Method for determining the content of 7-dehydrocholesterol in a yeast

Abstract

The present application relates to the technical field of biological chemical industry detection, and particularly relates to a method for determining the content of 7-dehydrocholesterol in yeast, which realizes efficient extraction and accurate determination of 7-DHC in yeast cells through the steps of yeast cell wall breaking treatment, ultrasonic-assisted extraction, high performance liquid chromatography-ultraviolet detection (HPLC-UV) quantitative analysis. The recovery rate of the method can reach 98.8%-99.4%, and the relative standard deviation (RSD) is less than or equal to 2.1%. The method has the advantages of simple operation, high sensitivity and good repeatability, and can be widely applied to the screening of 7-DHC yeast engineering bacteria, the monitoring of the fermentation process and the quality control of industrialized products.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection technology, specifically a method for determining the content of 7-dehydrocholesterol in yeast. Background Technology

[0002] 7-Dehydrocholesterol (7-DHC) is a precursor for the synthesis of vitamin D3 and has important applications in the pharmaceutical, food, and cosmetic fields. The production of 7-DHC through fermentation using genetically engineered yeast is currently a hot topic in industrialization research. However, yeast cell walls are dense, and 7-DHC is mainly found in intracellular lipid bodies. Conventional extraction methods suffer from low extraction efficiency and severe interference from impurities, leading to poor accuracy and reproducibility of detection results.

[0003] Currently, commonly used methods for 7-DHC determination mainly include colorimetry, gas chromatography, and high-performance liquid chromatography, but these methods have the following shortcomings:

[0004] 1. Colorimetric methods have low sensitivity and are easily interfered with by other sterols in yeast;

[0005] 2. Gas chromatography requires derivatization of 7-DHC, which is cumbersome and time-consuming.

[0006] 3. The existing high-performance liquid chromatography (HPLC) method directly uses saponification followed by extraction and washing with organic solvents, desolvation, and then volume adjustment with HPLC eluent before HPLC analysis of the content. This method is complicated, time-consuming, and results in 7-DHC loss during extraction, which cannot meet the requirements for precise quality control in industrialization.

[0007] Therefore, developing an efficient, accurate, and stable method for determining the 7-DHC content in yeast is of great significance to the development of the 7-DHC yeast fermentation industry. Summary of the Invention

[0008] In view of the problems existing in the prior art, the purpose of this invention is to provide a method for determining the content of 7-dehydrocholesterol in yeast, which solves the problems of low extraction efficiency, complicated extraction process and poor detection accuracy of the existing methods, and realizes efficient extraction and accurate quantification of 7-DHC in yeast.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A method for determining the 7-dehydrocholesterol content in yeast, specifically including the following steps:

[0011] 1) Yeast cell wall disruption treatment: Mix yeast cells with an ethanol aqueous solution of alkali, place the mixture in a sealed reaction tube, seal the reaction tube, and heat it to the saponification reaction temperature to carry out the reaction.

[0012] 2) Ultrasonic-assisted extraction: After the reaction is complete, remove the sealed reaction tube, add anhydrous ethanol solvent while it is still hot within 3-6 minutes, extract with ultrasound, cool to room temperature, and make up to volume with anhydrous ethanol for later use.

[0013] 3) HPLC-UV quantitative analysis: Take 20-25 μL of the prepared sample solution, use high performance liquid chromatography-ultraviolet detector to determine the peak area of ​​7-DHC in the sample solution, and substitute it into the standard curve equation to calculate the concentration of 7-DHC in the sample solution;

[0014] 4) Calculation of 7-dehydrocholesterol content: Substitute the 7-DHC concentration in the sample solution obtained above into the formula for calculating the 7-DHC content in yeast, 7-DHC content (mg / g stem cells) = (C×V×D) / m, and the 7-dehydrocholesterol content can be obtained.

[0015] Wherein, C: 7-DHC concentration in the sample solution (μg / mL).

