Method for determining pyrazine compounds in fermentation liquor

Abstract

The invention belongs to the technical field of detection, and particularly relates to a method for determining pyrazine compounds in fermentation liquor. The method comprises the following steps: (1) preparation of fermentation liquor: carrying out shake culture fermentation on microorganisms to obtain the fermentation liquor; (2) taking fermentation liquor, centrifuging, taking supernatant, diluting with a methanol solution, and then filtering to obtain a sample to be detected; and (3) analyzing the to-be-detected sample obtained in the step (2) by using an ultra-high performance liquid chromatography-high resolution mass spectrometry method. The method has the characteristics of simple pretreatment, short analysis time, no sample loss, low detection limit, high recovery rate, good stability and the like, can maximally reflect the real content of the pyrazine compounds in the sample, and can rapidly, simply and accurately determine the pyrazine compounds in the fermentation liquor.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to a method for determining pyrazine compounds in fermentation broth. Background Art

[0002] Pyrazine compounds are a class of trace compounds that make an important contribution to the formation of liquor flavor. They are key aroma substances in sauce-flavor liquor. They have baking and nutty aromas, providing liquor with a unique aroma and taste, and have functional properties that are beneficial to human health.

[0003] Liang Huizhen and others used high-temperature culture and solid-state fermentation to screen high-yield pyrazine strains from high-temperature Daqu, and identified them as Bacillus through morphological observation and molecular biology. Bacillus licheniformis can produce more than 70 metabolites, and can produce aromatic compounds, C4 compounds, pyrazine and organic acids through solid-state fermentation. It is the main microorganism that metabolizes pyrazine. Pyrazine can be produced by biological fermentation, which has broad application prospects.

[0004] At present, the detection method of pyrazine is generally gas chromatography-mass spectrometry (GC-MS) technology, which usually uses solvent extraction, purge and trap technology combined with chromatographic analysis. This method not only requires a large number of samples, but also has cumbersome sample pretreatment process, long analysis time, insufficient extraction, high quantitative limit and other disadvantages. It is not a good choice for large quantities of samples. Ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) technology uses ultra-high performance liquid chromatography as the injection system of mass spectrometry to separate complex chemical components; and uses mass spectrometry as a detector for quantitative and qualitative analysis. The use of ultra-high performance liquid chromatography-high resolution mass spectrometry technology to detect pyrazine compounds can well overcome the shortcomings of existing technologies. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for determining pyrazine compounds in a fermentation broth.

[0006] Another object of the present invention is to provide a method for detecting eight pyrazine compounds, namely 2-methylpyrazine, 2-ethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2,3,5-trimethylpyrazine, 2,3-diethylpyrazine, tetramethylpyrazine and 2,3-dimethyl-5-ethylpyrazine, in a fermentation broth.

[0007] The above-mentioned purpose of the present invention is achieved by the following technical means:

[0008] In one aspect, the present invention provides a method for determining pyrazine compounds in a fermentation broth, comprising the following steps:

[0009] (1) Preparation of fermentation broth;

[0010] (2) pretreatment of the fermentation liquid to obtain a sample to be tested;

[0011] (3) Analyze the sample obtained in step (2) using ultra-high performance liquid chromatography-high resolution mass spectrometry.

[0012] In some experimental schemes, the fermentation broth is a bacterial broth containing pyrazine compounds.

[0013] In some experimental schemes, the fermentation broth is a bacterial broth obtained by culturing microorganisms that produce pyrazine compounds.

[0014] In some experimental schemes, the step of preparing the fermentation broth includes: fermenting the microorganisms with shaking culture to obtain the fermentation broth.

[0015] In some experimental protocols, the shaking culture time is 10 to 100 hours.

[0016] In some experimental protocols, the shaking culture time is 40 to 90 hours.

[0017] In some experimental schemes, the rotation speed of the shaking culture is 10 r / min to 2000 r / min.

[0018] In some experimental protocols, the rotation speed of the shaking culture is 50 r / min to 500 r / min.

[0019] In some experimental schemes, the rotation speed of the shaking culture is 50 r / min to 300 r / min.

[0020] In some experimental protocols, the temperature of the shaking culture is 15°C to 45°C.

[0021] In some experimental protocols, the temperature of the shaking culture is 15°C to 40°C.

