CN115128020B - C in fermentation liquor21System for measuring steroid content, construction method and application thereof - Google Patents
C in fermentation liquor21System for measuring steroid content, construction method and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/3103—Atomic absorption analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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Abstract
The invention discloses a system for measuring content of C 21 steroid in fermentation broth, a construction method and application thereof. The invention takes pregnenolone as a standard substance to prepare a gradient standard solution, and determines the optimal determination wavelength through full-band scanning; and determining the optimal determination condition of the content determination of the C 21 steroid substance by adopting an orthogonal experimental design, and establishing a standard curve under the optimal determination condition. The optimal measurement wavelength, the optimal measurement conditions and the standard curve obtained in the construction process form the measurement system, and can be applied to the measurement of the content of the C 21 steroid substances in the endophyte fermentation liquor of plants (radix cynanchi bungei), the sample is prepared by the endophyte fermentation liquor to be measured, the absorbance of the sample is measured, and the content of the C 21 steroid substances in the sample can be obtained by analysis according to the established standard curve. The method for measuring the content of the C 21 steroid substances is simple to operate, high in detection speed, and good in precision, reproducibility, accuracy and stability.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine component analysis, and particularly relates to a system for measuring the content of C 21 steroid substances in fermentation broth, a construction method thereof and application of the measurement system in measuring the content of C 21 steroid substances in plant (bunge auriculate root) endophyte fermentation broth.
Background
The C 21 steroid substance has remarkable biological activity in the aspects of resisting tumor, regulating immunity, resisting oxidation, protecting liver, resisting depression and the like, and mainly exists in the plants such as the Mi, the Marsdenia tenacissima, the radix cynomorium, the Cynanchum otophyllum and the black vine. Radix Cynanchi auriculati is one of the plants of Asclepiadaceae, and the most active ingredient in its root tuber is C 21 steroid. Research shows that the plant endophyte can produce metabolic products identical or similar to that of a host, particularly medicinal plants can search and screen substances with medicinal activity from the endophyte metabolic products to replace rare and precious host plants with slow growth cycle, and the plant endophyte can be used as a novel medicine source to solve the problems of slow growth, resource shortage and the like of medicines to a certain extent. The microbial fermentation method for preparing the product has wide application prospect in agriculture, medicine and other industries.
The method for measuring the content of the C 21 steroid compound is more, and mainly comprises two large reaction systems of a vanillin colorimetric method and a mercuric sulfoacetate colorimetric method. Common colorimetric systems for vanillin colorimetry include vanillin-perchloric acid, vanillin-hydrochloric acid, and the like. Deng Zhiyong and the like, the vanillin-perchloric acid colorimetry is used for measuring the contents of total saponins and total triterpenic acid, but the vanillin-perchloric acid method has poor batch-to-batch precision, unstable perchloric acid property and easy decomposition in the experimental process; tian Jianhua established that the hydrochloric acid-vanillin colorimetric method is suitable for measuring the content of procyanidins, and hydrochloric acid is used as an oxidant of a reaction system, and has low sensitivity because of the chromogenic reaction with vanillin. The invention explores the determination of C 21 steroid compounds in fermentation liquor by a mercuric sulfoacetate colorimetric method in the early stage, and discovers that the problems of unstable color development, interference of basic culture medium components on sample detection and the like exist in fermentation liquor sample determination.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary purpose of the invention is to provide a system for measuring the content of C 21 steroid substances.
It is still another object of the present invention to provide a method for constructing the above-mentioned measurement system.
To overcome the disadvantages and shortcomings of the prior art, another object of the present invention is to provide the use of the above assay system for determining the content of C 21 steroid in a fermentation broth of an endophyte of a plant (cynanchum bungei).