[0016] V: Sample final volume (mL)

[0017] D: Dilution factor

[0018] m: Yeast cell mass (g).

[0019] As a further preferred embodiment of the present invention, in step 1), the ethanol aqueous solution of the alkali is either KOH ethanol solution or NaOH ethanol aqueous solution, and the mass ratio of alkali, water and ethanol is (1-5):(5-10):(5-10).

[0020] As a further preferred embodiment of the present invention, in step 1), the mass-to-volume ratio of the yeast cells to the ethanol aqueous solution of alkali is 1:(30-100)g / mL.

[0021] As a further preferred embodiment of the present invention, in step 1), the saponification reaction temperature is 50-100℃ and the reaction time is 1-5h.

[0022] As a further preferred embodiment of the present invention, in step 2), the anhydrous ultrasonic extraction is performed at a temperature of 40-50℃, a power of 200-300W, and an extraction time of 20-30min.

[0023] As a further preferred embodiment of the present invention, in step 3), the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is an Inertsil ODS-3 5um 4.6*250mm (up), the mobile phase is methanol, the flow rate is (1.5-1.8) mL / min, the detection wavelength is 282nm, the column temperature is 25-28℃, and the injection volume is 20-25μL.

[0024] As a further preferred embodiment of the present invention, in step 3), the standard curve equation is plotted as follows:

[0025] Accurately weigh 101 mg of 7-DHC standard, dissolve it in methanol and dilute to 100 mL to obtain a standard stock solution with a concentration of 1010 μg / mL.

[0026] Accurately measure 0.1 mL, 0.5 mL, 1.0 mL, 2.0 mL, and 5.0 mL of the standard stock solution, respectively, and dilute to 10 mL with methanol to obtain standard working solutions with concentrations of 10.1 μg / mL, 50.5 μg / mL, 101 μg / mL, 202 μg / mL, and 505 μg / mL.

[0027] Then, following the HPLC-UV chromatographic conditions described above, linear regression was performed with 7-DHC concentration (X, μg / mL) as the abscissa and peak area (Y) as the ordinate to obtain the standard curve equation:

[0028] Y = 33505X + 1097.4, R² = 0.9999.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] 1. Excellent extraction efficiency: The alkaline-ethanol solution saponification cell disruption process can achieve a cell disruption rate of over 99% for yeast cells, significantly improving the extraction and enrichment effect of 7-DHC target substances from the source;

[0031] 2. Streamlined and efficient process: The yeast cell wall breaking and saponification liquid is directly diluted with ethanol and brought to a fixed volume. Only simple filtration is required before it can be put into HPLC for separation and quantitative detection, eliminating complex intermediate purification steps;

[0032] 3. Stable and reliable test results: The spiked recovery rate of this method can reach 95.2%-98.7%, and the relative standard deviation (RSD) is ≤2.1%. The method exhibits excellent repeatability, precision and stability, and the test data is highly reliable.

[0033] 4. Simple operation and suitable for industrialization: No complex derivatization pretreatment is required, the overall detection cycle is short, the practical threshold is low, and it is suitable for high-volume, high-throughput sample detection scenarios, which can fully meet the rapid quality control needs in industrial production processes. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a high-performance liquid chromatogram of the yeast sample solution in this invention.

[0036] Figure 2 This is a high-performance liquid chromatogram of the 7-DHC standard in this invention.

[0037] Figure 3 This is the standard curve diagram of 7-DHC in this invention. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] A method for determining the 7-DHC content in yeast samples

[0041] 1) Sample processing

[0042] Take 100 mg of yeast cells, accurately weigh them, place them in a sealed reaction tube, put in a rotor of appropriate size, add 4 ml of KOH ethanol aqueous solution, seal the reaction tube and put it in a 93℃ water bath for 3 h.

