Detection method of tetramethylpyrazine

Abstract

The invention relates to the technical field of compound detection, in particular to a detection method of tetramethylpyrazine. The detection method comprises the step of detecting the content of tetramethylpyrazine in a to-be-detected sample by using an ion mobility spectrometry technology, wherein the to-be-detected sample is white spirit or distiller's yeast leach liquor. The detection method is accurate in detection and simple to operate, harmful solvents such as acetonitrile and a chromatographic column do not need to be used, and detection equipment is simplified.

Description

Technical Field

[0001] This invention relates to the field of compound detection technology, and more specifically, to a method for detecting tetramethylpyrazine. Background Technology

[0002] Tetramethylpyrazine (TTMP), also known as ligustrazine (CAS number 1124-11-4), is a distinctive nitrogen-containing heterocyclic compound in baijiu (Chinese white liquor) that contributes to both flavor and potential functionality. Its main contributions are nutty and roasted aromas, particularly prominent in traditional solid-state fermented baijiu such as soy sauce-flavored and strong-flavored varieties. It is a key component reflecting the style and quality of baijiu. Specifically, it contributes to both flavor and style shaping. (1) TTMP has unique aroma properties of nuts, roast, herbs and a slight soy sauce aroma. Its threshold content at 46% vol alcohol is 80073.16 μg / L. The high threshold makes it highly stable during the storage of baijiu and it is not easy for the flavor to be unbalanced due to volatilization. When a small amount is present, and the ratio of its concentration to the threshold is close to 1, the sensory intensity is low. It will not mask the dominant flavor, but will instead contribute a unique and complex layered flavor. In view of this, the present invention is proposed.

[0003] (2) Flavor function: As the main aroma auxiliary component: In Maotai-flavor liquor, it works synergistically with esters (such as ethyl hexanoate), phenols (such as 4-ethylguaiacol), and other pyrazines (such as 2,3,5-trimethylpyrazine) to enhance the style of "rich, elegant and delicate Maotai aroma". (3) Improve flavor harmony: neutralize the sweetness of some esters, alleviate the spiciness brought by high alcohol content, and enhance the "smooth and sweet aftertaste" of baijiu; (4) Enhance aroma persistence: TTMP has a high boiling point (190℃ ~ 193℃) and is not easily volatilized, which can prolong the time that the aroma of baijiu remains in the mouth and enhance the drinking experience.

[0004] It is evident that tetramethylpyrazine has a significant impact on the flavor of baijiu (Chinese liquor), therefore, its detection has a substantial influence on the evaluation of baijiu. Current technologies employ detection methods such as gas chromatography-fiber spectroscopy (GC-FID), gas chromatography-mass spectrometry (GC-MS / MS), high-performance liquid chromatography (HPLC-UV), ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), capillary electrophoresis (CE-UV), and nuclear magnetic resonance (NMR). However, these methods have several drawbacks, such as the use of toxic and harmful reagents like acetonitrile, ether, or methanol, or the need for chromatographic columns during the detection process, making the existing methods overly complex.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting tetramethylpyrazine. The detection method provided by the embodiments of this invention is accurate, simple to operate, and eliminates the need for harmful solvents such as acetonitrile and chromatographic columns, thus simplifying the detection equipment.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a method for detecting tetramethylpyrazine, comprising: detecting the content of tetramethylpyrazine in a sample to be tested using ion mobility spectrometry, wherein the sample to be tested is a liquor or a yeast extract.

[0008] In an optional embodiment, the liquor is liquor with an alcohol content of 50 degrees or higher (including 50 degrees).

[0009] In an optional embodiment, the liquor is either a sauce-flavored liquor or a light-flavored liquor.

[0010] In an optional embodiment, the step of forming the yeast extract includes: mixing 50-70% ethanol and yeast and ultrasonically extracting, with each 200ml of ethanol corresponding to 25-40g of yeast.

[0011] In an optional implementation, the sample to be tested is filtered before detection; Preferably, the method includes: distilling the yeast extract.

