CN114047155A - Method for rapidly detecting carotenoid - Google Patents

Method for rapidly detecting carotenoid Download PDF

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CN114047155A
CN114047155A CN202111131613.5A CN202111131613A CN114047155A CN 114047155 A CN114047155 A CN 114047155A CN 202111131613 A CN202111131613 A CN 202111131613A CN 114047155 A CN114047155 A CN 114047155A
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carotenoid
absorbance value
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lycopene
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汪志明
李翔宇
杨艳红
陆姝欢
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Cabio Biotech Wuhan Co Ltd
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Abstract

The invention provides a method for rapidly detecting carotenoid, which comprises the following steps: A. diluting the sample solution to an absorbance value of between 0.2 and 0.8 at the wavelength of 600nm and 472 nm; B. detecting absorbance values A600 and A472 of the diluted sample solution at the wavelengths of 600nm and 472nm, determining the carotenoid content in the diluted sample solution by using ultraviolet or high performance liquid chromatography, and establishing a standard curve among the absorbance values A600 and A472, the dilution times and the carotenoid content; C. diluting the sample solution to be detected by n ' times with water, and detecting absorbance values A600 ' and A472 ' of the diluted sample solution to be detected under the wavelengths of 600nm and 472 nm; substituting the A600 ', the A472 ' and the n ' into the standard curve to calculate the carotenoid content in the sample solution to be detected.

Description

Method for rapidly detecting carotenoid
Technical Field
The invention relates to the technical field of biology, in particular to a method for quickly detecting carotenoid.
Background
In the process of screening carotenoid high-flux strains and optimizing the process, a large number of samples need to be subjected to result detection. The existing carotenoid detection methods mostly adopt High Performance Liquid Chromatography (HPLC) and conventional ultraviolet-visible spectrophotometry (UV-VIS). The content of the carotenoid is detected by HPLC and UV-VIS methods through extracting the carotenoid in a sample and then detecting the carotenoid content in the sample. And the carotenoid extraction process in the sample is complicated, time-consuming and labor-consuming, so that the detection efficiency is low. Therefore, an efficient high-throughput detection method is needed to improve the detection efficiency.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting carotenoid, in particular to a method for rapidly determining the yield of the carotenoid in fermentation liquor.
In order to achieve the object, in a first aspect, the invention provides a method for rapidly detecting carotenoid, comprising the following steps:
A. diluting the sample solution with water until the absorbance values under the wavelength conditions of 600nm +/-2 nm and 472nm +/-2 nm are both between 0.2 and 0.8, and the dilution multiple is n;
B. detecting an absorbance value A600 of the diluted sample solution under the wavelength condition of 600nm +/-2 nm and an absorbance value A472 of the diluted sample solution under the wavelength condition of 472nm +/-2 nm by using an enzyme-labeling instrument, then detecting the carotenoid content in the diluted sample solution by using an ultraviolet or high performance liquid chromatography, and establishing a standard curve among the absorbance value A600, the absorbance value A472, the dilution times and the carotenoid content;
C. diluting the sample solution to be detected by n ' times with water, and detecting the absorbance value A600 ' of the diluted sample solution to be detected under the wavelength condition of 600nm +/-2 nm and the absorbance value A472 ' of the diluted sample solution under the wavelength condition of 472nm +/-2 nm by using an enzyme-labeling instrument, wherein the absorbance values of A600 ' and A472 ' are both between 0.2 and 0.8; substituting the A600 ', the A472 ' and the n ' into the standard curve to calculate the carotenoid content in the sample solution to be detected.
In the method, the sample solution is a fermentation solution.
In the method, before detecting the carotenoid content in the diluted sample solution by using ultraviolet or high performance liquid chromatography, the cell wall is broken by adopting a ball milling method.
The wall breaking method is ball milling. The main steps are (1) centrifugally removing fermentation liquor and reserving thalli; (2) mixing an extraction solvent and grinding beads, and grinding for multiple times until the thalli are colorless; (3) transferring the extraction solvent to a brown volumetric flask for dissolving, and detecting on a machine after film passing.