[0043] 2) Ultrasonic-assisted extraction

[0044] After the reaction is complete, remove the sealed reaction tube and add anhydrous ethanol to 20 mL within 5 minutes while it is still hot. Then, extract by ultrasonication at 40°C and 200W for 20 minutes.

[0045] 3) Set the volume

[0046] After cooling to room temperature, dilute to 25 ml with anhydrous ethanol, shake well, and obtain the sample solution to be tested;

[0047] 4) HPLC-UV analysis

[0048] 20 μL of the sample solution to be tested was injected for analysis, and the peak area was recorded as 3520609. Substituting this into the standard curve equation, the concentration of 7-DHC in the sample solution was calculated to be 105.04 μg / mL.

[0049] Next, substitute the 7-DHC concentration in the sample solution obtained above into the formula for calculating the 7-DHC content in yeast. The formula for calculating the 7-DHC content is: 7-DHC content (mg / g stem cells) = (C×V×D) / m

[0050] in:

[0051] C: Concentration of 7-DHC in the sample solution (μg / mL)

[0052] V: Sample final volume (mL)

[0053] D: Dilution factor

[0054] m: Yeast cell mass (g)

[0055] Calculations showed that the 7-DHC content in this yeast sample was 26.26 mg / g stem cells.

[0056] To verify the effect of the process parameters set in Example 1 on the recovery rate of 7-DHC extraction from yeast, the following comparative experiment was designed.

[0057] Comparative Example 1: Lowering the saponification reaction temperature

[0058] Experimental procedure: Accurately weigh 100 mg of yeast cells and place them in a sealed reaction tube. Add a rotor and 4 mL of KOH ethanol solution. After sealing the reaction tube, place it in an 80°C water bath for 3 h of constant temperature reaction. The remaining ultrasonic extraction, volume adjustment, and HPLC detection procedures are completely consistent with those in Example 1. Finally, calculate the 7-DHC content in the sample.

[0059] Experimental Results and Analysis: The 7-DHC content in the sample detected in this group was 18.35 mg / g stem cells, which was significantly lower than 26.26 mg / g in Example 1;

[0060] The results showed that the water bath temperature of 80℃ was too low, and the yeast cell wall proteins and lipid structures could not be fully broken down, resulting in a slow saponification reaction rate and incomplete reaction. A large amount of intracellular 7-DHC was encapsulated by cell tissue and could not be released, ultimately leading to a significant decrease in the extraction recovery rate. This proved that 93℃ is the optimal temperature to ensure sufficient cell wall disruption and saponification.

[0061] Comparative Example 2: Shortening the saponification reaction time

[0062] Experimental procedure: Accurately weigh 100 mg of yeast cells and place them in a sealed reaction tube. Add a rotor and 4 mL of KOH ethanol aqueous solution. After sealing the reaction tube, place it in a 93°C water bath for 1.5 h. The remaining ultrasonic extraction, volume adjustment, and HPLC detection procedures are completely consistent with those in Example 1. Finally, calculate the 7-DHC content in the sample.

[0063] Experimental Results and Analysis: The 7-DHC content in the sample detected in this group was 22.96 mg / g stem cells, which is lower than the optimal process detection result;

[0064] The results showed that when the saponification time was shortened to 1.5 h, the saponification and degradation of lipid impurities were incomplete, the cell wall was not completely broken, and some bound 7-DHC could not be released into the extract, resulting in insufficient extraction. This confirmed that a saponification time of 3 h can completely break down the cell wall and remove impurities, ensuring that 7-DHC is fully dissolved.

[0065] Comparative Example 3: Replacing the saponification lye solution system

[0066] Experimental procedure: Accurately weigh 100 mg of yeast cells and place them in a sealed reaction tube. Add a rotor and replace the original KOH ethanol aqueous solution with an equal concentration of NaOH ethanol aqueous solution, with the added volume remaining at 4 mL. After sealing, react in a water bath at 93°C for 3 h. The subsequent ultrasonic extraction, volume adjustment, instrument detection, and content calculation procedures are consistent with those in Example 1.