[0012] In an optional implementation, the conditions for ion mobility spectrometry include: an electrospray ion source voltage of 1600-2300V.

[0013] In an optional implementation, the conditions for ion mobility spectrometry also include: inlet temperature: 150-200°C; ion mobility tube temperature: 150-200°C.

[0014] In an optional implementation, the conditions for ion mobility spectrometry also include: positive ion mode, ion mobility tube voltage of 7000-9000V, ion gate width of 70-200µs, and ion gate voltage of 30-60V.

[0015] In an optional implementation, the conditions for ion mobility spectrometry further include: migration gas flow rate: 1.0-1.6 L / min; exhaust pump flow rate: 1.3-1.6 L / min; direct liquid injection flow rate: 0.8-1.3 μL / min.

[0016] In an optional embodiment, the limit of quantitation for tetramethylpyrazine is 0.5 mg / L, and the maximum detectable concentration for tetramethylpyrazine is 11.0 mg / L.

[0017] The present invention has the following advantages: The embodiments of the present invention employ ion mobility spectrometry to directly and rapidly detect the content of tetramethylpyrazine. This detection method does not use toxic or harmful reagents such as acetonitrile, diethyl ether, or methanol, thus improving the safety of the detection process. Furthermore, the detection process does not require chromatographic columns or other equipment, making the detection equipment simpler and simplifying the detection procedure. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the structure of the ion mobility spectrometry technology provided in an embodiment of the present invention; Figure 2 This is a standard curve diagram of tetramethylpyrazine provided in Example 1 of the present invention; Figure 3 The image shows the detection results of the sample to be tested provided in Embodiment 1 of the present invention. Figure 4 This is a graph showing the detection results of the sample to be tested provided in Embodiment 2 of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] In a first aspect, embodiments of the present invention provide a method for detecting tetramethylpyrazine, comprising: S1. Sample preparation; The sample tested in this embodiment of the invention (i.e., the sample to be tested) can be baijiu (Chinese white liquor) or koji extract. The baijiu is defined as baijiu with an alcohol content of 50% or higher (including 50%), such as sauce-aroma baijiu or light-aroma baijiu.

[0022] In the process of making baijiu, the higher the solid-state fermentation temperature, the longer the fermentation cycle, and the longer the storage time, the higher the TTMP content. The highest TTMP content is found in sauce-aroma baijiu, ranging from 1.00 to 5.00 mg / L (some high-end baijiu can reach over 8.0 mg / L), serving as an auxiliary component of the core flavor and highlighting the sauce aroma characteristics. Next is strong-aroma baijiu, with TTMP content ranging from 0.10 to 1.00 mg / L, playing a role in harmonizing flavors and enhancing aroma complexity. The content is relatively low in light-aroma baijiu, ranging from 0.05 to 0.30 mg / L, falling into the trace level, but enhancing the complexity of the aroma.

[0023] Specifically, the process of making baijiu is as follows: The precursors are amino acids (such as valine and leucine), sugars (glucose and maltose), and alkaloids from raw materials such as sorghum and wheat. Preferably, sorghum and wheat, which have high protein content, are selected as raw materials to increase the reserve of precursor substances.

[0024] Then, solid-state fermentation is carried out. During solid-state fermentation, Bacillus (such as Bacillus subtilis and Bacillus licheniformis) and yeast (such as Candida albicans) synthesize TTMP through "amino acid decarboxylation, reductive amination, and cyclization reactions". During solid-state fermentation, the fermentation temperature of the koji blocks can be appropriately increased (55℃~65℃) to screen for microbial strains that produce high levels of TTMP (such as Bacillus isolated from old koji).

