A method for determining the content of N-acetylglutamate in yeast culture

Abstract

The invention discloses a method for determining the content of N-acetylglutamate in yeast culture, which belongs to the technical field of chemical analysis. The method includes: pretreatment; extraction; purification; drawing and determination of a standard curve, and calculating the content of N-acetylglutamate in a yeast culture sample to be tested. The invention adopts a high performance liquid chromatography-ultraviolet detector to determine the content of N-acetylglutamate in yeast culture, which is suitable for use in laboratories of most feed companies, and can accurately determine the content of N-acetylglutamate in yeast culture, in order to provide a reference for further development and preparation of yeast culture, and also pave the way for the next step of studying the mechanism of action of N-acetylglutamate in animal bodies, which is of great significance for clarifying the nutrition and health care mechanism of yeast culture.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and more particularly to a method for determining the content of N-acetylglutamic acid in yeast culture. Background Art

[0002] Yeast Culture (YC), as a green additive, has become one of the ideal choices to replace antibiotics in the context of a comprehensive ban on antibiotics due to its rich nutrients and diverse biological functions. The nutrients of YC are complex and diverse, mainly including three main parts: yeast metabolites, live yeast and culture medium. In the feeding of ruminants, YC is particularly widely used, mainly due to its rich amino acids, vitamins, organic acids and bioactive substances. On the one hand, these nutrients can promote the absorption of nutrients in feed in animals, thereby improving the digestibility and utilization of nutrients; on the other hand, the nutrients in YC can also promote the proliferation of beneficial microorganisms in the intestine. When these microorganisms are fermented, short-chain fatty acids are produced, resulting in a decrease in intestinal pH, thereby inhibiting the proliferation of acid-sensitive Escherichia coli and Salmonella in the intestine, and ultimately improving the growth performance and immune function of animals. In addition, studies have also shown that in addition to the two polysaccharides β-glucan and mannan, other small molecule metabolites in yeast culture also have a certain immunomodulatory effect on the animal body.

[0003] YC contains a wide variety of metabolites, the contents of various components are not the same, and not all components have immune activity. Therefore, our research group used non-targeted metabolomics methods to analyze the small molecule metabolites of YC in our research group, combining factors such as VIP value, P value, FC value, FRD value, and positive regulatory effects on the ruminant body, and finally screened out 12 small molecule metabolites, among which N-acetyl glutamic acid (NAG) ranked first.

[0004] NAG is an amino acid derivative that can activate key enzymes in the synthesis of endogenous arginine, including carbamoyl phosphate synthetase I and pyrroline-5-carboxylate synthetase, and increase the activity of these enzymes. Studies have shown that the addition of NAG and its structural analogs to feed can effectively promote the synthesis of citrulline and arginine in the intestine of piglets, increase the expression level of intestinal nutrient transporter genes, thereby enhancing the digestion and absorption function of the intestine and improving the growth performance of piglets. However, there are relatively few reports on the detection method of NAG.

[0005] At present, no standard detection method for accurately measuring the NAG content in yeast culture has been established at home and abroad. Therefore, developing a method for measuring the NAG content in yeast culture has become a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a method for determining the content of N-acetylglutamate in yeast culture.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for determining the content of N-acetylglutamate in a yeast culture comprises the following steps:

[0009] (1) Pretreatment: crush the yeast culture sample to be tested and sieve it for later use;

[0010] (2) Extraction: Divide methanol into two parts, mix the pretreated sample and the first part of methanol, heat and ultrasonically extract, oscillate and mix once every period of time, centrifuge the extracted solution to obtain a supernatant, add the residue after centrifugation to the second part of methanol, vortex and oscillate to mix, centrifuge the mixture, combine the supernatant and transfer it to a volumetric flask, return the combined supernatant to room temperature, add ammonia water to adjust the pH ≥ 6.5, and dilute to volume with methanol to obtain an extract;