[0067] Experimental results and analysis: After treatment with NaOH ethanol aqueous solution of equal concentration, the 7-DHC content of the sample was 23.94 mg / g stem cells, which was lower than that of the KOH ethanol aqueous solution system;

[0068] The results showed that compared with NaOH, KOH ethanol aqueous solution had stronger permeability and cell wall disruption, better saponification and decomposition effect on yeast lipid complexes, more thorough removal of impurities, better protection of 7-DHC, and higher extraction recovery rate, verifying the superiority of using KOH ethanol aqueous solution in the specific examples.

[0069] Comparative Example 4: Lowering the ultrasonic extraction temperature

[0070] Experimental procedure: Accurately weigh 100 mg of yeast cells and complete the cell wall disruption saponification using the standard saponification process of Example 1 (4 mL KOH ethanol aqueous solution, 93℃ water bath for 3 h); after the reaction, adjust the volume to 20 mL and adjust the ultrasonic conditions to room temperature 25℃, 200W ultrasonication for 20 min; the cooling and volume adjustment, instrument detection and content calculation steps remain unchanged.

[0071] Experimental results and analysis: After ultrasonic extraction at room temperature (25℃), the 7-DHC content was found to be 24.05 mg / g stem cells, which was not as good as the extraction effect under constant temperature ultrasonic conditions (40℃).

[0072] The results showed that under low temperature conditions, the molecular activity of the extract was low, the swelling and peeling effect of residual cell debris was poor, the dissolution and diffusion rate of 7-DHC was slow, and the dissolution was insufficient, proving that constant temperature ultrasound at 40℃ can effectively improve the dissolution efficiency and extraction recovery rate of the target substance.

[0073] Comparative Example 5: Reducing the ultrasonic extraction power

[0074] Experimental procedure: The saponification reaction steps of the sample were exactly the same as in Example 1. After saponification, the volume was adjusted to 20 mL with ethanol, and the ultrasonic parameters were adjusted to 40℃, 100W and ultrasonic for 20 min. The remaining volume adjustment, HPLC injection detection and content calculation operations were the same as in Example 1.

[0075] Experimental results and analysis: After treatment with 100W low-power ultrasound, the 7-DHC content of the sample was 23.46 mg / g stem cells, and the recovery rate was significantly reduced.

[0076] The results showed that insufficient ultrasonic power resulted in a weak cavitation effect, which could not effectively break up the residual tiny cell fragments and make it difficult to promote the release of encapsulated 7-DHC, thus significantly weakening the auxiliary extraction effect. This confirmed that 200W ultrasonic power can achieve the best auxiliary cell disruption extraction effect.

[0077] Comparative Example 6: Shortening the duration of ultrasonic extraction

[0078] Experimental procedure: Strictly follow the saponification process in Example 1. After saponification, bring the volume of ethanol to 20 mL. Under standard conditions of 40℃ and 200W, adjust the ultrasonic extraction time to 10 min. The subsequent cooling and volume adjustment, instrument detection, and data calculation process remain unchanged.

[0079] Experimental results and analysis: After ultrasonic extraction for 10 min, the 7-DHC content was found to be 24.08 mg / g stem cells;

[0080] The results showed that if the sonication time was too short, the ultrasonic cavitation effect could not continue to act on the extraction system, the intracellular residual target material could not be completely released and dissolved, the extraction reaction did not reach equilibrium, resulting in a low final detection content. This proved that a sonication time of 20 min can ensure that 7-DHC is fully dissolved and enriched.

[0081] Comparative Example 7: Replace the solvent for volume adjustment

[0082] Experimental procedure: The sample saponification and ultrasonic extraction steps were exactly the same as in Example 1. After ultrasonication and cooling to room temperature, the volume-fixing solvent was replaced with 50% ethanol aqueous solution, and the volume was adjusted to 25 mL and shaken well. The subsequent sample injection detection and content calculation methods remained the same.

