A method for identifying Daqu

Abstract

The present application relates to the technical field of food microbial detection, and specifically to a method for identifying Daqu, comprising the following steps: (1) weighing a Daqu sample and filtering the Daqu sample to obtain a filtrate; (2) measuring the filtrate, adding magnetic beads to the filtrate, and incubating the filtrate to obtain an incubation solution; (3) removing the magnetic beads from the incubation solution and lysing the microorganisms on the magnetic beads to obtain a sample to be tested; (4) detecting the sample to be tested using matrix-assisted laser desorption ionization time-of-flight mass spectrometry; and identifying the type of Daqu based on characteristic peaks in the mass spectrum of the sample to be tested. The present application has the advantages of accurate detection results and high detection efficiency.

Description

Technical Field

[0001] The present application relates to the technical field of food microbial detection, and in particular to a method for identifying Daqu. Background Art

[0002] Maotai-flavor baijiu (Chinese liquor) is one of the major flavor types in my country, characterized by a full-bodied liquor body, a prominent Maotai aroma, a lingering aroma, and a long aftertaste. This unique flavor is closely related to its unique brewing process, which is imbued with a traditional charm. The high-temperature daqu (Chinese koji) fermented from wheat contains a variety of bacterial strains and enzymes. These enzymes play a crucial role in the production of Maotai-flavor baijiu, including inoculum, saccharification, fermentation, aroma production, and providing nutrient substrates. This crucial role is directly related to yield, flavor, and quality, making it the very soul of baijiu. During the preparation of Maotai-flavor daqu, variations in temperature, humidity, and atmospheric composition at different locations within the daqu storage warehouse lead to the production of daqu of varying degrees of fermentation. In actual production, daqu of varying degrees of fermentation are typically categorized by color as white, yellow, or black. Identifying the type of daqu used during the winemaking process is often necessary, necessitating the development of a method that can quickly and accurately identify the type of daqu.

[0003] Existing methods for identifying daqu types rely on data analysis based on its flavor components, combined with discriminant analysis. However, due to subtle differences in flavor components during fermentation, effective identification is often difficult. Summary of the Invention

[0004] In order to solve the above technical problems, the present application provides a Daqu identification method.

[0005] The present application provides a method for identifying Daqu using the following technical solutions:

[0006] A method for identifying Daqu comprises the following steps:

[0007] (1) Weighing a Daqu sample, filtering the Daqu sample to obtain a filtrate;

[0008] (2) measuring the filtrate, adding magnetic beads to the filtrate, and incubating to obtain an incubation solution;

[0009] (3) removing the magnetic beads from the incubation solution and lysing the microorganisms on the magnetic beads to obtain a sample to be tested;

[0010] (4) Detecting the sample to be tested by matrix-assisted laser desorption ionization time-of-flight mass spectrometry; and identifying the type of Daqu based on characteristic peaks in the mass spectrum of the sample to be tested.

[0011] Preferably, in the step (4), the matrix-assisted laser desorption ionization time-of-flight mass spectrometry is set to a linear positive ion mode to collect mass spectrometry signals with a mass-to-charge ratio in the range of 2000-20000 Daltons; the identification of the Daqu category according to the characteristic peaks in the mass spectrum of the sample to be tested is as follows: when the maximum intensity characteristic peak in the mass spectrum of the sample to be tested is near 4545m / z, the Daqu is black qu; when the maximum intensity characteristic peak in the mass spectrum of the sample to be tested is near 4916m / z, the Daqu is white qu; when the maximum intensity characteristic peak in the mass spectrum of the sample to be tested is near 4541m / z, and the larger intensity characteristic peak is near 3840m / z, the Daqu is yellow qu.

[0012] Preferably, in step (1), the filtering treatment of the Daqu sample includes: adding Tris-HCl buffer to the Daqu sample, mixing and filtering; the mass of the weighed Daqu sample is 1 to 3 g, and the volume of the added Tris-HCl buffer is 4 to 6 mL; preferably, the mass of the weighed Daqu sample is 2 g, and the volume of the added Tris-HCl buffer is 5 mL.

[0013] Preferably, in step (2), adding magnetic beads to the filtrate comprises: adding Tris-HCl buffer and magnetic beads to the filtrate; the volume of the filtrate measured is 150 μL to 250 μL, and the sum of the volumes of the Tris-HCl buffer and the filtrate is 1 mL; preferably, the volume of the filtrate measured is 200 μL.

[0014] Preferably, in step (2), the magnetic beads are prepared by co-incubating magnetic particles modified with mannose lectin and streptavidin.

[0015] Preferably, in step (2), the amount of magnetic beads added is 15-25 μL; preferably, the amount of magnetic beads added is 20 μL.