[0025] Meanwhile, high-temperature koji making (such as 60℃~65℃ for sauce-flavored baijiu), long-term fermentation (1 year), and long-term storage (3 years or more) (promoting Maillard reaction and secondary microbial metabolism) can all significantly increase TTMP content. Ideally, extending the fermentation cycle in the fermentation pit (≥180 days), maintaining a micro-anaerobic and high-humidity environment within the pit, and promoting microbial metabolism are also beneficial. Furthermore, using earthenware jars for storage, controlling the storage temperature (15℃~25℃) and humidity (60%~70%), enhances TTMP content and flavor harmony through the aging process.

[0026] It should be noted that the liquor provided in this embodiment of the invention is liquor that can be purchased on the market.

[0027] Furthermore, filtering the liquor before testing removes particulate matter and high-boiling-point substances from the sample that may damage the instrument, interfere with testing, or affect the stability of the results. This avoids wear and contamination of core components, extends the instrument's lifespan, reduces maintenance costs, and also reduces the generation of random interfering ions from unstable components, which can cause signal fluctuations and ensure the stability of subsequent tests.

[0028] Furthermore, the preparation of the yeast extract includes: mixing 50-70% ethanol and yeast and ultrasonically extracting, with 25-40g of yeast corresponding to every 200ml of ethanol.

[0029] In addition, the yeast extract was distilled and filtered before testing to ensure the accuracy of the test results.

[0030] S2, Detection; Tetramethylpyrazine was detected using ion mobility spectrometry (IMS) to obtain a standard curve. Specifically, tetramethylpyrazine standard was dissolved in 50-70% ethanol and serially diluted to prepare solutions of 1.04, 2.08, 5.20, and 10.40 mg / L for detection, followed by calibration of the detection curve.

[0031] The principle of ion mobility spectrometry is as follows: a liquid sample is mixed with a solvent and introduced into the system for ionization. The resulting ion-solvent clusters then enter the desolvation region. Ions selected by an ion-gated pulse are accelerated in an electric field of 8-10 kV. During acceleration, the ions collide with drifting gases (usually air or N2) along their path. Finally, these ions strike a Faraday detector, and the charge sensed by the detector is converted into a signal intensity in volts. (See schematic diagram below.) Figure 1 .

[0032] The S1-treated sample was analyzed using ion mobility spectrometry (IMS). The IMS conditions included: an electrospray ion source voltage of 1600-2300V (e.g., any value between 1600V, 1800V, 1900V, 2000V, 2100V, 2200V, 2300V, etc.); an inlet temperature of 150-200℃; and an ion migration tube temperature of 150-200℃ (e.g., any value between 150℃, 160℃, 170℃, 180℃, 190℃, 200℃, etc.). Ion migration tube voltage: 7000-9000V; for example, any value between 7000V, 7500V, 8000V, 8500V, 9000V, etc.; ion gate width: 70-200µs; for example, any value between 70µs, 80µs, 90µs, 100µs, 110µs, 120µs, 130µs, 140µs, etc.; ion gate voltage: 30-60V; for example, any value between 30V, 35V, 40V, 45V, 50V, 55V, 60V, etc.; migration gas flow rate: 1.0-1.6 L / min; for example, any value between 1.3-1.5 L / min, such as 1.3 L / min, 1.4 L / min, 1.5 L / min, etc. Exhaust pump flow rate: 1.3-1.6 L / min; for example, any value between 1.3-1.6 L / min such as 1.3 L / min, 1.4 L / min, 1.5 L / min, 1.6 L / min, etc. Direct liquid injection flow rate: 0.8-1.3 μL / min; for example, any value between 0.8 μL / min, 0.9 μL / min, 1.0 μL / min, 1.1 μL / min, 1.2 μL / min, 1.3 μL / min, etc. Spectrum length: 20-35 ms; Acquisition time: 25-35 s.

[0033] Furthermore, the limit of quantitation (LOQ) for tetramethylpyrazine is 0.5 mg / L, and the maximum detectable concentration is 11.0 mg / L. Above this concentration, a supersaturation peak will appear, making it impossible to detect the specific concentration. Therefore, near this concentration, dilution should be performed for secondary confirmation, with the minimum dilution factor being the optimal choice for quantification.

