Screening method of astaxanthin high-yield microbial strain

Abstract

The invention discloses a screening method of astaxanthin high-yield microbial strains, which comprises the following steps: step 1, roughly quantifying the astaxanthin yield of a to-be-identified strain by thin layer chromatography to obtain a strain meeting requirements; 2, carrying out enlarged culture on the strain obtained in the step 1, detecting the astaxanthin content by adopting a high performance liquid chromatography to obtain the astaxanthin yield of the strain, and screening the strain meeting the yield requirement; according to the method, the practicability of the thin-layer chromatography technology in trace analysis is expanded, and a new way for efficiently and rapidly screening and optimizing mutant strains is provided for the field of biotechnology.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial screening, and in particular to a method for screening a high-astaxanthin-producing microbial strain. Background Art

[0002] Natural astaxanthin's numerous biological activities, including antioxidant, anti-tumor, cardiovascular disease prevention, and immune-enhancing properties, have been widely recognized. Chemical synthesis, extraction, and microbial fermentation are currently the primary methods for obtaining astaxanthin. Chemical synthesis is complex, produces numerous byproducts, and results in low safety and bioactivity. Extraction methods face challenges such as low production, numerous impurities, ambiguous technical approaches, and high costs, all while remaining unsustainable. Microbial fermentation offers the advantages of natural safety, cost-effectiveness, environmental friendliness, and sustainability. Therefore, the synthesis of astaxanthin based on microbial fermentation is becoming one of the most effective approaches for industrial production. However, the low astaxanthin yields of wild-type microbial strains preclude large-scale industrial production. Mutagenesis methods, such as ARTP-based atmospheric pressure and room temperature plasma mutagenesis, can effectively create mutant strain libraries.

[0003] However, strain screening typically uses traditional liquid chromatography, a method that suffers from drawbacks such as long single-sample testing time (≥30 minutes), strong equipment dependence, and high testing costs, which severely restrict the efficiency of screening high-yield strains. For example, patent application No. 2010101504229 discloses an astaxanthin-producing strain and its screening method and application. Using the mutagenized strain N1806-04 of Phaffia rhodozyma as the starting strain, Phaffia rhodozyma protoplasts were prepared enzymatically, and then astaxanthin-producing strains were selected through methods such as NTG mutagenesis and β-ionone screening. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a method for screening a microbial strain with high astaxanthin production.

[0005] The technical solution adopted in the present invention is:

[0006] A method for screening a high-yielding microbial strain of astaxanthin comprises the following steps:

[0007] Step 1: Use thin layer chromatography to roughly quantify the astaxanthin production of the strain to be identified to obtain a strain that meets the requirements;

[0008] Step 2: Expand and culture the strain obtained in step 1, detect the astaxanthin content by high performance liquid chromatography, obtain the astaxanthin yield of the strain, and screen the strain that meets the yield requirements.

[0009] Furthermore, the microbial strain is Phaffia rhodozyma.

[0010] Furthermore, the rough quantitative method in step 1 is as follows:

[0011] extracting the fermentation liquid of the strain to obtain a crude extract;

[0012] Spot the crude extract on an activated silica gel plate, dry it, place it in a developing tank, and use a developing agent for adsorption to separate the components;

[0013] The astaxanthin content in the fermentation broth was calculated using a standard curve.

[0014] Furthermore, the developing agent is a mixture of n-hexane and acetone in a volume ratio of 7:3.

[0015] Furthermore, the extraction process of the fermentation broth of the strain is as follows:

[0016] The fermentation broth was centrifuged at 4000 rpm for 3 min, and the supernatant was removed;

[0017] The obtained bacteria were washed by centrifugation with ultrapure water and the supernatant was removed by centrifugation;

[0018] Add hydrochloric acid, mix thoroughly, and place in boiling water bath for 3 minutes. After cooling, remove the supernatant.

[0019] Extract and disperse the cells by ultrasonication for 1 to 2 minutes. Centrifuge at 4°C and collect the supernatant to obtain the desired crude extract.