In one embodiment of the present invention, the wall breaking method is:
taking a bacterium solution into a centrifugal tube, centrifuging and then discarding a supernatant; adding tetrahydrofuran and grinding beads, cooling the bacterial liquid, grinding for multiple times at intervals of 3s in a grinder at 70Hz for 90s, and extracting until the thallus is colorless; the solution was transferred to a brown flask (dilution factor determined according to concentration) and the beads and tube walls were washed repeatedly and collected in the same flask to constant volume. The upper organic layer was extracted with a disposable syringe and passed through a membrane (0.2 μm); and (6) performing detection on the machine.
Preferably, the carotenoid is lycopene.
More preferably, the sample solution is a lycopene-producing saccharomyces cerevisiae fermentation broth. The equation corresponding to the standard curve is:
lycopene content of 0.3739 × (A472-A600) × n +0.0015
Wherein the unit of lycopene content is g/L.
The preparation method of the lycopene-producing saccharomyces cerevisiae fermentation liquor comprises the following steps:
(1) strain activation
The strain (recombinant saccharomyces cerevisiae, preservation number CGMCC No.13013) is subjected to plate streaking in a slant culture medium and then is subjected to activation culture at 30 ℃ for 24-36 h.
The slant culture medium is as follows: 20g/L of glucose, 30g/L of yeast extract and 20g/L of agar. Sterilizing at 121 deg.C for 20 min.
(2) Seed culture
The activated and cultured strains are transferred into a seed culture medium and cultured for 16h at 30 ℃ and 250rpm to be used as seed liquid.
The seed culture medium is as follows: 20g/L of glucose and 30g/L of yeast extract. Sterilizing at 121 deg.C for 20 min.
(3) Flask-shaking enlarged fermentation
Inoculating the seed liquid obtained in the step (2) into a shake flask fermentation culture medium, wherein the inoculation amount is 5% v/v, and culturing at 30 ℃ and 250rpm for 120 h.
The shake flask fermentation medium is as follows: 40g/L of glucose, 30g/L of yeast extract and 10g/L of D-galactose. Sterilizing at 121 deg.C for 20 min.
(4) Fermenting in a fermentation tank
Performing shake flask fermentation on the OD obtained in the step (3)600Inoculating the seed solution of 20 percent into a fermentation medium according to the inoculation amount of 5 percent v/v, wherein the liquid filling amount of the fermentation tank is 4L/7L, the components of the fermentation medium are 40g/L glucose and 80g/L yeast extract, the culture temperature is 30 ℃, the rotation speed is 250rpm, and the fermentation is carried out for 120 h; and D-galactose is added into the mixture after fermentation for 48 hours to ensure that the final concentration is 10 g/L. And anhydrous ethanol is fed-batch at 52h of fermentation, and the concentration of the ethanol in the fermentation liquor is kept below 10 g/L.
In the invention, the product of the saccharomyces cerevisiae only contains lycopene and has no interference of other carotenoids, so the ultraviolet spectrophotometry and the high performance liquid chromatography can be suitable. The determination conditions can be determined by referring to detection conditions in various national standards and group standards, such as ultraviolet spectrophotometer method and high performance liquid chromatography in GB 1886.78-2016; for example, the reference conditions of liquid chromatography in GB/T22249-.
In one embodiment of the present invention, the lycopene content is determined by uv-spectrophotometry, wherein the diluent solvent is tetrahydrofuran and the absorption wavelength is 472 nm.
In another embodiment of the invention, the determination is performed by using a high performance liquid chromatography method, wherein the high performance liquid chromatography detection method specifically comprises the following steps:
(1) chromatographic conditions
A chromatographic column: suplex PKB-100 (Supelco); 250X 4.6mm, 5 μm;
wavelength: 472 nm;
flow rate: 0.5 ml/min;
sample introduction volume: 10 mu L of the solution;
column temperature: at 30 ℃.
(2) Mobile phase formulation and conditions
Phase A: methanol.
Phase B: 50mg of BHT (2, 6-di-tert-butyl-p-cresol) was weighed into a 1L volumetric flask and dissolved in 20mL of isopropanol. 0.2mL of N, N-diisopropylethylamine, 25mL of 0.2% ammonium acetate solution, 455mL of acetonitrile, 450mL of methanol was added and the solution was brought to room temperature and diluted to the mark with methanol.