The invention is realized in such a way that a method for constructing a system for measuring the content of C 21 steroid substances in fermentation broth comprises the following steps:
(1) Preparation of standard solutions
Dissolving a pregnenolone standard substance into ethanol to obtain mother liquor, diluting the mother liquor with ethanol to obtain standard substance mother liquor with set concentration, and taking standard substance mother liquor with different volumes in a test tube within the range of 0.2-4.0 mL to obtain standard solutions with different pregnenolone contents;
(2) Determination of the optimal measurement wavelength
Volatilizing the standard solution in the step (1) through water bath heating, adding 0.4mL of 5%wt vanillin-glacial acetic acid and 0.6mL of concentrated sulfuric acid into a volatilized test tube, uniformly mixing, adding a plug, heating and reacting for 30min through a water bath at 60 ℃, stopping the reaction through the ice water bath, adding 10mL of glacial acetic acid, uniformly mixing, adopting full-band scanning, and determining the optimal determination wavelength of each standard solution according to absorption peaks under different wavelengths;
(3) Determination of optimal measurement conditions
Detecting the influence of different water bath temperatures, reaction time and vanillin-glacial acetic acid concentration on the content measurement of the C 21 steroid substances in the step (2) through orthogonal test design, measuring the absorbance of the standard solution under the condition of the optimal measurement wavelength determined in the step (2), and determining the optimal measurement condition of the C 21 steroid substances according to the correlation between the concentration of the standard solution and the absorbance value under different reaction conditions;
(4) Preparation of a Standard Curve
And (3) volatilizing the standard solution in the step (1) through water bath heating, reacting to the end under the optimal measurement condition determined in the step (3), stopping the reaction in an ice water bath, adding 10mL of glacial acetic acid, uniformly mixing, taking the ethanol solution without pregnenolone as a blank control, measuring the absorbance under the optimal measurement wavelength condition in the step (2), taking the absorbance as an ordinate, taking the content of C 21 steroid as an abscissa, and drawing a standard curve.
Preferably, in step (1), the concentration of the standard stock solution is 0.15mg/mL; the mass volume ratio of pregnenolone to ethanol in the mother liquor is 0.75g:5mL.
Preferably, in step (1), the volume of each standard stock solution is 0.2mL, 0.5mL, 1mL, 1.5mL, 2.0mL, 4.0mL.
Preferably, in the step (2) and the step (4), the water bath temperature for volatilizing the standard solution is 70 ℃.
Preferably, in step (2), the optimal measurement wavelength is 450nm.
Preferably, in step (3), a one-factor and orthogonal experiment is performed to optimize the color development temperature, the color development time and the concentration of the color developer, and the optimal measurement conditions are: to the tube after the volatilization, 0.4mL 7% by weight vanillin-glacial acetic acid and 0.6mL concentrated sulfuric acid were added, and the mixture was stirred and stoppered, and the mixture was heated in a water bath at 75℃for 24 minutes.
The invention further discloses a measurement system obtained by the construction method, wherein the measurement system comprises an optimal measurement wavelength, optimal measurement conditions and a standard curve.
Preferably, the optimal measurement wavelength is 450nm, and the optimal measurement conditions are: to the tube after the volatilization, 0.4mL 7% by weight vanillin-glacial acetic acid and 0.6mL concentrated sulfuric acid were added, and the mixture was stirred and stoppered, and the mixture was heated in a water bath at 75℃for 24 minutes.
The invention further discloses an application of the measuring system in measuring the content of C 21 steroid substances in endophyte fermentation broth, which comprises the following steps:
(1) Preparation of sample solutions for sample preparation
Culturing endophyte with C 21 steroid substance synthesizing capacity to a secondary metabolism stage, collecting fermentation liquor, centrifuging, taking 1mL supernatant to react under the optimal determination condition until the reaction is finished, stopping the reaction by ice water bath, adding 10mL glacial acetic acid, and uniformly mixing to obtain a sample solution;
(2) Determination of content of steroid substances in auriculate root endophyte fermentation liquor C 21
And measuring absorbance of the sample solution at the optimal measurement wavelength, and analyzing the content of C 21 steroid substances in the auriculate root endophyte fermentation liquor by referring to the standard curve.
Preferably, the plant endophyte is a bunge auriculate root endophyte.
The invention overcomes the defects of the prior art and provides a system for measuring the content of C 21 steroid substances in fermentation broth, and a construction method and application thereof. The construction process of the measuring system comprises the following steps: preparing a gradient standard solution by taking pregnenolone as a standard substance, and determining the optimal determination wavelength through full-band scanning; determining the optimal determination conditions (including optimal temperature, optimal reaction time and optimal vanillin-glacial acetic acid concentration) of the content determination of the C 21 steroid substances by adopting an orthogonal experimental design; under the optimal measurement conditions, a standard curve is established. The optimal measurement wavelength, the optimal measurement conditions and the standard curve obtained in the construction process form the measurement system, and can be applied to the measurement of the content of the C 21 steroid substances in the endophyte fermentation liquor of plants (radix cynanchi bungei), the sample is prepared by the endophyte fermentation liquor to be measured, the absorbance of the sample is measured, and the content of the C 21 steroid substances in the sample can be obtained by analysis according to the established standard curve.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects: compared with the prior art, in the construction process of the determination system, the reaction system is vanillin-concentrated sulfuric acid according to the characteristics of the components of the fermentation broth, and the reaction conditions are further optimized, and the determination method can be suitable for determining the content of C 21 steroid substances in various types of microorganism fermentation broth based on LB, potato dextrose water and Gao's first-order culture medium, and has the advantages of simple operation, high detection speed, and good precision, reproducibility, accuracy and stability.