[0034] In summary, the detection method provided by the present invention has the following advantages: (1) On the one hand, it saves a lot of time in preparing reagents, and on the other hand, it does not require toxic and harmful organic reagents such as acetonitrile, ether, and methanol. The detection method is more environmentally friendly and more beneficial to human health.

[0035] (2) The analysis speed is extremely fast, far exceeding GC and LC. IMS does not require column equilibration and gradient elution. The ion migration separation time is only in the second range (0.1~10.0 s / sample), and the detection and analysis cycle is only 1 min. In contrast, the GC analysis cycle is usually in the minute range (10~60 min / sample), and the LC analysis cycle is 20~90 min / sample (including mobile phase equilibration and gradient elution).

[0036] (3) Sample pretreatment is simple and the matrix is ​​more resistant. Baijiu samples only need simple filtration (to remove particulate matter), and the sample can be detected by transferring the filtered sample with the injection needle. There is no need for the complex derivatization (partial components) of GC (gas chromatograph) and the deep purification of solid phase extraction (SPE) of LC (liquid chromatograph). GC requires a high alcohol matrix, which can easily contaminate the chromatographic column, so it is necessary to split or dilute (reduce detection sensitivity) before injection. LC will have a decrease in column efficiency due to the high concentration of ethanol and large molecular impurities in baijiu, so strict purification is required. Compared with GC and LC, IMS saves a lot of time and consumable costs, and is green and low-carbon.

[0037] (4) IMS operates at atmospheric pressure and does not require a high-pressure carrier gas system, long chromatographic column and complex temperature control module of GC.

[0038] (5) Low operating and maintenance costs and lower entry barrier. IMS is a portable, miniaturized (desktop / portable) and lightweight (weighing only a few kilograms) device, while GC is mostly a large device weighing tens of kilograms. It does not require the carrier gas cylinder of GC (saving gas procurement costs) and the mobile phase of LC (methanol / acetonitrile and other organic solvents), only an air generator is needed to provide the carrier gas source. Maintenance only requires periodic cleaning of the ionization source and migration tube; while GC requires continuous procurement of high-purity nitrogen, and the cost of replacing the chromatographic column is as high as several thousand yuan per column; LC consumes a large amount of organic reagents such as acetonitrile and methanol, and the chromatographic column is also contaminated by the large molecular impurities in the liquor matrix that easily clog the column bed.

[0039] The equipment and reagents used in the detection of this invention are as follows: Instrumentation: GA2200 ESI-HPIMS ion mobility spectrometer, Suzhou Yuezhi Biotechnology Co., Ltd.; Vislon Analysis software, version 2.3.1.23, Excellims, USA; LA230S electronic balance, Sartorius; DIRECT 8 ultrapure water system, Merck, Germany. Tetramethylpyrazine standard, 98% purity, was purchased from Merck AG, Germany; anhydrous ethanol (chromatographic grade), >99.9% purity, was purchased from Beijing Dicoma Technology Co., Ltd. The test water was prepared using a DIRECT 8 pure water system.

[0040] The starter culture is prepared using existing methods, which will not be detailed in the embodiments of this invention. Specifically, it includes low-temperature starter culture and bran starter culture. Low-temperature starter culture is prepared using barley and peas, with the maximum temperature controlled at 40-50℃ during the starter culture preparation process, such as stubble starter culture, post-fire starter culture, and red heart starter culture. Bran starter culture is prepared using wheat bran and peas as raw materials, inoculated with pure microorganisms, with the maximum temperature controlled at 37-50℃ during the starter culture preparation process.

[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0042] Example 1 This invention provides a method for detecting tetramethylpyrazine, comprising: 10.4 mg of tetramethylpyrazine standard (purchased from Merck, Germany) was dissolved in 10 mL of 60% ethanol and then filtered to form the standard solution. 20 μL of the standard solution was transferred using a syringe needle and analyzed in an ion mobility spectrometer (GA2200ESI-HPIMS).