[0016] Preferably, the average particle size of the magnetic beads is 250 nm to 500 nm; preferably, the average particle size of the magnetic beads is 500 nm.

[0017] Preferably, in step (2), the incubation time is greater than or equal to 30 min; preferably, the incubation time is 30 min.

[0018] Preferably, in step (3), the lysis treatment includes: adding formic acid solution to the magnetic beads for lysis to obtain a lysate, and then adding acetonitrile to the lysate, the volume of the formic acid solution added is 4 to 6 μL, and the lysis time is greater than or equal to 5 minutes; preferably, the volume of the formic acid solution added is 5 μL, and the lysis time is 5 minutes.

[0019] Preferably, the volume of acetonitrile added to the lysate is 4-6 μL; preferably, the volume of acetonitrile added to the lysate is 5 μL.

[0020] This application has the following beneficial technical effects:

[0021] This application enriches the microorganisms in Daqu, then performs a pyrolysis treatment, uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect the pyrolysis substances, and judges whether the detected Daqu belongs to black qu, yellow qu or white qu based on the position of the characteristic peak with the largest intensity in the mass spectrum. This can more quickly and accurately identify the type of Daqu, thereby improving the brewing quality of liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the mass spectrum of the first detection of white koji in Example 1;

[0023] Figure 2 This is the mass spectrum of the first detection of black koji in Example 1;

[0024] Figure 3 This is the mass spectrum of the first detection of yellow koji in Example 1;

[0025] Figure 4 This is the mass spectrum of the second detection of white koji in Example 1;

[0026] Figure 5 This is the mass spectrum of the second detection of black koji in Example 1;

[0027] Figure 6 This is the mass spectrum of the second detection of yellow koji in Example 1;

[0028] Figure 7 is the mass spectrum of the white koji detected in Example 2;

[0029] Figure 8 is the mass spectrum of black koji detected in Example 2;

[0030] Figure 9 is the mass spectrum of yellow koji detected in Example 2;

[0031] Figure 10 is the mass spectrum of the white koji detected in Example 3;

[0032] Figure 11 is the mass spectrum of black koji detected in Example 3;

[0033] Figure 12 is the mass spectrum of yellow koji detected in Example 3;

[0034] Figure 13 This is the mass spectrum of the first detection of white koji in comparative example 1;

[0035] Figure 14 This is the mass spectrum of the first detection of black koji in Comparative Example 1;

[0036] Figure 15 This is the mass spectrum of the first detection of yellow koji in Comparative Example 1;

[0037] Figure 16 This is the mass spectrum of the second detection of Baiqu in Comparative Example 1;

[0038] Figure 17 This is the mass spectrum of the second detection of black koji in Comparative Example 1;

[0039] Figure 18 This is the mass spectrum of the second detection of yellow koji in Comparative Example 1. DETAILED DESCRIPTION

[0040] The present application is further described below with reference to the accompanying drawings and examples.

[0041] Reagents: Tris-HCl buffer (concentration of 50 mmol / L), formic acid solution (concentration of 70%), acetonitrile (acetonitrile is pure acetonitrile).

[0042] Instrument: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, model: M-Discover 100Excellence; manufacturer: Zhuhai Meihua Medical Technology Co., Ltd.

[0043] The present application provides a method for identifying Daqu, comprising the following steps:

[0044] (1) Weigh a Daqu sample, filter the Daqu sample, and obtain a filtrate.

[0045] Specifically, after the Daqu block is crushed, 1 to 3 g of the Daqu sample is weighed and placed in a 50 mL centrifuge tube, 4 to 6 mL of Tris-HCl buffer is added to the Daqu sample, and after vigorous shaking for 3 minutes with an oscillator to fully mix it, a double layer of medical gauze is used for suction filtration to obtain a filtrate. In this application, the Daqu sample is specifically selected to be 2 g, and the Tris-HCl buffer is specifically selected to be 5 mL. Tris-HCl buffer is tris(hydroxymethyl)aminomethane hydrochloride, and the cation in this application is Ca 2+ The pH value can be arbitrarily selected from 5 to 9. In this application, the specific pH value is 7.4.

[0046] (2) Measure the filtrate, add magnetic beads to the filtrate, and incubate to obtain an incubation solution.