[0020] Furthermore, before the extraction, the pH is adjusted to 7, and then ethyl acetate is used for extraction, and ultrasonic dispersion is performed after concentration.

[0021] Furthermore, the process of detecting astaxanthin content by high performance liquid chromatography in step 2 is as follows:

[0022] The fermentation broth was subjected to cell disruption, concentrated using an extractant, filtered using a 0.22 μm filter membrane, and then the astaxanthin content was detected using high performance liquid chromatography.

[0023] Furthermore, the mobile phase used in the high performance liquid chromatography method is a mixture of acetonitrile and water in a volume ratio of 9:1 in the first pump; and a mixture of methanol and isopropanol in a volume ratio of 6:4 in the second pump.

[0024] The flow rate was 1 mL / min and the detection wavelength was 470 nm.

[0025] Furthermore, the content is detected in combination with the standard curve in step 1 and step 2.

[0026] The beneficial effects of the present invention are:

[0027] The present invention is applicable to a high-throughput detection method for microbial strains and is applicable to the rapid detection of traditional mutagenic microbial mutant strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the separation effect of the primary screening developer in an embodiment of the present invention, A is the result under indoor natural light, and B is the result of the UV analyzer.

[0029] Figure 2 Schematic diagram of the standard curve of astaxanthin content used in the examples of the present invention.

[0030] Figure 3 Schematic diagram of the detection results of repeated screening in an embodiment of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] A method for screening a high-yielding microbial strain of astaxanthin comprises the following steps:

[0033] Step 1: Use thin layer chromatography to roughly quantify the astaxanthin production of the strain to be identified to obtain a strain that meets the requirements;

[0034] The rough quantitative method is as follows:

[0035] extracting the fermentation liquid of the strain to obtain a crude extract;

[0036] Spot the crude extract on an activated silica gel plate, dry it, place it in a developing tank, and use a developing agent for adsorption to separate the components;

[0037] The astaxanthin content in the fermentation broth was calculated using a standard curve.

[0038] The developing solvent was a mixture of n-hexane and acetone in a volume ratio of 7:3.

[0039] The extraction process of the strain fermentation broth is as follows:

[0040] The fermentation broth was centrifuged at 4000 rpm for 3 min, and the supernatant was removed;

[0041] The obtained bacteria were washed by centrifugation with ultrapure water and the supernatant was removed by centrifugation;

[0042] Add hydrochloric acid, mix thoroughly, and place in boiling water bath for 3 minutes. After cooling, remove the supernatant.

[0043] Extract and disperse the cells by ultrasonication for 1 to 2 minutes. Centrifuge at 4°C and collect the supernatant to obtain the desired crude extract.

[0044] Before extraction, the pH was adjusted to 7, and then ethyl acetate was used for extraction, and after concentration, ultrasonic dispersion was performed.

[0045] Step 2: Expand the culture of the strain obtained in step 1, detect the astaxanthin content by high performance liquid chromatography, obtain the astaxanthin yield of the strain, and screen the strain that meets the yield requirements

[0046] The process of detecting astaxanthin content by high performance liquid chromatography is as follows:

[0047] The fermentation broth was subjected to cell disruption, concentrated using an extractant, filtered using a 0.22 μm filter membrane, and then the astaxanthin content was detected using high performance liquid chromatography.

[0048] The mobile phase used in the HPLC method is a mixture of acetonitrile and water in a volume ratio of 9:1 in the first pump; a mixture of methanol and isopropanol in a volume ratio of 6:4 in the second pump;

[0049] The flow rate was 1 mL / min and the detection wavelength was 470 nm.

[0050] Example

[0051] The present method targets the screening of Phaffia rhodozyma. Although P. rhodozyma is a natural substrate for astaxanthin production, wild-type strains have low yields and are prone to degeneration, making large-scale industrial production challenging. The present method generates a series of strains through mutagenesis, which are then rapidly screened using the present method to identify strains with high astaxanthin production.