[0022] In some experimental schemes, the medium for the shaking culture is a fermentation medium.

[0023] In some experimental protocols, the fermentation medium contains 1.0-100.0 g / L peptone.

[0024] In some experimental schemes, the fermentation medium contains 1.0-100.0 g / L diammonium hydrogen phosphate.

[0025] In some experimental schemes, the fermentation medium contains 1.0-100.0 g / L yeast extract powder.

[0026] In some experimental schemes, the fermentation medium contains 1.0-500.0 g / LD (+)-anhydrous glucose.

[0027] In some experimental protocols, the microorganism is Bacillus licheniformis.

[0028] In some experimental protocols, the microorganism is Bacillus licheniformis.

[0029] In some experimental protocols, the microorganism is Bacillus licheniformis ATCC 14580.

[0030] In some experimental schemes, the pretreatment step of the fermentation broth includes: taking the fermentation broth, centrifuging it, taking the supernatant, diluting it with a methanol solution and then filtering it.

[0031] In some experimental schemes, the centrifugal rotation speed is 5000-20000 r / min.

[0032] In some experimental schemes, the centrifugal rotation speed is 8000-15000 r / min.

[0033] In some experimental protocols, the centrifugation time is 1 to 20 minutes.

[0034] In some experimental protocols, the centrifugation time is 1 to 10 minutes.

[0035] In some experimental schemes, the volume fraction of the methanol solution is 5-50%.

[0036] In some experimental schemes, the volume fraction of the methanol solution is 10-40%.

[0037] In some experimental protocols, the methanol solution is chromatography grade methanol.

[0038] In some experimental schemes, the pyrazine compound includes one or more of 2-methylpyrazine, 2-ethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2,3,5-trimethylpyrazine, 2,3-diethylpyrazine, tetramethylpyrazine and 2,3-dimethyl-5-ethylpyrazine.

[0039] In some experimental schemes, the filter membrane used for filtration is a 0.22 μm filter membrane.

[0040] In some experimental schemes, the filter membrane is a microporous filter membrane.

[0041] In some experimental schemes, in the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the ultra-high performance liquid chromatography conditions are: chromatographic column: liquid chromatography column; column oven: 20-60°C; constant flow: column flow rate is 0.1-0.5mL / min; injection volume: 1-5μL; mobile phase A: formic acid aqueous solution, mobile phase B: formic acid-acetonitrile solution; gradient elution conditions: 0-1.0min, 93%A; 1.0-12.0min, 93%-87%A; 12.0-12.5min, 87%-0%A; 12.5-15.0min, 0%A; 15.0-15.5min, 0-93%A; 15.5-19.0min, 93%A.

[0042] The volume percentage of mobile phase A refers to the volume percentage of mobile phase A in the mixture of mobile phase A and mobile phase B.

[0043] In some experimental schemes, the ultra-high performance liquid chromatography conditions are: chromatographic column: Thermo Hypersil Gold chromatographic column; column oven: 30-40°C; constant flow: column flow rate is 0.2-0.3 mL / min; injection volume: 1-2 μL; mobile phase A: 0.01-0.5% formic acid aqueous solution, mobile phase B: formic acid-acetonitrile (0.1-5:1000, V / V); gradient elution conditions: 0-1.0 min, 93% A; 1.0-12.0 min, 93%-87% A; 12.0-12.5 min, 87%-0% A; 12.5-15.0 min, 0% A; 15.0-15.5 min, 0-93% A; 15.5-19.0 min, 93% A.

[0044] In some experimental schemes, the ultra-high performance liquid chromatography conditions are: chromatographic column: Thermo Hypersil Gold, 150mm×2.1mm, 1.9μm; column oven: 35°C; constant flow: column flow rate is 0.3mL / min; injection volume: 2μL; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: formic acid-acetonitrile (1:1000, V / V); gradient elution conditions: 0-1.0min, 93%A; 1.0-12.0min, 93%-87%A; 12.0-12.5min, 87%-0%A; 12.5-15.0min, 0%A; 15.0-15.5min, 0-93%A; 15.5-19.0min, 93%A.