[0011] (3) Purification: Activate the solid phase extraction column with methanol and water in turn, take the extract obtained in step (2) and slowly pass it through the solid phase extraction column, rinse with ammonia water, pump it to near dryness, then wash it with methanol, and finally elute it with formic acid methanol solution, collect the eluate in a water bath, evaporate the solution to dryness, add ultrapure water to reconstitute it, filter it, and obtain the test solution;

[0012] (4) Drawing and determination of standard curve:

[0013] Dissolve the N-acetylglutamate standard in ultrapure water to prepare a 1 mg / mL standard stock solution. Dilute the standard stock solution into a standard series working solution with concentrations of 1, 5, 10, 25, 50, 100, 250, and 500 μg / mL, respectively. The standard series working solution and the test solution are measured by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0014] Column: Acclaim C30;

[0015] Mobile phase: a mixed solution of 0.05% phosphoric acid aqueous solution and methanol in a volume ratio of 95:5;

[0016] Flow rate: 1.0 mL / min;

[0017] Column temperature: 35°C;

[0018] Detector: UV detector;

[0019] Detection wavelength: 215nm;

[0020] Injection volume: 10 μL;

[0021] The target peak of the standard series working solution is analyzed to obtain the chromatographic peak area, and the standard curve is drawn with the concentration of the standard series working solution as the horizontal coordinate and the chromatographic peak area of ​​the standard series working solution as the vertical coordinate to obtain the linear regression equation. The target peak of the test solution is analyzed to obtain the chromatographic peak area, and the content of N-acetylglutamate in the yeast culture sample to be tested is calculated through the linear regression equation.

[0022] Furthermore, in step (1), the yeast culture to be tested is sampled by a five-point method, crushed, passed through a 60-mesh sieve, and stored at -20°C for later use.

[0023] Furthermore, in step (2), the mass of the sample to be tested after pretreatment is 3-10 g, the volume of the first part of methanol is 20-90 mL, the volume of the second part of methanol is 10 mL, 400 μL of 5% ammonia water is added to adjust the pH to ≥ 6.5, and the volume is fixed with methanol to 30-100 mL.

[0024] Further, in step (2), the pretreated sample to be tested and the first part of methanol are mixed and heated at 50-60°C for ultrasonic extraction for 30 minutes, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, and the mixture is oscillated and mixed every 5 minutes. The extracted solution is centrifuged at a speed of 4000r / min for 10 minutes to obtain a supernatant. The residue after centrifugation is mixed and added to the second part of methanol and vortexed and mixed. The power of the vortex oscillation is 50W, the speed is 1800-2800r / min, the oscillation mode is touched, and the vortex oscillation time is more than 1 minute. After vortex oscillation mixing, the mixture is centrifuged at a speed of 7000r / min for 5 minutes, and the supernatant is combined.

[0025] Furthermore, in step (3), 5 mL of methanol and 5 mL of water are used to activate the solid phase extraction column in sequence, 2 mL of the obtained extract is slowly passed through the solid phase extraction column, rinsed with 3 mL of 5% ammonia water, pumped to near dryness, then washed with 3 mL of methanol, and finally eluted with 6 mL of 2% formic acid methanol solution, the eluate is collected in a 65°C water bath, after the solution is evaporated to dryness, 2 mL of ultrapure water is added for reconstitution, and filtered through a 0.22 μm filter membrane to obtain the test solution.

[0026] Furthermore, in step (3), the solid phase extraction cartridge is AMICROM MAX, specification: 60 mg / 3 mL, and the obtained extract is slowly passed through the solid phase extraction cartridge at a flow rate of less than 1 mL / min.

[0027] Furthermore, in step (4), the specification of the chromatographic column: Acclaim C30 is 4.6 mm*250 mm, 5 μm.