The mobile phase gradient elution procedure was as follows:
Figure BDA0003280733000000031
in a second aspect, the present invention provides the use of said method for screening high carotenoid producing strains.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention improves the detection efficiency of the carotenoid in the fermentation liquor and solves the problem of low efficiency caused by detection limitation in the processes of strain screening and process optimization.
Drawings
FIG. 1 is a standard curve of lycopene production in a preferred embodiment of the invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
In the following examples, the lycopene-producing Saccharomyces cerevisiae is a homologous recombinant yeast strain introduced with crtB, crtI, crtE and crtY genes, and the preservation number of the strain is CGMCC No. 13013. Saccharomyces cerevisiae with the preservation number CGMCC No.13013 is disclosed in CN 106566779B.
Example 1 high throughput screening of high Carotenoid-producing strains
This example provides a method for screening high-yield carotenoid strains in high throughput (i.e., a method for rapidly determining the carotenoid yield in fermentation broth), comprising the following steps:
process for producing lycopene by fermentation of saccharomyces cerevisiae
The fermentation process comprises the following steps:
1. strain activation
The strain (recombinant saccharomyces cerevisiae, preservation number CGMCC No.13013) is subjected to plate streaking in a slant culture medium and then is subjected to activation culture at 30 ℃ for 24-36 h.
The slant culture medium is as follows: 20g/L of glucose, 30g/L of yeast extract and 20g/L of agar. Sterilizing at 121 deg.C for 20 min.
2. Seed culture
The activated and cultured strains are transferred into a seed culture medium and cultured for 16h at 30 ℃ and 250rpm to be used as seed liquid.
The seed culture medium is as follows: 20g/L of glucose and 30g/L of yeast extract. Sterilizing at 121 deg.C for 20 min.
3. Flask-shaking enlarged fermentation
And (3) inoculating the seed solution obtained in the step (2) into a shake flask fermentation medium, wherein the inoculation amount is 5% v/v, and culturing at 30 ℃ and 250rpm for 120 h.
The shake flask fermentation medium is as follows: 40g/L of glucose, 30g/L of yeast extract and 10g/L of D-galactose. Sterilizing at 121 deg.C for 20 min.
4. Fermenting in a fermentation tank
Shaking the flask obtained in step 3 to ferment to obtain OD600Inoculating the seed solution of 20 percent into a fermentation medium according to the inoculation amount of 5 percent v/v, wherein the liquid filling amount of the fermentation tank is 4L/7L, the components of the fermentation medium are 40g/L glucose and 80g/L yeast extract, the culture temperature is 30 ℃, the rotation speed is 250rpm, and the fermentation is carried out for 120 h; and D-galactose is added into the mixture after fermentation for 48 hours to ensure that the final concentration is 10 g/L. And anhydrous ethanol is fed-batch at 52h of fermentation, and the concentration of the ethanol in the fermentation liquor is kept below 10 g/L.
Second, ultraviolet detection method for lycopene
1. 5mL of the fermentation liquid is added into a 10mL centrifuge tube, centrifuged at 13000rpm for 1min, and then the fermentation liquid is discarded.
2. 1mm and 2mm of zirconia grinding beads were added, respectively, in a total volume of about 1ml, followed by 5ml of tetrahydrofuran. Refrigerating in a refrigerator at-20 deg.C for 15min to cool the bacteria liquid.
3. And (3) placing the centrifuge tube into a full-automatic sample rapid grinding instrument for grinding, wherein the grinding parameters are set to 70Hz for 90s, and the suspension is carried out for 3 s.
4. After grinding for 8 times, taking out the centrifuge tube, and then, the extract is clear and transparent, and the thallus is colorless. Collecting extractive solution tetrahydrofuran in 25ml brown volumetric flask, then repeatedly washing the centrifuge tube and grinding bead with tetrahydrofuran until colorless, collecting tetrahydrofuran for washing in volumetric flask, and fixing volume.
5. Diluting with tetrahydrofuran to a certain multiple n0The absorbance x at 472nm was measured so that the absorbance value was between 0.2 and 0.8.
6. The lycopene content y is calculated.
Standard curve: y (mg/L) ═ 2.8575x-0.0546, R2＝1.0000
y(g/L)＝(2.8575x-0.0546)n0×25/5
Establishing a standard curve
The United states Biotek microplate reader, model Synergy LX, was used.