[0045] In some experimental schemes, in the ultra performance liquid chromatography-high resolution mass spectrometry method, the high resolution mass spectrometry conditions are: heated electrospray ionization source; sheath gas flow rate: 5-50 arb; auxiliary gas flow rate: 5-50 arb; spray voltage: 2.0-4.0 kV; ion source temperature: 200-350°C; capillary temperature: 200-350°C; positive ion scanning acquisition mode of Full MS scanning, resolution: 70,000; scanning range: 50-1000 m / z.

[0046] In some experimental schemes, the high-resolution mass spectrometry conditions are: heated electrospray ionization source; sheath gas flow rate: 30arb; auxiliary gas flow rate: 10arb; spray voltage: 2.0kV; ion source temperature: 350°C; capillary temperature: 300°C; positive ion scanning acquisition mode using Full MS scanning, resolution: 70,000; scanning range: 70-200m / z.

[0047] In some experimental schemes, the qualitative analysis of the pyrazine compounds is performed by matching the retention time and accurate mass of the pyrazine standard in combination with the secondary characteristic fragment ions.

[0048] In some experimental schemes, the method further comprises drawing a standard curve using a standard substance of the pyrazine compound, thereby calculating the content of the pyrazine compound in the sample to be tested.

[0049] On the other hand, the present invention also provides the application of the above method in detecting the type or content of pyrazine compounds in fermentation broth.

[0050] Compared with the prior art, the present invention has the following beneficial technical effects:

[0051] The present invention enables the separation of eight pyrazine compounds within 19 minutes, and has a good linear relationship within the detection range of 8 to 1808 μg / L. The correlation coefficient R 2 All of them were greater than 0.999, the lowest limits of quantification were less than 0.1 μg / L, and the lowest limits of detection were lower than 0.1 μg / L, which could reflect the true content of pyrazine compounds in the samples to the greatest extent and could quickly, simply and accurately determine pyrazine compounds in the fermentation broth. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the chromatogram of a mixed standard solution of eight pyrazine compounds.

[0053] Figure 2 The secondary characteristic fragment ion patterns of 8 pyrazine compounds.

[0054] Figure 3 This is a standard curve diagram of 8 pyrazine compounds.

[0055] Figure 4 The chromatogram of the fermentation broth sample to be tested.

[0056] Figure 5 Chromatograms of eight pyrazine compounds analyzed using a ThermoAccucore C18 column.

[0057] Figure 6 The chromatograms are as follows: 0.1% formic acid aqueous solution and 0.1% acetonitrile aqueous solution as mobile phase A and B respectively.

[0058] Figure 7 The chromatograms are when pure water solution and 0.1% acetonitrile water solution are used as mobile phase A and B respectively.

[0059] Figure 8 Chromatograms of eight pyrazine compounds analyzed by gradient elution condition 1 for comparison.

[0060] Fig. 9 Chromatograms of 8 pyrazine compounds analyzed by gradient elution condition 2 for comparison. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0062] In the examples herein, the main reagents are: chromatographic grade methanol and acetonitrile, from Merck, Germany; mass spectrometry grade formic acid, from Fisher, USA; diammonium hydrogen phosphate of analytical grade, item number: R009469-500g, from Shanghai Yien Chemical Technology Co., Ltd.; peptone, item number: 01-001, from Beijing Aoboxing Biotechnology Co., Ltd.; yeast extract powder, item number: 4304391-02, from OXOID, UK; D(+)-anhydrous glucose of analytical grade, item number: HH7180P2O7UQ, from Sinopharm Chemical Reagent Co., Ltd.

[0063] In the examples herein, the main instruments are: high performance liquid chromatograph Ultimate 3000, Dionex, USA; high resolution mass spectrometer Q-Exactive Focus, Thermo Fisher, USA.

[0064] In the examples herein, the standard substances: 2-methylpyrazine, 2-ethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2,3,5-trimethylpyrazine, 2,3-diethylpyrazine, tetramethylpyrazine, 2,3-dimethyl-5-ethylpyrazine, with a purity greater than 99.0%, are from Aladin Corporation.

[0065] In the examples herein, the formula of the fermentation medium is 30.0 g / L peptone, 30.0 g / L diammonium hydrogen phosphate, 10.0 g / L yeast extract powder, 50.0 g / L D(+)-anhydrous glucose, with water as the dissolving matrix, pH 7.0, and high-pressure steam sterilization at 115°C for 15 min.

[0066] In the examples herein, the 0.1% formic acid aqueous solution and the 0.1% acetonitrile aqueous solution are formic acid:pure water (1:1000, V / V) and acetonitrile:pure water (1:1000, V / V), respectively.