[0028] Furthermore, in step (4), the calculation formula for the content of N-acetylglutamate in the yeast culture sample to be tested is:

[0029]

[0030] Where:

[0031] - The content of N-acetylglutamate in the yeast culture sample to be tested, in milligrams per kilogram, mg / kg;

[0032] -The concentration of N-acetylglutamate chromatographic peak area corresponding to the test solution, in micrograms per milliliter, μg / mL;

[0033] -The volume of the extract after constant volume, in milliliters, mL;

[0034] -The volume of the extract, in milliliters (mL);

[0035] -The volume of ultrapure water added for reconstitution, in milliliters, mL;

[0036] -The mass of the sample to be tested after pretreatment, in grams, g.

[0037] Furthermore, each yeast culture sample was subjected to multiple replicate measurements, and the measurement results were expressed as the arithmetic mean of the N-acetylglutamate content in the multiple replicate samples.

[0038] Furthermore, each yeast culture sample was subjected to three replicate measurements, and the measurement results were expressed as the arithmetic mean of the N-acetylglutamate content in the three replicate samples.

[0039] The beneficial effects of the present invention are as follows: the present invention adopts a high performance liquid chromatography-ultraviolet detector to determine the content of N-acetylglutamate in yeast culture, is suitable for use in laboratories of most feed companies, can accurately determine the content of N-acetylglutamate in yeast culture, so as to provide a reference for further development and preparation of yeast culture, and also pave the way for the next step of studying the mechanism of action of N-acetylglutamate in animal bodies, which is of great significance for clarifying the nutrition and health care mechanism of yeast culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 A is the effect diagram of purification without column purification, and B is the effect diagram of purification with column purification.

[0041] Figure 2 A is an extraction effect diagram when water is used as the extraction solvent, and B is an extraction effect diagram when methanol is used as the extraction solvent.

[0042] Figure 3 A is the chromatogram of N-acetylglutamate (NAG), and B is the standard curve.

[0043] Figure 4 The influence of weighing samples of different weights on the test results. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] 1. Instruments and equipment

[0046] Agilent 1260 InfinityⅡ liquid chromatography system (Agilent, USA); chromatographic column Acclaim C30 (4.6mm*250mm, 5μm) (Thermo Fisher Scientific (China) Co., Ltd.); ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.); electronic analytical balance (accuracy 0.0001g, BCE124-1CCN, Sartorius Scientific Instrument (Beijing) Co., Ltd.); Milli-Q ultrapure water system (Millipore, Germany); electric constant temperature water bath (Shanghai Pudong Rongfeng Scientific Instrument Co., Ltd.); TDZ5 desktop low-speed centrifuge (Hunan Hercy Instrument Equipment Co., Ltd.); low-temperature high-speed centrifuge (Sorvall ST4 Plus, Thermo Fisher Scientific, USA); vortex mixer (Shanghai Qite Analytical Instrument Co., Ltd.); pH meter (accuracy 0.01, Mettler-Toledo Technology (China) Co., Ltd.).

[0047] 2. Reagents and Materials

[0048] N-acetylglutamate standard (purity ≥99%) was purchased from Shanghai Aladdin Technology Co., Ltd.; analytical grade acetonitrile (purity ≥99.9%) and formic acid (purity ≥99.9%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; analytical grade ammonia (25%-28%) and phosphoric acid (purity ≥99.9%) were purchased from Tianjin Kemao Chemical Reagent Co., Ltd.; chromatographic grade methanol (purity ≥99.9%) was purchased from Fisher, USA; MAX mixed anion exchange solid phase extraction column (AMICROM MAX, 60 mg / 3mL) was purchased from Hangzhou Weimipai Technology Co., Ltd.