[0083] Experimental results and analysis: After dilution with 50% ethanol aqueous solution, the sample showed slight turbidity and precipitation. The content of 7-DHC was found to be 22.61 mg / g stem cells, and the detection accuracy and recovery rate decreased significantly.

[0084] The results showed that the aqueous system caused the saponified lipid impurities to re-precipitate, interfering with the dissolution of the target analyte. At the same time, some 7-DHC was adsorbed into the impurity precipitate and could not be detected by the instrument, which greatly reduced the detection value and verified the necessity of adjusting the volume with anhydrous ethanol.

[0085] Comparative Example 8: Elimination of the ultrasound-assisted extraction step

[0086] Experimental procedure: Accurately weigh 100 mg of yeast sample, complete the standard saponification reaction at 93℃ for 3 h, add anhydrous ethanol to 20 mL while hot, do not perform ultrasonic extraction, cool directly to room temperature, and make up to 25 mL with anhydrous ethanol; shake well and then test on the instrument to calculate the 7-DHC content.

[0087] Experimental Results and Analysis: After eliminating the ultrasonic extraction step, the 7-DHC content was only 20.03 mg / g stem cells, which was the lowest among all experimental groups;

[0088] The results showed that saponification alone could not completely remove intracellular bound 7-DHC, leaving a large amount of the target substance in the cell residue. The cell wall disruption and dissolution effect without ultrasound assistance was extremely poor, which fully demonstrates the key synergistic effect of ultrasound extraction on improving the extraction recovery rate of 7-DHC.

[0089] Comparative Example 9: Using a non-sealed saponification reaction

[0090] Experimental procedures: Sample weighing, reagent addition, reaction temperature and duration were consistent with those in Example 1, only the reaction conditions were changed: the sealing operation was removed, and an open water bath was used for 3 hours; the subsequent ultrasonic extraction, volume adjustment, detection and calculation steps remained completely unchanged.

[0091] Experimental results and analysis: After open saponification reaction, the 7-DHC content was found to be 24.19 mg / g stem cells;

[0092] The results showed that during the open high-temperature reaction, the continuous evaporation of ethanol solvent led to an increase in the concentration of the saponification liquid and an imbalance in the reaction system. At the same time, 7-DHC was easily exposed to air and underwent slight oxidative degradation, resulting in the loss of the target substance. Sealed reaction can effectively avoid this problem and ensure the stability of the reaction system and the integrity of the target substance.

[0093] Comparative Example 10: Ethanol added after saponification and cooling.

[0094] Experimental procedure: After completing the saponification reaction at 93℃ for 3 hours, wait for the reaction tube to cool completely to room temperature, then add anhydrous ethanol to make up to 20 mL, followed by sonication at 40℃ and 200W for 20 minutes; the remaining procedures for volume adjustment, instrument detection, and content calculation are the same as in Example 1.

[0095] Experimental results and analysis: After cooling and treatment with ethanol, the 7-DHC content was found to be 21.97 mg / g stem cells;

[0096] The results showed that after the saponification system cooled, the saponification products and lipid impurities quickly solidified and agglomerated, tightly encapsulating some 7-DHC in the solid residue. Subsequent ultrasonic and volume adjustment treatments could not fully dissolve it, resulting in serious loss of the target substance. This confirmed that adding alcohol while hot can effectively disperse the saponification system and ensure the full dissolution of 7-DHC.

[0097] To further verify the stability and reliability of the measurement method in this invention, the following experimental method was set up for verification.

[0098] 1. Recovery rate test

[0099] Three concentrations of 7-DHC standards (low, medium, and high) were added to yeast samples with known 7-DHC content, and the results were determined according to the method of this invention. The recovery rates were calculated, and the results are as follows:

[0100] When the spiked concentration was 10.1 μg / mL, the measured concentration was 10.01 μg / mL, and the recovery rate reached 99.1%.