[0067] In this article, the term "rejuvenation" refers to the measure of isolating and selectively culturing a declining bacterial species (population) so that the non-declining individuals can reproduce in large numbers and become a purebred population again.

[0068] Example 1 Quantitative detection of eight pyrazine compounds in a standard solution using UPLC-HRMS

[0069] Ultra-high performance liquid chromatography-high resolution mass spectrometry was used to quantitatively detect eight pyrazine compounds. The specific conditions are as follows:

[0070] (1) Chromatographic conditions:

[0071] Chromatographic column: Thermo Hypersil Gold (150mm×2.1mm, 1.9μm); column oven: 35°C; constant flow: column flow rate is 0.3mL / min; injection volume is 2μL; mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is formic acid-acetonitrile (1:1000, V / V).

[0072] Gradient elution conditions: 0-1.0 min, 93% A; 1.0-12.0 min, 93%-87% A; 12.0-12.5 min, 87%-0% A; 12.5-15.0 min, 0% A; 15.0-15.5 min, 0-93% A; 15.5-19.0 min, 93% A.

[0073] The volume percentage of mobile phase A refers to the volume percentage of mobile phase A in the mixture of mobile phase A and mobile phase B.

[0074] (2) Mass spectrometry conditions:

[0075] A heated electrospray ionization source was used; sheath gas flow rate: 30 arb; auxiliary gas flow rate: 10 arb; spray voltage: 2.0 kV; ion source temperature: 350°C; capillary temperature: 300°C. Full MS scan was used in positive ion scanning acquisition mode, resolution: 70,000; scanning range: 70-200 m / z.

[0076] (3) Preparation of mixed solutions of 8 pyrazine compound standards:

[0077] Accurately weigh 8 pyrazine standards using a 1 / 10,000 balance, dilute and fix to 100 mL with a 20% by volume methanol solution to prepare a standard solution. The specific concentrations are shown in Table 1.

[0078] Table 1 Concentrations of 8 pyrazine compounds in the standard solution

[0079]

[0080] (4) Qualitative analysis of 8 pyrazine compounds:

[0081] Take 1 mL of the standard solution in step (3) and dilute it to 10 mL with 20% methanol solution. Under the above-mentioned ultra-high performance liquid chromatography-high-resolution mass spectrometry conditions, qualitative analysis is achieved by matching the retention time and accurate mass of the pyrazine standard and combining the secondary characteristic fragment ions. The results are as follows: Figure 1 and Figure 2 shown.

[0082] (5) Quantification of 8 pyrazine compounds:

[0083] Take 1mL, 0.5mL, 0.2mL, 0.1mL, 0.05mL, and 0.02mL of the standard solution prepared in step (3) above, dilute to 10mL with 20% methanol solution, and dilute to a series of standard gradient concentrations for later use. Draw a standard curve with mass concentration (X, μg / L) as the horizontal axis and characteristic ion peak area (Y) as the vertical axis, as shown in the figure. Figure 3 The specific parameters are shown in Table 2.

[0084] Table 2 Standard curve parameters of 8 pyrazine compounds

[0085]

[0086] (6) Repeatability experiment:

[0087] Take 0.1 mL of the standard solution in step (3) and dilute to 10 mL with 20% methanol solution. Under the above conditions, the content of pyrazine compounds was determined, and the repeatability of the determination results of 8 pyrazine compounds was calculated. The relative standard deviation (RSD) was less than 10%, indicating that the method had good repeatability. The specific results are shown in Table 3.

[0088] Table 3 Repeatability experimental data results of 8 pyrazine compounds

[0089]

[0090] (7) Limit of detection (LOD) and limit of quantification (LOQ)

[0091] Take 0.02 mL of the standard solution in step (3) and dilute to 10 mL with 20% methanol solution. Under the above conditions, the content of pyrazine compounds was determined. The limit of detection (LOD) is the sample mass concentration corresponding to the signal intensity / baseline noise (S / N) = 3:1; the limit of quantification (LOQ) is the sample mass concentration corresponding to the signal intensity / baseline noise (S / N) = 10:1.