[0049] Example 1

[0050] The method for determining the content of N-acetylglutamate in a yeast culture comprises the following steps:

[0051] (1) Pretreatment: The yeast culture to be tested is sampled using the five-point method, crushed, passed through a 60-mesh sieve, and stored at -20°C for later use;

[0052] (2) Extraction: Divide methanol into two parts and accurately weigh 4.0000 g ( ) The pretreated sample was placed in a 100mL conical flask, 30mL of methanol was added to the sample, and the mixture was heated at 60℃ for 30min for ultrasonic extraction. The ultrasonic power was 500W and the ultrasonic frequency was 40KHz. The solution was oscillated and mixed every 5min. The extracted solution was centrifuged at a speed of 4000r / min for 10min to obtain a supernatant. 10mL of methanol was added to the residue after centrifugation and vortexed to mix. The power of the vortexing was 50W and the speed was 1800r / min. The oscillation mode was touched and the vortexing time was 1min. After vortex mixing, the mixture was centrifuged at a speed of 7000r / min for 5min, and the supernatants were combined. After the combined supernatant was restored to room temperature, 400μL of 5% ammonia water was added to adjust the pH to 7, and the volume was fixed with methanol to 40mL ( ), to obtain an extract;

[0053] (3) Purification: Activate a solid phase extraction cartridge (AMICROM MAX, specification: 60 mg / 3 mL) with 5 mL of methanol and 5 mL of water, take 2 mL ( ) The extract obtained in step (2) was passed through the solid phase extraction column at a flow rate of 0.8 mL / min, eluted with 3 mL of 5% ammonia water, pumped to near dryness, then washed with 3 mL of methanol, and finally eluted with 6 mL of 2% formic acid methanol solution. The eluate was collected and added to a 10 mL beaker and placed in a 65°C water bath in a fume hood. After the solution was evaporated to dryness, 2 mL ( ) Add ultrapure water to a small beaker for re-dissolution, and filter through a 0.22 μm filter membrane to obtain the test solution;

[0054] (4) Drawing and determination of standard curve:

[0055] Weigh 10 mg of N-acetylglutamate standard into a 10 mL centrifuge tube, add ultrapure water to dissolve and dilute to volume to prepare a 1 mg / mL standard stock solution, store at 4 °C, dilute the standard stock solution to a standard series working solution with a concentration of 1, 5, 10, 25, 50, 100, 250, 500 μg / mL, prepare it before use, and measure the standard series working solution and the test solution by high performance liquid chromatography. The high performance liquid chromatography conditions are as follows:

[0056] Chromatographic column: Acclaim C30, 4.6mm*250mm, 5μm;

[0057] Mobile phase: a mixed solution of 0.05% phosphoric acid aqueous solution and methanol in a volume ratio of 95:5;

[0058] Flow rate: 1.0 mL / min;

[0059] Column temperature: 35°C;

[0060] Detector: UV detector;

[0061] Detection wavelength: 215nm;

[0062] Injection volume: 10 μL;

[0063] The target peak of the standard series working solution was analyzed to obtain the chromatographic peak area. The standard curve was drawn with the concentration of the standard series working solution as the horizontal coordinate and the chromatographic peak area of ​​the standard series working solution as the vertical coordinate to obtain the linear regression equation. The target peak of the test solution was analyzed to obtain the chromatographic peak area. The concentration corresponding to the chromatographic peak area of ​​the test solution N-acetylglutamic acid was calculated by the linear regression equation ( );

[0064] The calculation formula for the content of N-acetylglutamate in the yeast culture sample to be tested is:

[0065]

[0066] Where:

[0067] - The content of N-acetylglutamate in the yeast culture sample to be tested, in milligrams per kilogram, mg / kg;

[0068] -The concentration of N-acetylglutamate chromatographic peak area corresponding to the test solution, in micrograms per milliliter, μg / mL;

[0069] -The volume of the extract after constant volume, in milliliters, mL;

[0070] -The volume of the extract, in milliliters (mL);

[0071] -The volume of ultrapure water added for reconstitution, in milliliters, mL;

[0072] -The mass of the sample to be tested after pretreatment, in grams, g.

[0073] The yeast culture samples in this example were measured three times, and the measurement results were expressed as the arithmetic mean of the content of N-acetylglutamate in the three repeated measurement samples.