Drawings
FIG. 1 is a graph showing the result of optimizing the optimum wavelength under the measurement conditions of the present invention;
FIG. 2 is an optimized result of the optimum developing temperature in the measurement conditions of the present invention;
FIG. 3 is an optimization result of the optimal color development time in the measurement conditions of the present invention;
FIG. 4 is a standard curve of C 21 steroid concentration in the assay of the invention;
FIG. 5 is a graph showing the measurement of the content of C 21 steroid in fermentation broth of endophyte in the example of the present invention;
FIG. 6 is a graph showing the results of determining the content of C 21 steroid in a fermentation broth of endophytic fungi in an embodiment of the present invention;
FIG. 7 shows the results of determining the content of C 21 steroid in fermentation broth of actinomycetes endophyte in the examples of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The determination of the optimal wavelength comprises the following specific operation steps:
(1) Preparation of standard solutions
0.75G of pregnenolone standard substance is weighed and dissolved in 5mL of ethanol to prepare mother liquor, 100 mu L of mother liquor is taken and added into 100mL of ethanol to prepare 0.15mg/mL of standard substance mother liquor; and respectively taking 0.2mL, 0.5 mL mL, 1mL, 1.5mL, 2.0mL and 4.0mL of standard substance mother liquor, and shaking uniformly to obtain standard solutions with different pregnenolone contents.
(2) Determination of optimal wavelength
Taking standard solutions with different concentrations in the step (1), volatilizing the standard solutions in a test tube in a water bath at 70 ℃, sequentially adding 0.4 mL of 5% vanillin-glacial acetic acid and 0.6mL of concentrated sulfuric acid, uniformly mixing, adding a plug, and carrying out water bath at 60 ℃ for 30min. At the end of the water bath, the reaction was immediately transferred to an ice-water bath, quenched and 10mL of glacial acetic acid was added and mixed well. And adopting full-band scanning, and determining the optimal measurement wavelength according to absorption peaks at different wavelengths.
As a result, see FIG. 1, the optimum wavelength was determined to be 450nm.
Example 2
Orthogonal experiments were performed based on the results of example 1 to determine the optimal assay conditions, with the following steps:
Orthogonal test designs were used (see table 1). The effect of different water bath temperatures, times, vanillin-glacial acetic acid concentrations on the determination of the C 21 steroid was studied, the absorbance was measured at 450nm, and the optimal determination conditions of the C 21 steroid were determined.
TABLE 1 orthogonal experimental design for optimal measurement conditions
The optimal measurement conditions for obtaining the C 21 steroid substance according to the orthogonal experimental result are as follows: the reaction temperature is 80 ℃, the reaction time is 30min, and the concentration of vanillin-glacial acetic acid is 7% wt, so that the measurement effect is relatively good.
TABLE 2 results of orthogonal experiments for optimal measurement conditions
As can be seen from the best orthogonal experimental results (see Table 2), the color reaction parameters are: the experimental determination was relatively good at a vanillin-glacial acetic acid concentration of 7% wt in a water bath at 80℃for 30min, but the R 2 value did not reach 0.99 when a standard curve was established.
Example 3
On the basis of the orthogonal experiment, a single factor experiment is further set, and reaction parameters are optimized: color development temperature and color development time. The experiment design shows that the color development temperature is 65 ℃,70 ℃,75 ℃ and other 3 gradients, and the color development time is 25min,30min and 35min. The experiment shows that when the color development temperature is 75 ℃ and the color development time is 25min, the R 2 value is closest to 0.99 when the standard curve is established. On the basis, the color development time is optimized, and 3 gradients of 21min,24min,27min and the like are designed. As shown in FIGS. 2 and 3, the color reaction parameters after further optimization are water bath at 75 ℃ for 24min, and the established standard curve R 2 reaches 0.9958.