[0092] In this embodiment, the detection limit (LOD) of the eight pyrazine compounds is 0.021 μg / L to 12.164 μg / L, and the quantification limit (LOQ) is 0.070 μg / L to 40.545 μg / L. The specific calculation results are shown in Table 4.

[0093] Table 4 Calculation results of limit of detection (LOD) and limit of quantification (LOQ) of 8 pyrazine compounds

[0094]

[0095] Example 2 Quantitative detection of eight pyrazine compounds in fermentation broth using UPLC-HRMS

[0096] The ultra-high performance liquid chromatography-high resolution mass spectrometry described in Example 1 was used to quantitatively detect eight pyrazine compounds in the fermentation broth, specifically comprising the following steps:

[0097] (1) Fermentation medium configuration:

[0098] The fermentation medium was prepared according to the following concentrations: peptone 30.0 g / L, diammonium phosphate 30.0 g / L, yeast extract powder 10.0 g / L, D(+)-anhydrous glucose 50.0 g / L, with water as the dissolving matrix, pH 7.0, and high-pressure steam sterilization at 115°C for 15 min.

[0099] (2) Fermentation broth preparation:

[0100] After activating and rejuvenating Bacillus licheniformis ATCC 14580, the culture was shaken in a fermentation medium at 200 r / min at 37° C. for 72 hours. After the fermentation was completed, the content of pyrazine compounds in the fermentation broth was detected by ultra-high performance liquid chromatography-high resolution mass spectrometry.

[0101] (3) Pretreatment of samples to be tested:

[0102] Take 2 mL of fermentation broth, centrifuge at 1,2000 r / min for 5 min, take the supernatant, dilute it with 20% methanol solution by an appropriate multiple, filter it through a 0.22 μm microporous filter membrane, and inject it directly.

[0103] (4) Sample determination:

[0104] The determination conditions and methods are the same as those in Example 1. The chromatogram of the obtained fermentation broth sample is as follows: Figure 4 The peak area of ​​the pyrazine compounds was substituted into the standard curve to calculate the content of each pyrazine compound in the fermentation broth sample. In the fermentation broth sample of this example, 2,3,5-trimethylpyrazine was 169.765 μg / L, tetramethylpyrazine was 1172.681 μg / L, and the other 6 pyrazine compounds were not detected.

[0105] (5) Determination of the recovery rate of 8 pyrazine compounds in fermentation broth:

[0106] The determination conditions and methods were the same as those in Example 1. A certain concentration of 8 pyrazine standards was added to the fermentation broth (fermentation broth: standard = 1:1, V / V) to determine the spike recovery rate. The sample was repeated 3 times to determine its relative standard deviation (RSD). The specific results are shown in Table 5. As can be seen from Table 5, this method has good accuracy and precision for quantitative pyrazine compounds, and can meet the qualitative and quantitative analysis of pyrazine compounds in fermentation broth.

[0107] Table 5 Experimental results of recoveries of 8 pyrazine compounds

[0108]

[0109] Note: “ND” means not detected.

[0110] Comparative Example 1 Effect of chromatographic column on the determination results of pyrazine compounds

[0111] Take 1 mL of the standard solution (same as in Example 1) and dilute to 10 mL with 20% methanol solution. Thermo Accucore C18 column was used to detect and analyze 8 pyrazine compounds as a comparative method. The other determination conditions were the same as in Example 1. The specific results are shown in Figure 5 shown.

[0112] In the mixed pyrazine standard solution system, the chromatograms of the eight pyrazine compounds showed broad peaks and even tailing. The eight pyrazine compounds are all alkyl-substituted compounds, among which 2-ethylpyrazine and 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine and 2,3,5-trimethylpyrazine, 2,3-diethylpyrazine and tetramethylpyrazine and 2,3-dimethyl-5-ethylpyrazine are three groups of isomers. The retention times of isomers are very close, making it difficult to achieve effective separation. Through the selection of chromatographic columns, the best separation effect of the eight pyrazine compounds was finally obtained on the Thermo Hypersil Gold column, see Figure 1 .

[0113] Comparative Example 2 Effect of mobile phase on the determination results of pyrazine compounds

[0114] Take 1 mL of the standard solution (same as in Example 1) and dilute to 10 mL with 20% methanol solution. Use two different mobile phase systems from the present invention to detect and analyze eight pyrazine compounds as a comparative method:

[0115] (1) Phase A: 0.1% formic acid aqueous solution, Phase B: 0.1% acetonitrile aqueous solution;

[0116] (2) Phase A: pure aqueous solution, Phase B: 0.1% acetonitrile aqueous solution.