[0074] The specific data of the concentration c (μg / mL) corresponding to the chromatographic peak area of ​​N-acetylglutamate in the three repeated determinations of the test solution are: 25.93888 μg / mL, 24.73688 μg / mL, 26.40075 μg / mL, and the contents obtained after conversion formula are: 254.859 mg / kg, 244.907 mg / kg, 261.620 mg / kg;

[0075] The NAG content measured in Example 1 was 253.795±8.407 mg / kg, RSD=0.033%.

[0076] Effect test

[0077] 1. Data Statistical Analysis

[0078] The peak area results of all results on the liquid chromatogram were obtained by automatic integration of Agilent 1100 ChemStation; the data results were preliminarily sorted and analyzed by Excel 2021. GraphPad Prism 9.5.1 was used to analyze and plot the obtained data.

[0079] 2. Results and Analysis

[0080] 2.1 Results of the selection of not passing through the column and passing through the column

[0081] Comparative Example 1

[0082] In Comparative Example 1, the purification step (3) was omitted and the extract was directly used as the test solution. The remaining steps and parameters were the same as those in Example 1.

[0083] The extraction effect of NAG in yeast culture by not passing through the column in Comparative Example 1 and passing through the column in Example 1 was tested. Figure 1 As shown, Figure 1 Figure A in the figure is a picture showing the effect of purification without column purification, and Figure B is a picture showing the effect of purification with column purification. The chromatogram without column purification has many impurities, the peak shape of NAG is incomplete, and the peak shape of nearby peaks greatly interferes with the peak shape of NAG; compared with the chromatogram without column purification, the chromatogram with column purification has relatively fewer impurities, the peak shape of NAG is complete, and the peak shape of nearby peaks has little interference with the peak shape of NAG. Therefore, the present invention uses the column purification method to purify the sample.

[0084] 2.2 Results of extraction solvent selection

[0085] Comparative Example 2

[0086] In Comparative Example 2, except that the methanol in step (2) is replaced by water, the remaining steps and parameters are the same as those of Example 1.

[0087] In this experiment, the extraction effect of NAG from yeast culture was compared using water and methanol as extraction solvents. The results showed that (see Figure 2 ), Figure 2 Figure A is the extraction effect diagram of water as the extraction solvent, and Figure B is the extraction effect diagram of methanol as the extraction solvent. When water is used as the extraction solvent, there are many miscellaneous peaks, and the miscellaneous peaks interfere with each other, resulting in a large peak area concentrated near a time point, and a small NAG peak area; when methanol is used as the extraction solvent, the peak shape is relatively independent, and there is no phenomenon of a large concentrated peak area. Compared with the NAG peak area extracted by water, the NAG peak area extracted by methanol is larger. Therefore, methanol was finally selected as the extraction solvent in this experiment.

[0088] 2.3 Linear Relationship

[0089] In Example 1, the NAG chromatograms of NAG standard and test group samples are shown in Figure 3 Figure A. By drawing a NAG standard curve ( Figure 3 Figure B shows that there is a good linear relationship between NAG concentration and chromatographic peak area in the concentration range of 1-500 ng / μL, with a fitting equation of y=1.714x-4.150 and a correlation coefficient of R²=1.000. The detection limit (LOD) corresponding to the signal-to-noise ratio (S / N) of 3 was calculated to be 0.672 mg / L. Similarly, the quantification limit (LOQ) corresponding to the signal-to-noise ratio (S / N) of 10 was calculated to be 2.239 mg / L. These results show that this method has high precision and accuracy and can be used as a standard method for quantitative analysis.

[0090] 2.4 Method precision test

[0091] Three NAG standard working solutions of different concentrations were selected and measured on the machine according to the experimental conditions of Example 1. Each standard working solution was measured 6 times, and the average value of the peak area and the relative standard deviation were calculated.

[0092] Method precision test results

[0093] The results of the method precision test are listed in Table 1. The RSDs were all less than 1%, indicating that the instrument maintained excellent analytical performance under the current chromatographic conditions.