[0052] A method for screening a high-yielding microbial strain of astaxanthin comprises the following steps:

[0053] Step 1: Use thin layer chromatography to roughly quantify the astaxanthin production of the strain to be identified to obtain a strain that meets the requirements;

[0054] Transfer a loopful of the strain to be screened into a 15mL shake tube containing 3mL of YPD liquid medium and incubate in a shaker at 22°C and 250 rpm for 24 hours for activation. Take a certain amount of primary seeds and inoculate them into a 100mL Erlenmeyer flask containing 10mL of YPD medium at a 1% inoculum. Incubate at 22°C and 250 rpm for 48 hours, then transfer to 16°C and 250 rpm for 24 hours.

[0055] Place the culture medium in a 2 mL centrifuge tube, centrifuge at 4000 rpm for 3 minutes, and remove the supernatant; wash the obtained bacteria once with 1 mL of ultrapure water, and remove the supernatant by centrifugation; then add 1 mL of 3 mol / L hydrochloric acid, mix thoroughly, place in a boiling water bath for 3 minutes, quickly cool on ice for 3 minutes, and centrifuge to remove the supernatant; add 1.8 mL of acetone for extraction, ultrasonicate for 1-2 minutes to disperse the cells, centrifuge at 4°C, take the supernatant and place it in a new 2 mL centrifuge tube. The supernatant is the astaxanthin extract.

[0056] After activating the silica gel plate at 70°C for 1.5 hours, spot the astaxanthin standard and the crude extract sample to be analyzed (set 3 parallels as controls), repeat the spotting 10 times for each point, and place the silica gel plate in a developing tank after it is completely dried; due to the elution of the developing agent and the adsorption of silica gel, the components in the sample will gradually separate, and the developing agent will be taken out when it rises to 2 cm from the silica gel plate; after the development is completed, it will be immediately placed in a UV analyzer at a wavelength of 254 nm to take pictures and record; use JTLC software for preliminary screening analysis, calculate the astaxanthin content of the red yeast fermentation broth by the standard curve, and obtain the strain that meets the requirements (select the strain with astaxanthin content greater than the quantitative value).

[0057] Step 2: Expand and culture the strain obtained in step 1, detect the astaxanthin content by high performance liquid chromatography, obtain the astaxanthin yield of the strain, and screen the strain that meets the yield requirements.

[0058] Strains that passed the initial screening were expanded to the logarithmic phase using YPD medium at 22°C. After 24 hours of shake flask culture, the fermentation experiment was repeated, with three replicates per sample, to screen for fast-growing strains. A 1% inoculum of these strains was inoculated into a 100 mL Erlenmeyer flask containing 10 mL of YPD medium and incubated at 22°C and 250 rpm in a shaker. After 48 hours, the temperature was adjusted to 16°C and cultured for an additional 24 hours before sampling.

[0059] The cells of different fermentation broths were broken, and the crude extracts were moderately concentrated using an extraction agent, and then separated using a 0.22 μm microporous filter membrane. The astaxanthin components in different fermentation broths were detected and compared with the astaxanthin standard curve to calculate the astaxanthin content in different fermentation broths.

[0060] The astaxanthin standard and fermentation crude extract were filtered through a 0.22 μm microporous filter membrane, and the astaxanthin content was detected by HPLC. According to the national standard GB / T 31520-2015, a Shimadzu LC-2050 high-performance liquid chromatograph and an Amethyst C18-H column (Saifen Technology Co., Ltd., 4.6.150 mm, 5 μm) were used for analysis.

[0061] The chromatographic conditions and elution procedures are shown in Tables 1 and 2.

[0062] Table 1. Chromatographic conditions

[0063]

[0064] Table 2. Elution program

[0065]

[0066]

[0067] The method for preparing the astaxanthin standard curve is as follows: dissolve 5 mg of astaxanthin standard in acetone and dilute it to 50 mL. Under the action of ultrasound, the above solution will be evenly dispersed to obtain an astaxanthin solution with a concentration of 0.1 mg / mL, and filter it using a 0.22 μm organic filter membrane. Dilute the above astaxanthin standard solution with acetone to a concentration of 0.001, 0.002, 0.004, 0.006, 0.008, and 0.01 mg / mL. Under this condition, perform 3 parallel injections on each sample and draw a standard curve between its corresponding peak area and concentration. Figure 2 shown.