Finally, the reaction system of the vanillin colorimetric method is determined as follows: the reaction temperature was 75℃and the reaction time was 24 min% by weight, the vanillin-glacial acetic acid concentration being 7% by weight.
The optimal color reaction conditions after optimization are as follows: standard solutions with different pregnenolone content gradients are selected, volatilized in a water bath at 70 ℃ in a test tube, reacted under the optimized optimal measurement conditions, and added with 0.4mL 7% wt vanillin-glacial acetic acid and 0.6mL sulfuric acid which are prepared at present, shaken uniformly, plugged and water-bath at 75 ℃ for 24 min. Immediately after the reaction, the mixture was transferred into an ice-water bath for 10min, taken out, 10mL of glacial acetic acid was added, the mixture was shaken well, and the mixture was allowed to stand for 20min, and the OD value was measured at 450nm (see FIG. 4).
Effect examples
1. Linear relationship investigation
The absorbance values were determined at a wavelength of 450nm for the pregnenolone standard solutions of different concentrations obtained under the "preparation of standard solution". Linear regression is performed by taking the pregnenolone concentration (X) as an abscissa and the absorbance (Y) as an ordinate to obtain a regression equation Y=0.0604X-0.0015 (R 2 = 0.9958), and the result shows that the linear relationship of pregnenolone is good in the range of 0 mg/mL-1.2 mg/mL.
2. Precision experiments
The pregnenolone standard solution with the concentration of 0.03mg/mL is taken, the measurement is continuously carried out for 3 times, the absorbance is recorded, the RSD value is calculated to be 3.208%, the experimental precision is good, and the result is shown in Table 3.
Table 3 precision experimental determination
3. Stability test
Three parallel groups were set up to obtain pregnenolone standard solution with concentration of 0.15mg/mL, different time gradients were set up, and absorbance (A value) was measured and recorded at regular time. The results are shown in Table 4. The table shows that the light absorption value of the standard solution is stable within 96 hours, and the method is proved to have good stability within 96 hours.
Table 4 stability test assay
4. Sample recovery rate experiment
And (3) measuring the absorbance value according to the optimized optimal color development condition, selecting standard substances with different concentrations, adding 0.6mL of equal amount of fermentation liquor, measuring the corresponding absorbance value, and calculating the sample adding recovery rate. As is clear from Table 5, the average value of the recovery rate of the sample was 74.01%, which proves that the sample recovery rate of the method was good.
TABLE 5 sample recovery test results
Application examples
The method for measuring the content of C 21 steroid in the fermentation liquid of the auriculate root endophyte comprises the following specific operation steps:
(1) Preparation of sample solutions for sample preparation
3 Types of endophytes (bacteria, fungi and actinomycetes) screened from different tissue parts of the coastal bunge auriculate root in a laboratory are taken as materials, the endophytes are respectively cultured on 3 different types of basic culture media such as LB, potato dextrose water, gaoshan No. one and the like, fermentation liquor is periodically collected, the fermentation liquor is centrifuged, 1mL of supernatant is taken, 0.4mL of 7%wt vanillin-glacial acetic acid and 0.6mL of concentrated sulfuric acid are sequentially added, the mixture is uniformly mixed and plugged, the water bath at 75 ℃ is carried out for 24min, the reaction is immediately transferred to an ice water bath, the reaction is terminated, 10mL of glacial acetic acid is added, and the mixture is uniformly mixed, thus obtaining the sample solution.