[0094] Table 1 Method precision test results

[0095]

[0096] 2.5 Sample repeatability test

[0097] The sample to be tested in Example 1 was selected and treated according to the pretreatment method of Example 1. The sample to be tested was repeated 6 times and measured on a chromatography machine under the chromatographic conditions of Example 1 to calculate the average value and relative standard deviation of the peak area.

[0098] Sample repeatability test results

[0099] As shown in Table 2, the results of the sample repeatability test show that the chromatographic peak area obtained by multiple measurements of the sample does not change much, and the RSD is less than 1%, indicating that the random error of the test is small. Therefore, this method has high reliability and effectiveness.

[0100] Table 2 Sample repeatability test results

[0101]

[0102] 2.6 Spike recovery test

[0103] The raw material of the yeast culture in Example 1 was selected as a blank group sample after pretreatment for the spike recovery test. An equal amount of the blank group sample was accurately weighed, and three different weights of standard samples were added respectively. Six replicates were made for each spike level. The samples were processed and tested according to the test method of Example 1. The results were statistically analyzed, and the average value and relative standard deviation of the peak area were calculated.

[0104] Spike recovery test results

[0105] The test results were summarized and the spike recovery was calculated. The results are shown in Table 3. The samples were spiked in the range of 0.5-2.1 mg, and the average recovery was 90.139%-103.955%, with RSDs less than 1%. This shows that the method has high applicability and feasibility and meets the requirements of routine testing.

[0106] Table 3 Spiked recovery test results

[0107]

[0108] 2.7 Application of the method

[0109] According to the determination method of Example 1, another 7 different yeast culture samples were analyzed, each sample was repeated 3 times, and the results were expressed as arithmetic mean values.

[0110] The data in Table 4 show that NAG was present in all eight yeast cultures, ranging from 67.888±2.716 mg / kg to 504.674±7.358 mg / kg, with RSDs of less than 1%.

[0111] Table 4 Detection results of N-acetylglutamate in different yeast cultures

[0112]

[0113] Example 2

[0114] The yeast culture in Example 1 was measured. Except for step (2), the other steps and parameters were the same as those in Example 1.

[0115] Step (2) extraction in Example 2: Divide methanol into two parts, accurately weigh 3.0000g of the pretreated sample to be tested in a 100mL conical flask, add 20mL of methanol and mix with the sample to be tested, heat and ultrasonically extract at 58°C for 30min, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, and oscillate and mix once every 5min, centrifuge the extracted solution at a centrifugal speed of 4000r / min, and the centrifugal time is 10min to obtain a supernatant. Add 10mL of methanol to the centrifuged residue, vortex and oscillate to mix, the vortex oscillation power is 50W, the speed is 1800r / min, touch the oscillation mode, and the vortex oscillation time is: 1min. After vortex mixing, centrifuge the mixture at a centrifugal speed of 7000r / min, and the centrifugal time is 5min, and combine the supernatants. After the combined supernatant returned to room temperature, 400 μL of 5% ammonia water was added to adjust the pH to 7.5, and the volume was fixed with methanol to 30 mL to obtain an extract.

[0116] The yeast culture samples in this example were measured three times, and the measurement results were expressed as the arithmetic mean of the content of N-acetylglutamate in the three repeated measurement samples.

[0117] The specific data of the concentration c (μg / mL) corresponding to the chromatographic peak area of ​​N-acetylglutamate in the three repeated determinations of the test solution are: 14.12269 μg / mL, 14.57563 μg / mL, 14.69060 μg / mL, and the contents obtained after conversion formula are: 139.950 mg / kg, 144.438 mg / kg, 145.578 mg / kg;

[0118] The test results are: 143.322±2.975mg / kg, RSD=0.021%.

[0119] Example 3

[0120] The yeast culture in Example 1 was measured. Except for step (2), the other steps and parameters were the same as those in Example 1.