[0047] Specifically, 150 μL to 250 μL of the filtrate in step (1) is measured, Tris-HCl buffer and 15 to 25 μL of magnetic beads are added to the filtrate, and the mixture is shaken and incubated for more than 30 minutes to obtain an incubation solution. At this time, the microorganisms in the Daqu are enriched on the surface of the magnetic beads. When the magnetic beads are less than 15 μL, it is not conducive to the enrichment of Daqu microorganisms, and an accurate and representative Daqu mass spectrum cannot be obtained; when the magnetic beads are higher than 25 μL, the amount of magnetic beads is too much, resulting in waste of magnetic beads and increasing the detection cost. The volume of the filtrate selected for measurement in this application is 200 μL, the sum of the volume of Tris-HCl buffer added and the volume of the filtrate is 1 mL, the volume of the magnetic beads selected for addition is 20 μL, and the incubation time is selected to be 30 minutes. An incubation time of less than 30 minutes will result in fewer characteristic peaks of the mass spectrum detected, and the mass spectrum cannot be effectively used for Daqu type identification. The magnetic beads in this application are prepared by co-incubating magnetic particles modified with mannose lectin and streptavidin (Fc-MBL). The average particle size of the magnetic beads is 250nm to 500nm, and the average particle size of the magnetic beads in this application is specifically selected to be 500nm. The particle size of 500nm is within the particle size range of universal microorganisms and is therefore more suitable for capturing microorganisms, that is, more conducive to microbial enrichment. When the particle size of the magnetic beads is less than 250nm or greater than 500nm, the capture effect of the microorganisms will be reduced, thereby reducing the microbial enrichment effect.

[0048] (3) removing the magnetic beads from the incubation solution and performing a lysis treatment on the microorganisms on the magnetic beads to obtain a sample to be tested.

[0049] Specifically, use a magnet to take out the magnetic beads from the incubation solution, wash the magnetic beads three times with 1 mL of sterile water each time, and then use a magnet to adsorb the magnetic beads to separate the magnetic beads from the washing liquid. Then add 4 to 6 μL of formic acid solution to the magnetic beads for lysis for more than 5 minutes, use a magnet to adsorb and separate the magnetic beads to obtain a lysate, add 4 to 6 μL of acetonitrile to the lysate, and mix well to obtain a sample to be tested. In this application, the volume of formic acid solution is specifically selected to be 5 μL, and the lysis time is specifically selected to be 5 minutes. If the lysis time is less than 5 minutes, the enriched Daqu microorganisms may not be completely lysed, resulting in a low mass spectrometry detection signal and an inability to effectively identify the type of Daqu. The volume of acetonitrile is specifically selected to be 5 μL.

[0050] (4) Detecting the sample to be tested by matrix-assisted laser desorption ionization time-of-flight mass spectrometry; and identifying the type of Daqu based on characteristic peaks in the mass spectrum of the sample to be tested.

[0051] Specifically, 1 μL of the sample to be tested was taken as a target, and after natural drying, it was covered with an equal volume of α-cyano-4-hydroxycinnamic acid (CHCA) matrix. After the matrix was dried, matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used for detection. The instrument laser intensity was set to 25%, the cumulative number of laser shots was 180, and Escherichia coli (E. coli ATCC 8739) was used as the quality control bacteria. Mass spectrometric signals with a mass-to-charge ratio range of 2000-20000 Daltons were collected in linear positive ion mode, and the type of Daqu was identified according to the position of the characteristic peaks in the mass spectrum.

[0052] This application enriches the microorganisms in Daqu, then performs a cracking treatment, uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect the cracked substances, and judges whether the detected Daqu belongs to black qu, yellow qu or white qu based on the position of the characteristic peak with the largest intensity in the mass spectrum. It can more quickly and accurately identify the type of Daqu, thereby improving the brewing quality of liquor. In the case of characteristic spectral signals, the classification boundaries between Daqu with different fermentation degrees are clearer. In the actual production process, the mixing ratio of different types of Daqu can be evaluated and adjusted more objectively and accurately, which is conducive to the optimization of the later brewing process.

[0053] Example 1

[0054] A method for identifying Daqu comprises the following steps:

[0055] (1) After crushing the Daqu block, weigh 2 g of Daqu sample and put it into a 50 mL centrifuge tube. Add 5 mL of Tris-HCl buffer to the Daqu sample, shake it vigorously for 3 minutes to fully mix it, and then filter it with double-layer medical gauze to obtain a filtrate.

[0056] (2) Measure 200 μL of the filtrate in step (1), add Tris-HCl buffer and 20 μL of magnetic beads to the filtrate, and incubate at 1000 rpm for 30 minutes to obtain an incubation solution. At this time, the microorganisms in the Daqu are enriched on the surface of the magnetic beads.

[0057] (3) Wash the magnetic beads three times with 1 mL of sterile water each time, then use a magnet to adsorb the magnetic beads and separate them from the liquid. Then add 5 μL of formic acid solution to the magnetic beads and lyse them for 5 minutes. Use a magnet to adsorb and separate the magnetic beads to obtain a lysate. Add 5 μL of acetonitrile to the lysate and mix thoroughly to obtain the sample to be tested.