Taking fermentation liquor of the saccharomyces cerevisiae for producing the lycopene as a sample, respectively detecting an absorbance value under the condition of 600nm wavelength and an absorbance value under the condition of 472nm wavelength, diluting until the absorbance value A600 under the condition of 600nm wavelength and the absorbance value A472 under the condition of 472nm wavelength are both between 0.2 and 0.8, recording the absorbance values A600 and A472 and a dilution multiple n, and establishing a standard curve (shown in figure 1) between the absorbance value A600, the absorbance value A472, the dilution multiple n and the carotenoid content by using the lycopene yield obtained in the step two.
The formula for lycopene content is thus obtained:
lycopene content of 0.3739 × (A472-A600) × n +0.0015
Wherein the unit of lycopene content is g/L.
Fourth, sample detection
When high-throughput screening of high-yield lycopene strains or process optimization is carried out, an enzyme-labeling instrument is used for high-throughput detection of absorbance values of samples under the wavelength conditions of 600nm and 472nm, dilution times are recorded, and the absorbance values are substituted into the standard curve, so that the lycopene yield of the fermentation liquid sample can be calculated (table 1).
The method comprises the following specific steps:
1) taking the fermentation liquor of the saccharomyces cerevisiae for producing lycopene, diluting the fermentation liquor with water until the absorbance value under the condition of 600nm wavelength and the absorbance value under the condition of 472nm wavelength are between 0.2 and 0.8, and diluting by a multiple n';
2) detecting an absorbance value A600 'of the diluted sample under the condition of 600nm wavelength and an absorbance value A472' of the diluted sample under the condition of 472nm wavelength by using an enzyme-labeling instrument;
3) substituting the detection result into the standard curve to calculate the lycopene yield of the fermentation liquid sample.
Taking lycopene fermentation broth of different batches to carry out rapid detection simultaneously, and comparing with HPLC measured values, and the results are shown in Table 2.
TABLE 1
Figure BDA0003280733000000051
Figure BDA0003280733000000061
TABLE 2
Figure BDA0003280733000000062
The absolute difference of the two methods is within 10 percent, which shows that the rapid detection method is established effectively. The method is also suitable for the rapid detection of other carotenoids, such as fermentation broth for producing beta-carotene.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (7)

1. The method for rapidly detecting the carotenoid is characterized by comprising the following steps of:
A. diluting the sample solution with water until the absorbance values under the wavelength conditions of 600nm +/-2 nm and 472nm +/-2 nm are both between 0.2 and 0.8, and the dilution multiple is n;
B. detecting an absorbance value A600 of the diluted sample solution under the wavelength condition of 600nm +/-2 nm and an absorbance value A472 of the diluted sample solution under the wavelength condition of 472nm +/-2 nm by using an enzyme-labeling instrument, detecting the carotenoid content in the diluted sample solution by using ultraviolet or high performance liquid, and establishing a standard curve among the absorbance value A600, the absorbance value A472, the dilution times and the carotenoid content;
C. diluting the sample solution to be detected by n ' times with water, and detecting the absorbance value A600 ' of the diluted sample solution to be detected under the wavelength condition of 600nm +/-2 nm and the absorbance value A472 ' of the diluted sample solution under the wavelength condition of 472nm +/-2 nm by using an enzyme-labeling instrument, wherein the absorbance values of A600 ' and A472 ' are both between 0.2 and 0.8; substituting the A600 ', the A472 ' and the n ' into the standard curve to calculate the carotenoid content in the sample solution to be detected.
2. The method of claim 1, wherein the sample solution is a fermentation broth.
3. The method according to claim 1, wherein the cell wall is broken by ball milling before detecting the carotenoid content in the diluted sample solution by ultraviolet or high performance liquid chromatography.
4. The method of claim 3, wherein the carotenoid is lycopene.
5. The method of claim 4, wherein the sample solution is a lycopene-producing Saccharomyces cerevisiae fermentation broth.
6. The method of claim 5, wherein the standard curve corresponds to the equation:
lycopene content of 0.3739 × (A472-A600) × n +0.0015
Wherein the unit of lycopene content is g/L.
7. Use of the method of any one of claims 1 to 6 for screening of a high carotenoid-producing strain.
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