[0121] Step (2) extraction in Example 3: Divide methanol into two parts, accurately weigh 10.0000g of the pretreated sample to be tested in a 200mL conical flask, add 90mL of methanol, mix, heat and ultrasonically extract at 56°C for 30min, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, and the solution after extraction is centrifuged at a speed of 4000r / min for 10min to obtain a supernatant. Add 10mL of methanol to the residue after centrifugation and vortex oscillate to mix, the power of the vortex oscillation is 50W, the speed is 1800r / min, the oscillation mode is touched, and the vortex oscillation time is 1min. After vortex mixing, centrifuge the mixture at a speed of 7000r / min for 5min, and combine the supernatants. After the combined supernatant is restored to room temperature, add 400μL of 5% ammonia water to adjust the pH to 6.5, and dilute with methanol to a constant volume of 100mL to obtain an extract.

[0122] The yeast culture samples in this example were measured three times, and the measurement results were expressed as the arithmetic mean of the content of N-acetylglutamate in the three repeated measurement samples.

[0123] The specific data of the concentration c (μg / mL) corresponding to the chromatographic peak area of ​​N-acetylglutamate in the three repeated determinations of the test solution are: 24.31575 μg / mL, 24.97443 μg / mL, 25.47558 ​​μg / mL, and the contents obtained after conversion formula are: 240.959 mg / kg, 247.486 mg / kg, 252.452 mg / kg;

[0124] The test results were: 246.966±5.764 mg / kg, RSD=0.023%.

[0125] illustrate:

[0126] Yeast culture is a product prepared by solid-state fermentation and drying process using Saccharomyces cerevisiae as the main fermentation strain. Its production process includes: inoculating the liquid-cultured strain into the fermentation matrix according to a certain ratio, solid-state stacking fermentation after sufficient mixing, and drying after fermentation. During the fermentation process, the distribution of metabolites produced by the strain may be uneven. In order to avoid deviations caused by local unevenness during sample weighing, the present invention adopts a five-point sampling method for sample collection.

[0127] The present invention tests the effect of different sampling weights (3g, 4g, 10g) on ​​the NAG concentration in the sample. The results show (see Figure 4), compared with the sampling weight of 3g, the NAG concentration in the sample was significantly increased when the sampling weight was 4g and 10g (p≤0.0001), but there was no significant difference between the sampling weights of 4g and 10g (p>0.05). Based on the considerations of reducing sample loss, reducing experimental material consumption and improving sample processing efficiency, the present invention uses 4g as the sampling weight in subsequent experiments to perform analysis and testing of other yeast culture samples.

[0128] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for determining the content of N-acetylglutamate in a yeast culture, characterized in that: The following steps are involved: (1) Pretreatment: The yeast culture sample to be tested is crushed and sieved for later use; (2) Extraction: Methanol is divided into two parts, the pretreated sample to be tested and the first part of methanol are mixed and heated for ultrasonic extraction, and the mixture is shaken and mixed once every period of time. The extracted solution is centrifuged to obtain a supernatant, and the residue after centrifugation is added to the second part of methanol and vortexed and shaken to mix. The mixture is centrifuged, and the supernatant is combined and transferred to a volumetric flask. After the combined supernatant is restored to room temperature, ammonia water is added to adjust the pH ≥ 6.5, and the volume is fixed with methanol to obtain an extract; (3) Purification: Activate the solid phase extraction column with 5 mL of methanol and 5 mL of water in turn, take 2 mL of the extract obtained in step (2) and slowly pass it through the solid phase extraction column at a flow rate of less than 1 mL / min, rinse with 3 mL of 5% ammonia water, pump until it is almost dry, then wash with 3 mL of methanol, and finally elute with 6 mL of 2% formic acid methanol solution, collect the eluate in a 65°C water bath, evaporate the solution to dryness, add 2 mL of ultrapure water to reconstitute it, and filter it through a 0.22 μm filter membrane to obtain a test solution; The solid phase extraction cartridge is AMICROM MAX, specification: 60 mg / 3 mL; (4) Drawing and determination of standard curve: Dissolve the N-acetylglutamate standard in ultrapure water to prepare a 1 mg / mL standard stock solution, and dilute the standard stock solution into a series of standard working solutions with concentrations of 1, 5, 10, 25, 50, 100, 250, and 500 μg / mL, respectively; The standard series working solution and the test solution were measured by high performance liquid chromatography, and the high performance liquid chromatography conditions were: Column: Acclaim C30; Mobile phase: a mixed solution of 0.05% phosphoric acid aqueous solution and 95:5 methanol by volume; Flow rate: 1.0 mL / min; Column temperature: 35 °C; Detector: UV detector; Detection wavelength: 215nm; Injection volume: 10 μL; The target peak of the standard series working solution is analyzed to obtain the chromatographic peak area, and the standard curve is drawn with the concentration of the standard series working solution as the horizontal coordinate and the chromatographic peak area of ​​the standard series working solution as the vertical coordinate to obtain the linear regression equation. The target peak of the test solution is analyzed to obtain the chromatographic peak area, and the content of N-acetylglutamate in the yeast culture sample to be tested is calculated through the linear regression equation.