[0117] The remaining measurement conditions are the same as those in Example 1.

[0118] like Figure 6 and Figure 7 As shown in the figure, the use of the above two mobile phase systems for detection results in the 8 pyrazine compounds emitting peaks earlier. Although the peak shapes are good, the retention times are relatively close, making it difficult to completely and effectively separate them. Under the mobile phase system conditions of the method of the present invention, the 8 pyrazine compounds can obtain good separation effects, as shown in the figure. Figure 1 .

[0119] Comparative Example 3 Effect of elution conditions on the determination results of pyrazine compounds

[0120] Take 1 mL of the standard solution (same as in Example 1) and dilute to 10 mL with 20% methanol solution. Change the gradient elution conditions to detect and analyze 8 pyrazine compounds as a comparative method. The specific parameters are shown in Table 6. The other determination conditions are the same as in Example 1. Figure 8 and Fig. 9 As shown, it can be clearly seen that under the two comparative gradient elution conditions 1 and elution condition 2, the peak shapes of the eight pyrazine compounds are sharp and have a high response, but the peak elution times are relatively close, making it difficult to achieve effective separation.

[0121] Table 6 Specific parameters of mobile phase A under gradient elution conditions

[0122]

[0123] Comparative Example 4 Effect of extraction solvent on the determination results of pyrazine compounds

[0124] Take 2mL of fermentation broth (same as Example 2), centrifuge at 1,2000r / min for 5min, take the supernatant, use pure water, 20% methanol solution by volume and 20% ethanol solution by volume as dilution and extraction solvent, filter through 0.22μm microporous filter membrane, inject directly, and the other determination conditions are the same as Example 1. Using high concentration of extraction solvent to dilute the fermentation broth will produce turbidity and affect the initial mobile phase of analysis, so it is not considered. The results are shown in Table 7. The method of the present invention uses 20% methanol solution by volume as the extraction solvent, and the detection result of pyrazine compounds in the fermentation broth has the highest content and the best extraction effect.

[0125] Table 7 Detection results of pyrazine compound content in fermentation broth extracted with different solvents (μg / L)

[0126]

Claims

1. A method for determining pyrazine compounds in a fermentation broth, characterized in that: The following steps are involved: (1) Preparation of fermentation broth; (2) pretreatment of the fermentation liquid to obtain a sample to be tested; (3) Analyze the sample obtained in step (2) using ultra-high performance liquid chromatography-high resolution mass spectrometry.

2. The measuring method according to claim 1, characterized in that The fermentation broth is a bacterial broth containing pyrazine compounds; Preferably, the fermentation broth is a bacterial broth obtained by culturing a microorganism that produces pyrazine compounds; Preferably, the step of preparing the fermentation broth in step (1) comprises: fermenting the microorganisms by shaking culture to obtain the fermentation broth; Preferably, the shaking culture time is 10 to 100 hours; Preferably, the shaking culture time is 40 to 90 hours; Preferably, the rotation speed of the shaking culture is 10 r / min to 2000 r / min; Preferably, the rotation speed of the shaking culture is 50 r / min to 500 r / min; Preferably, the rotation speed of the shaking culture is 50 r / min to 300 r / min; Preferably, the shaking culture temperature is 15°C to 45°C; Preferably, the shaking culture temperature is 15°C to 40°C; Preferably, the medium for the shaking culture is a fermentation medium; Preferably, the fermentation medium contains 1.0 to 100.0 g / L peptone; Preferably, the fermentation medium contains 1.0 to 100.0 g / L diammonium hydrogen phosphate; Preferably, the fermentation medium contains 1.0-100.0 g / L yeast extract powder; Preferably, the fermentation medium contains 1.0 to 500.0 g / LD (+)-anhydrous glucose; Preferably, the microorganism is Bacillus licheniformis; Preferably, the microorganism is Bacillus licheniformis; Preferably, the microorganism is Bacillus licheniformis ATCC 14580.