(2) Determination of content of steroid substances in auriculate root endophyte fermentation liquor C 21
The absorbance of the pretreated sample is measured at 450nm, the content of C 21 steroid substances in the fermentation liquor of the auriculate root endophyte is analyzed by comparing with the established standard curve, the results are shown in figures 5-7, and 3 strains with high-efficiency synthesis capacity of C 21 steroid substances are finally screened out: the endophytic bacteria X-32 (bacillus cereus X-32) (with a collection number of CCTCCNO: M20211165, a collection address of the China center for type culture Collection of Wuhan, jiu, wuhan, gmbH, with a collection number of 430072), endophytic fungi Z-44 (colletotrichum glomerum Z-44, colletotrichum gloeosporioides Z-44) (with a collection number of CCTCCNO: M20211164, a collection address of the China center for type culture collection of 2021, wuhan, wuchan, GY, with a collection address of 430072, a collection address of the Wuhan, GY, with a collection address of 430072), endophytic actinomyces F-45 (Rhizobium, gmbH, with a collection address of 430072, a collection address of the China center for type culture collection of Wuhan, gmbH, with a collection number of 2021, and Wuhan, with a collection address of 430072). The application of the invention provides support for the development and application of the bunge auriculate root endophyte source C 21 steroid substances to a certain extent.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (9)
1. The construction method of the system for measuring the content of C 21 steroid in fermentation broth is characterized by comprising the following steps:
(1) Preparation of standard solutions
Dissolving a pregnenolone standard substance into ethanol to obtain mother liquor, diluting the mother liquor with ethanol to obtain standard substance mother liquor with set concentration, and taking standard substance mother liquor with different volumes in a test tube within the range of 0.2-4.0 mL to obtain standard solutions with different pregnenolone contents;
(2) Determination of the optimal measurement wavelength
Volatilizing the standard solution in the step (1) through water bath heating, adding 0.4mL of 5%wt vanillin-glacial acetic acid and 0.6mL of concentrated sulfuric acid into a volatilized test tube, uniformly mixing, adding a plug, heating and reacting for 30min through a water bath at 60 ℃, stopping the reaction through the ice water bath, adding 10mL of glacial acetic acid, uniformly mixing, adopting full-band scanning, and determining the optimal determination wavelength of each standard solution according to absorption peaks under different wavelengths;
(3) Determination of optimal measurement conditions
Detecting the influence of different water bath temperatures, reaction time and vanillin-glacial acetic acid concentration on the content measurement of the C 21 steroid substances in the step (2) through orthogonal test design, measuring the absorbance of the standard solution under the condition of the optimal measurement wavelength determined in the step (2), and determining the optimal measurement condition of the C 21 steroid substances according to the correlation between the concentration of the standard solution and the absorbance value under different reaction conditions;
(4) Preparation of a Standard Curve
And (3) volatilizing the standard solution in the step (1) through water bath heating, reacting to the end under the optimal measurement condition determined in the step (3), stopping the reaction in an ice water bath, adding 10mL of glacial acetic acid, uniformly mixing, taking the ethanol solution without pregnenolone as a blank control, measuring the absorbance under the optimal measurement wavelength condition in the step (2), taking the absorbance as an ordinate, taking the content of C 21 steroid as an abscissa, and drawing a standard curve.
2. The method for constructing an assay system according to claim 1, wherein in the step (1), the concentration of the standard mother liquor is 0.15mg/mL; the mass volume ratio of pregnenolone to ethanol in the mother liquor is 0.75g:5mL.
3. The method of constructing an assay system according to claim 1, wherein in the step (1), the volume of each standard mother solution is 0.2mL, 0.5mL, 1mL, 1.5mL, 2.0mL, or 4.0mL.
4. The method of constructing a measurement system according to claim 1, wherein in the step (2) and the step (4), the water bath temperature for volatilizing the standard solution is 70 ℃.
5. The method of constructing an assay system according to claim 1, wherein in the step (2), the optimal measurement wavelength is 450nm.
6. The method of constructing an assay system according to claim 1, wherein in the step (3), a single factor and orthogonal experiment is performed to optimize the color development temperature, the color development time and the concentration of the color developing agent, and the optimum measurement conditions are: to the tube after the volatilization, 0.4mL 7% by weight vanillin-glacial acetic acid and 0.6mL concentrated sulfuric acid were added, and the mixture was stirred and stoppered, and the mixture was heated in a water bath at 75℃for 24 minutes.
7. The measurement system according to any one of claims 1 to 6, wherein the measurement system comprises the optimal measurement wavelength, the optimal measurement condition, and a standard curve.
8. Use of the assay system according to claim 7 for determining the content of C 21 steroid in a fermentation broth of endophytes, characterized in that the use comprises the steps of:
(1) Preparation of sample solutions for sample preparation
Culturing endophyte with C 21 steroid substance synthesizing capacity to a secondary metabolism stage, collecting fermentation liquor, centrifuging, taking 1mL supernatant to react under the optimal determination condition until the reaction is finished, stopping the reaction by ice water bath, adding 10mL glacial acetic acid, and uniformly mixing to obtain a sample solution;
(2) Determination of content of steroid substances in auriculate root endophyte fermentation liquor C 21
And measuring absorbance of the sample solution at the optimal measurement wavelength, and analyzing the content of C 21 steroid substances in the auriculate root endophyte fermentation liquor by referring to the standard curve.
9. The use according to claim 8, wherein the plant endophyte is a bunge auriculate root endophyte.
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