2. A method for determining the content of N-acetylglutamate in a yeast culture according to claim 1, characterized in that: In step (1), the yeast culture to be tested is sampled by the five-point method, crushed, passed through a 60-mesh sieve, and stored at -20°C for later use.

3. A method for determining the content of N-acetylglutamate in yeast culture according to claim 1, characterized in that: In step (2), the mass of the sample to be tested after pretreatment is 3-10 g, the volume of the first part of methanol is 20-90 mL, the volume of the second part of methanol is 10 mL, 400 μL of 5% ammonia water is added to adjust the pH to ≥ 6.5, and the volume is fixed with methanol to 30-100 mL.

4. A method for determining the content of N-acetylglutamate in yeast culture according to claim 1, characterized in that: In step (2), the pretreated sample to be tested and the first part of methanol are mixed and heated at 50-60°C for ultrasonic extraction for 30 minutes, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, and the mixture is oscillated and mixed every 5 minutes. The extracted solution is centrifuged at a speed of 4000r / min for 10 minutes to obtain a supernatant. The residue after centrifugation is mixed and added to the second part of methanol for vortex oscillation. The power of the vortex oscillation is 50W, the speed is 1800-2800r / min, the oscillation mode is touched, and the vortex oscillation time is more than 1 minute. After vortex oscillation mixing, the mixture is centrifuged at a speed of 7000r / min for 5 minutes, and the supernatant is combined.

5. The method for determining the content of N-acetylglutamate in yeast culture according to claim 1, characterized in that: In step (4), the chromatographic column: Acclaim C30 has a specification of 4.6 mm*250 mm, 5 μm.

6. A method for determining the content of N-acetylglutamate in yeast culture according to claim 1, characterized in that: In step (4), the calculation formula for the content of N-acetylglutamate in the yeast culture sample to be tested is: Where: X i - The content of N-acetylglutamate in the yeast culture sample to be tested, in milligrams per kilogram, mg / kg; c- the concentration of N-acetylglutamate chromatographic peak area corresponding to the test solution, in micrograms per milliliter, μg / mL; V-volume of the extract after constant volume, in milliliters, mL; V1-the volume taken from the extract, in milliliters, mL; V2-the volume of ultrapure water added for reconstitution, in milliliters, mL; m- The mass of the sample to be tested after pretreatment, in grams, g.

7. A method for determining the content of N-acetylglutamate in yeast culture according to claim 1, characterized in that: Each yeast culture sample was subjected to multiple replicate measurements, and the measurement results were expressed as the arithmetic mean of the N-acetylglutamate content in the multiple replicate samples.

8. The method for determining the content of N-acetylglutamate in yeast culture according to claim 1, characterized in that: Each yeast culture sample was tested three times, and the test results were expressed as the arithmetic mean of the N-acetylglutamate content in the three repeated samples.

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