 mm, 5 μm, and the model of the chromatographic column is SunFire. Mobile phase A is acetonitrile, mobile phase B is ultrapure water. The volume flow rate of gradient elution is 0.4 mL / min, the column temperature is 30 °C, the analysis time is 45 min, the detection wavelength is 195 nm, and the injection volume is 10 μL. Among them, the gradient elution program is shown in Table 3.

[0085] Comparative Example 1

[0086] The difference between Comparative Example 1 and Example 1 is only that the change rate of mobile phase A and mobile phase B in gradient elution is different. That is, during the elution process from 5 min to 10 min, the proportion of acetonitrile is increased to 90% at a rate of 6% per minute, and during 20 min - 30 min, the proportion of acetonitrile is decreased to 80% at a rate of 2% per minute.

[0087] Comparative Example 2

[0088] The difference between Comparative Example 1 and Example 1 is only that the volume fractions of mobile phase A and mobile phase B in gradient elution are different. Among them, the gradient elution program of Comparative Example 2 is shown in Table 4.

[0089] Comparative Example 3

[0090] The difference between Comparative Example 3 and Example 1 is only that in Comparative Example 3, gas chromatography is used for quantitative testing of sclareol, sclareodiol and sclareolide in the catalytic solution to be tested.

[0091]

[0092]

[0093]

[0094]

[0095] As Figures 9 to 11 shown, the linear regression equation of sclareodiol is: f(x) = 742423 × x + 71662.8, the linear regression equation of sclareolide is: f(x) = 984438 × x + 32782.6, and the linear regression equation of sclareol is: f(x) = 6166940 × x + 3454480. Figure 12 The peak areas of sclareodiol, sclareolide and sclareol in are 814086, 581974 and 16651732 respectively. According to the peak areas of sclareol, sclareodiol and sclareolide in the high performance liquid chromatogram of the catalytic solution to be tested and the linear regression equations of the corresponding standard curves, the contents of sclareol, sclareodiol and sclareolide in the catalytic solution to be tested are calculated, and the results are shown in Table 5.

[0096]

[0097] As shown in Table 5, the contents of sclareol, sclareodiol, and sclareolide detected in Example 1 and Comparative Example 3 in the catalytic solution to be tested were basically the same, indicating that the quantitative detection results of sclareol, sclareodiol, and sclareolide in the catalytic solution to be tested by using high performance liquid chromatography combined with a gradient elution program and ultraviolet detection means in the present invention are accurate and can be used for separating and detecting sclareol, sclareodiol, and sclareolide in the catalytic solution to be tested.

[0098] such as Figures 7 to 8 and Figure 12 shown, the retention times of sclareol, sclareodiol, and sclareolide in the liquid chromatograms of the single-concentration mixed standard solution, the gradient-concentration mixed standard solution, and the catalytic solution to be tested are consistent, and the peak resolution of each component is good, and sclareol, sclareodiol, and sclareolide can be clearly distinguished.

[0099] In Comparative Example 1, due to the too fast gradient change rate, the peak resolution of sclareol, sclareodiol, and sclareolide decreased significantly, and some peaks overlapped, resulting in the peak shape becoming less sharp and symmetric. However, in Example 1, the peak resolution of the chromatogram is good, the three substances can be clearly distinguished, the peak shape is sharp and symmetric, and the analysis time is also maintained at 45 min, indicating that the gradient elution rate in Example 1 is superior in terms of ensuring peak resolution and peak shape.

[0100] In the gradient elution program of Comparative Example 2, after changing the volume fractions of mobile phase A and mobile phase B, although the peak resolution of sclareol and sclareodiol changed, the peak of sclareolide showed tailing, and the overall analysis time was extended to 50 min. However, in Example 1, the peak resolution of sclareol, sclareodiol, and sclareolide is moderate, the peak shape is normal, and the analysis time is stable at 45 min, indicating that the volume fractions of mobile phase A and mobile phase B in each elution stage in the gradient elution program in Example 1 are more advantageous in terms of separation effect and analysis efficiency.

[0101] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0102] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A method for the separation and detection of sclareol, sclareolide and sclareodiol, characterized in that, Comprising: Preparing a mixed standard solution configured with sclareol, sclareolide, and sclareodiol; Detecting the mixed standard solution by high performance liquid chromatography (HPLC) to obtain standard curves and linear regression equations corresponding to sclareol, sclareolide, and sclareodiol; Separating and detecting sclareol, sclareolide, and sclareodiol in the catalytic solution to be tested by using the high performance liquid chromatography method; Wherein, the catalytic solution to be tested is obtained by using a fungus with the preservation number ATCC 20624 to catalyze the sclareol to generate the sclareolide and the sclareodiol. The chromatographic column of the high performance liquid chromatography method is a C18 column, and the detection wavelength of the ultraviolet detector is any value in the range of 190 nm - 200 nm. Acetonitrile is used as mobile phase A and ultrapure water is used as mobile phase B for gradient elution. The gradient elution program is as follows: at 0 - 5 min, the volume fraction of mobile phase A is 60% and the volume fraction of mobile phase B is 40%; at 5 min - 10 min, the volume fraction of mobile phase A increases from 60% to 90% and the volume fraction of mobile phase B decreases from 40% to 10%; at 10 min - 20 min, the volume fraction of mobile phase A is 90% and the volume fraction of mobile phase B is 10%; at 20 min - 30 min, the volume fraction of mobile phase A decreases from 90% to 80% and the volume fraction of mobile phase B increases from 10% to 20%; at 30 min - 45 min, the volume fraction of mobile phase A decreases from 80% to 60% and the volume fraction of mobile phase B increases from 20% to 40%.

2. The separation and detection method according to claim 1, wherein: The specification of the chromatographic column is 4.6 mm × 250 mm, 5 μm.

3. The separation and detection method according to claim 2, wherein: The column temperature of the chromatographic column is any value in the range of 25°C - 35°C.

4. The separation and detection method according to any one of claims 1 - 3, wherein: The volume flow rate of the gradient elution is any value in the range of 0.3 mL / min - 0.6 mL / min.

5. The separation and detection method according to claim 4, wherein: The step of using a fungus with the preservation number ATCC 20624 to catalyze the sclareol to generate the sclareolide and the sclareodiol in the catalytic solution to be tested further includes: Preparing a cell suspension of fungus ATCC 20624; Adding the sclareol solution to the basic inorganic salt maintenance solution to prepare a mixed solution; Add the cell suspension to the mixed solution to prepare a reaction solution, and the cell suspension is set to have an OD inoculated into the reaction solution 600 of any value from 28 to 32; Controlling the reaction solution to react at a first preset temperature for a first preset time at a preset rotation speed; Subjecting the reaction solution to extraction, centrifugation, and filtration in sequence to prepare the catalytic solution to be tested.

6. The separation and detection method according to claim 5, wherein: The first preset temperature is any value in the range of 20°C - 28°C, and the first preset time is any value in the range of 60 h - 80 h.

7. The separation detection method according to claim 6, wherein The step of preparing the cell suspension of fungus ATCC 20624 further includes: Add a solution of sclareol at a preset concentration to a yeast malt medium containing the fungus ATCC 20624 to prepare a culture solution, and culture it for a second preset time under a second preset temperature condition; Centrifuge and wash the culture solution in sequence; Resuspend the culture solution in the basic inorganic salt maintenance solution to prepare the cell suspension.

8. The separation and detection method according to claim 7, wherein The second preset temperature is any value in the range of 20°C - 28°C, and the second preset time is any value in the range of 36h - 60h.

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