[0068] Figure 1 The results show the separation effect of the strain initial screening process in this example. It can be seen from the results that thin layer chromatography has a good performance for initial screening of strains with relatively high astaxanthin production. Its convenient and rapid screening method greatly improves the efficiency of the experiment.

[0069] Figure 3 This is the result of rescreening after the initial screening. As can be seen from the figure, the required mutagenic strain can be obtained through accurate screening.

[0070] The present invention first spots astaxanthin standards of different concentrations on a silica gel plate for thin-layer chromatography, and determines the chromatographic peak corresponding to each concentration by drawing a standard curve. After obtaining the target band of astaxanthin in the fermentation broth, JTLC software is used to analyze the results, and the astaxanthin content in the fermentation broth is roughly quantified to quickly screen out the target strain with relatively high astaxanthin production. The astaxanthin content in the fermentation broth is accurately quantified by high-performance liquid chromatography to screen out the best mutagenic bacteria. The present invention expands the practicality of thin-layer chromatography technology in trace analysis, and also provides a new approach for efficient and rapid screening and optimization of mutant strains in the field of biotechnology.

Claims

1. A method for screening a high-yielding microbial strain of astaxanthin, characterized in that: The following steps are involved: Step 1: Use thin layer chromatography to roughly quantify the astaxanthin production of the strain to be identified to obtain a strain that meets the requirements; Step 2: Expand and culture the strain obtained in step 1, detect the astaxanthin content by high performance liquid chromatography, obtain the astaxanthin yield of the strain, and screen the strain that meets the yield requirements.

2. The method for screening a high-yielding microbial strain of astaxanthin according to claim 1, wherein The microbial strain is Phaffia rhodozyma.

3. The method for screening a high-yielding microbial strain of astaxanthin according to claim 1, wherein The rough quantitative method in step 1 is as follows: extracting the fermentation liquid of the strain to obtain a crude extract; Spot the crude extract on an activated silica gel plate, dry it, place it in a developing tank, and use a developing agent for adsorption to separate the components; The astaxanthin content in the fermentation broth was calculated using a standard curve.

4. The method for screening a high-yielding microbial strain of astaxanthin according to claim 3, wherein: The developing solvent is a mixture of n-hexane and acetone in a volume ratio of 7:

3.

5. The method for screening a high-yielding microbial strain of astaxanthin according to claim 3, wherein: The extraction process of the strain fermentation broth is as follows: The fermentation broth was centrifuged at 4000 rpm for 3 min, and the supernatant was removed; The obtained bacteria were washed by centrifugation with ultrapure water and the supernatant was removed by centrifugation; Add hydrochloric acid, mix thoroughly, and place in boiling water bath for 3 minutes. After cooling, remove the supernatant. Extract and disperse the cells by ultrasonication for 1 to 2 minutes. Centrifuge at 4°C and collect the supernatant to obtain the desired crude extract.

6. The method for screening a high-yielding microbial strain of astaxanthin according to claim 5, wherein: Before the extraction, the pH is adjusted to 7, and then ethyl acetate is used for extraction, and after concentration, ultrasonic dispersion is performed.

7. The method for screening a high-yielding microbial strain of astaxanthin according to claim 1, wherein: The process of detecting astaxanthin content by high performance liquid chromatography in step 2 is as follows: The fermentation broth was subjected to cell disruption, concentrated using an extractant, filtered using a 0.22 μm filter membrane, and then the astaxanthin content was detected using high performance liquid chromatography.

8. The method for screening a high-yielding microbial strain of astaxanthin according to claim 7, wherein: The mobile phase used in the high performance liquid chromatography method is a mixture of acetonitrile and water in a volume ratio of 9:1 in the first pump; and a mixture of methanol and isopropanol in a volume ratio of 6:4 in the second pump. The flow rate was 1 mL / min and the detection wavelength was 470 nm.

9. The method for screening a high-yielding microbial strain of astaxanthin according to claim 1, wherein: In the steps 1 and 2, the content is detected in combination with the standard curve.

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