3. The measuring method according to claim 1, characterized in that The fermentation broth pretreatment step includes: taking the fermentation broth, centrifuging, taking the supernatant, diluting it with a methanol solution and then filtering it; Preferably, the centrifugal speed is 5000-20000 r / min; Preferably, the centrifugal speed is 8000-15000 r / min; Preferably, the centrifugation time is 1 to 20 minutes; Preferably, the centrifugation time is 1 to 10 min; Preferably, the volume fraction of the methanol solution is 5 to 50%; Preferably, the volume fraction of the methanol solution is 10-40%; Preferably, the methanol solution is chromatography grade methanol.

4. The measuring method according to claim 1, characterized in that The pyrazine compounds include one or more of 2-methylpyrazine, 2-ethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2,3,5-trimethylpyrazine, 2,3-diethylpyrazine, tetramethylpyrazine and 2,3-dimethyl-5-ethylpyrazine.

5. The assay method according to claim 3, wherein The filter membrane used for the filtration is a 0.22 μm filter membrane; Preferably, the filter membrane is a microporous filter membrane.

6. The measuring method according to claim 1, characterized in that In the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the ultra-high performance liquid chromatography conditions are: chromatographic column: liquid chromatography column; column oven: 20-60°C; constant flow: column flow rate is 0.1-0.5mL / min; injection volume: 1-5μL; mobile phase A: formic acid aqueous solution, mobile phase B: formic acid-acetonitrile solution; gradient elution conditions: 0-1.0min, 93%A; 1.0-12.0min, 93%-87%A; 12.0-12.5min, 87%-0%A; 12.5-15.0min, 0%A; 15.0-15.5min, 0-93%A; 15.5~19.0min, 93%A; the volume percentage of mobile phase A refers to the volume percentage of mobile phase A in the mixture of mobile phase A and mobile phase B; Preferably, the ultra-high performance liquid chromatography conditions are as follows: chromatographic column: Thermo Hypersil Gold chromatographic column; column oven: 30-40°C; constant flow: column flow rate is 0.2-0.3 mL / min; injection volume: 1-2 μL; mobile phase A: 0.01-0.5% formic acid aqueous solution, mobile phase B: formic acid-acetonitrile (0.1-5:1000, V / V); gradient elution conditions: 0-1.0 min, 93% A; 1.0-12.0 min, 93%-87% A; 12.0-12.5 min, 87%-0% A; 12.5-15.0 min, 0% A; 15.0-15.5 min, 0-93% A; 15.5~19.0min, 93%A; Preferably, the ultra-high performance liquid chromatography conditions are: chromatographic column: Thermo Hypersil Gold, 150mm×2.1mm, 1.9μm; column oven: 35°C; constant flow: column flow rate is 0.3mL / min; injection volume: 2μL; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: formic acid-acetonitrile (1:1000, V / V); gradient elution conditions: 0-1.0min, 93%A; 1.0-12.0min, 93%-87%A; 12.0-12.5min, 87%-0%A; 12.5-15.0min, 0%A; 15.0-15.5min, 0-93%A; 15.5~19.0min, 93%A.

7. The measuring method according to claim 1, characterized in that In the ultra-high performance liquid chromatography-high resolution mass spectrometry method, the high resolution mass spectrometry conditions are: heated electrospray ionization source; sheath gas flow rate: 5-50arb; auxiliary gas flow rate: 5-50arb; spray voltage: 2.0-4.0kV; ion source temperature: 200-350°C; capillary temperature: 200-350°C; FullMS scanning positive ion scanning acquisition mode, resolution: 70,000; scanning range: 50-1000m / z; Preferably, the high-resolution mass spectrometry conditions are: heated electrospray ionization source; sheath gas flow: 30arb; auxiliary gas flow: 10arb; spray voltage: 2.0kV; ion source temperature: 350°C; capillary temperature: 300°C; positive ion scanning acquisition mode using Full MS scanning, resolution: 70,000; scanning range: 70-200m / z.

8. The assay method according to claim 1, wherein The qualitative analysis of the pyrazine compounds is performed by matching the retention time and accurate mass of the pyrazine standard and combining the secondary characteristic fragment ions for qualitative analysis.

9. The measuring method according to claim 1, characterized in that The method further comprises drawing a standard curve using a standard substance of the pyrazine compound, thereby calculating the content of the pyrazine compound in the sample to be tested.

10. Use of the determination method according to any one of claims 1 to 9 in detecting the type or content of pyrazine compounds in fermentation broth.

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