[0043] The detection conditions were as follows: In positive ion mode, the spectrum length was 25 ms; the acquisition time was 30 s; the ion gate width was 130 μs; the ion gate voltage was 40 V; the ion migration tube voltage was 8000 V; the electrospray ion source voltage was 1900 V; the inlet temperature was 180 °C; the ion migration tube temperature was 180 °C; the migration gas flow rate was 1.50 L / min; the exhaust pump flow rate was 1.45 L / min; and the direct liquid injection flow rate was 1.00 μL / min.

[0044] The standard curve for this tetramethylpyrazine was obtained; the results are shown in [link to standard curve]. Figure 2 .

[0045] A 63-degree light-aroma baijiu (self-brewed) was filtered to obtain the filtrate. 20 μL of the filtrate was transferred using a syringe and analyzed using an ion mobility spectrometer (GA2200 ESI-HPIMS). The detection conditions were the same as above. Results are shown below. Figure 3 .

[0046] according to Figure 2 and Figure 3 It can be seen that the tetramethylpyrazine content of the light-aroma baijiu No. 3 is 4.97 mg / L.

[0047] Example 2 Mix 200ml of 60% ethanol and 25g of yeast (self-developed) and extract by ultrasonication for 30min. Then, distill the yeast extract to obtain a distillate. Finally, filter the distillate.

[0048] 20 μL of the filtrate was transferred using a syringe and analyzed in an ion mobility spectrometer (GA2200 ESI-HPIMS). The detection conditions were the same as in Example 1. Results are shown below. Figure 4 .

[0049] according to Figure 2 and Figure 4 It can be seen that the tetramethylpyrazine content in the 17# yeast extract was 1.62 mg / L.

[0050] Validity of test data The ion mobility spectrometry detection technique provided in this embodiment of the invention was used to detect the same sample, and the detection data were then compared. The results are as follows:

[0051]

[0052] Based on the above results, it can be seen that ion mobility spectrometry can obtain tetramethylpyrazine data within 1 minute, which is comparable to that obtained by GC detection over nearly an hour, with a relative deviation within ±20%, indicating that the data is reliable.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting tetramethylpyrazine, characterized in that, include: The content of tetramethylpyrazine in the test sample was detected by ion mobility spectrometry. The test sample was baijiu (Chinese liquor) or koji extract.

2. The detection method according to claim 1, characterized in that, The liquor in question is liquor with an alcohol content of 50 degrees or higher.

3. The detection method according to claim 1, characterized in that, The liquor in question is either a sauce-flavored liquor or a light-flavored liquor.

4. The detection method according to claim 1, characterized in that, The steps for forming the yeast extract include: mixing 50-70% ethanol and yeast and extracting by ultrasonication, with 25-40g of yeast corresponding to every 200ml of ethanol.

5. The detection method according to claim 4, characterized in that, The sample to be tested is filtered before detection; Preferably, the method includes: distilling the yeast extract.

6. The detection method according to claim 1, characterized in that, The conditions for ion mobility spectrometry include: an electrospray ion source voltage of 1600-2300V.

7. The detection method according to claim 6, characterized in that, The conditions for ion mobility spectrometry also include: inlet temperature: 150-200℃; ion mobility tube temperature: 150-200℃.

8. The detection method according to claim 7, characterized in that, The conditions for ion mobility spectrometry also include: ion mobility tube voltage 7000-9000V; ion gate width 70-200µs; ion gate voltage 30-60V.

9. The detection method according to claim 8, characterized in that, The conditions for ion mobility spectrometry also include: migration gas flow rate: 1.0-1.6 L / min; exhaust pump flow rate: 1.3-1.6 L / min; direct liquid injection flow rate: 0.8-1.3 μL / min.

10. The detection method according to claim 1, characterized in that, The limit of quantitation for tetramethylpyrazine is 0.5 mg / L, and the maximum detectable concentration for tetramethylpyrazine is 11.0 mg / L.

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