[0101] When the spiked concentration was 101.0 μg / mL, the measured concentration was 99.75 μg / mL, and the recovery rate reached 98.8%.

[0102] When the spiked concentration was 300.5 μg / mL, the measured concentration was 298.62 μg / mL, and the recovery rate reached 99.4%.

[0103] The average recovery rate was 99.1%, and the RSD was 0.3%.

[0104] 2. Repeatability test

[0105] The same yeast sample was measured six times in parallel according to the method of the present invention. The results of 7-DHC content determination were 12.5 mg / g, 12.7 mg / g, 12.6 mg / g, 12.4 mg / g, 12.7 mg / g and 12.5 mg / g, respectively. The average value was 12.6 mg / g and the RSD was 1.1%, indicating that the method has good repeatability.

[0106] 3. Stability test

[0107] The sample solution was taken and analyzed at 0h, 2h, 4h, 8h, 12h and 24h respectively. The peak area RSD was 0.8%, indicating that the sample solution had good stability within 24h.

[0108] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for determining the content of 7-dehydrocholesterol in yeast, characterized in that, Specifically, the steps include the following: 1) Yeast cell wall disruption treatment: Mix yeast cells with an ethanol aqueous solution of alkali, place the mixture in a sealed reaction tube, seal the reaction tube, and heat it to the saponification reaction temperature to carry out the reaction. 2) Ultrasonic-assisted extraction: After the reaction is complete, remove the sealed reaction tube, add anhydrous ethanol solvent while it is still hot within 3-6 minutes, extract with ultrasound, cool to room temperature, and make up to volume with anhydrous ethanol for later use. 3) HPLC-UV quantitative analysis: Take 20-25 μL of the prepared sample solution, use high performance liquid chromatography-ultraviolet detector to determine the peak area of ​​7-DHC in the sample solution, and substitute it into the standard curve equation to calculate the concentration of 7-DHC in the sample solution; 4) Calculation of 7-dehydrocholesterol content: Substitute the 7-DHC concentration in the sample solution obtained above into the formula for calculating the 7-DHC content in yeast, 7-DHC content (mg / g stem cells) = (C×V×D) / m, and the 7-dehydrocholesterol content can be obtained. Wherein, C: 7-DHC concentration (μg / mL) in the sample solution. V: Sample final volume (mL) D: Dilution factor m: Yeast cell mass (g).

2. The method for determining the 7-dehydrocholesterol content in yeast according to claim 1, characterized in that, In step 1), the ethanol aqueous solution of the alkali is either KOH ethanol aqueous solution or NaOH ethanol aqueous solution, and the mass ratio of alkali, water and ethanol is (1-5):(5-10):(5-10).

3. The method for determining the 7-dehydrocholesterol content in yeast according to claim 1, characterized in that, In step 1), the mass-to-volume ratio of the yeast cells to the alkali ethanol aqueous solution is 1:(30-100)g / mL.

4. The method for determining the 7-dehydrocholesterol content in yeast according to claim 1, characterized in that, In step 1), the saponification reaction temperature is 50-100℃ and the reaction time is 1-5h.

5. The method for determining the 7-dehydrocholesterol content in yeast according to claim 1, characterized in that, In step 2), the anhydrous ultrasonic extraction is performed at a temperature of 40-50℃, a power of 200-300W, and an extraction time of 20-30min.

6. The method for determining the 7-dehydrocholesterol content in yeast according to claim 1, characterized in that, In step 3), the chromatographic conditions for high performance liquid chromatography are as follows: the column is an Inertsil ODS-3 5um 4.6*250mm (up), the mobile phase is methanol, the flow rate is (1.5-1.8) mL / min, the detection wavelength is 282nm, the column temperature is 25-28℃, and the injection volume is 20-25μL.

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