[0058] (4) Take 1 μL of the sample to be tested and spot it on the target. After it is naturally dried, cover it with an equal volume of CHCA matrix. After the matrix is ​​dried, matrix-assisted laser desorption ionization time-of-flight mass spectrometry is used for detection. The laser intensity of the instrument is set to 25%, the cumulative number of laser shots is 180, and Escherichia coli (E. coli ATCC 8739) is used as the quality control bacteria. Mass spectrometry signals with a mass-to-charge ratio of 2000-20000 Daltons are collected in the linear positive ion mode. The type of unknown Daqu is identified based on the position of the maximum intensity characteristic peak of the mass spectrum.

[0059] The black koji, white koji and yellow koji of known daqu types were detected according to Example 1. Specifically, the same daqu was subjected to two mass spectrometry tests according to the above method. The mass spectra were as follows: Figures 1 to 6 As shown, Figures 1 to 3 These are the mass spectra of white, black, and yellow koji in the first test; Figures 4-6 The mass spectra of white koji, black koji and yellow koji in the second test are shown in Figure 2. Figures 1 to 6 It can be seen that the maximum intensity characteristic peak in the black koji sample is around 4545m / z; the maximum intensity characteristic peak in the white koji sample is around 4916m / z; the maximum intensity characteristic peak in the yellow koji sample is around 4541m / z, and the larger intensity characteristic peak is around 3840m / z.

[0060] When detecting a sample of unknown Daqu type, the detection is performed according to steps (1) to (4), and the type of the unknown Daqu is identified based on the position of the maximum intensity characteristic peak of the mass spectrum. When the maximum intensity characteristic peak in the mass spectrum of the unknown type Daqu sample is detected to be near 4545m / z, the Daqu is black qu; when the maximum intensity characteristic peak in the mass spectrum of the unknown type Daqu sample is detected to be near 4916m / z, the Daqu sample is white qu; when the maximum intensity characteristic peak in the mass spectrum of the unknown type Daqu sample is detected to be near 4541m / z, and the larger intensity characteristic peak is near 3840m / z, the Daqu sample is yellow qu.

[0061] Example 2

[0062] The difference between Example 2 and Example 1 is that in Example 2, Fc-MBL magnetic beads are replaced with carboxyl magnetic beads, the concentration of the carboxyl magnetic beads is 10 mg / mL, and the average particle size is 500 nm.

[0063] The black, white and yellow koji of known Daqu types were detected according to the detection method in Example 2, and the mass spectra were as follows: Figures 7-9 As shown, Figure 7 is the mass spectrum of Baiqu in Example 2, Figure 8 is the mass spectrum of the black song in Example 2, Figure 9 It is the mass spectrum of yellow song in Example 2.

[0064] Example 3

[0065] The difference between Example 3 and Example 1 is that in Example 3, Fc-MBL magnetic beads are replaced with hydroxy magnetic beads, the concentration of the hydroxy magnetic beads is 10 mg / mL, and the average particle size is 500 nm.

[0066] The black, white and yellow koji of known daqu types were detected according to the detection method in Example 3, and the mass spectra were as follows: Figures 10-12 As shown, Figure 10 is the mass spectrum of Baiqu in Example 3, Figure 11 is the mass spectrum of the black koji in Example 3, Figure 12 It is the mass spectrum of yellow song in Example 3.

[0067] Combined with Examples 1 to 3 and Figures 1 to 12 As shown in the figure, it can be seen that although the effect of Fc-MBL magnetic beads in the identification of white koji and black koji is similar to that of carboxyl magnetic beads and hydroxyl magnetic beads, Fc-MBL magnetic beads can obtain more and concentrated characteristic peaks in the identification of yellow koji. This shows that the microorganisms in yellow koji are more easily enriched by Fc-MBL magnetic beads, and more characteristic peaks can be obtained after the microorganisms are lysed. This also shows that the use of Fc-MBL magnetic beads in Example 1 of the present application to enrich the microorganisms in Daqu has a better effect and is more conducive to identifying the type of Daqu.

[0068] Comparative Example 1

[0069] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not use Fc-MBL magnetic beads to enrich the microorganisms in Daqu.

[0070] Specifically, (1) after crushing the Daqu block, 2 g of the Daqu sample was weighed and placed in a 50 mL centrifuge tube, 5 mL of Tris-HCl buffer was added to the Daqu sample, and the mixture was vigorously shaken for 3 min to fully mix it, and then filtered using a double-layer medical gauze to obtain a filtrate.

[0071] (2) Take 5 μL of the filtrate obtained in step (1), add 5 μL of formic acid solution (70%) to the filtrate, mix well, then add 5 μL of acetonitrile solution, mix again to obtain the sample to be tested.

[0072] (3) Take 1 μL of the sample to be tested and spot it on the target. After it is naturally dried, cover it with an equal volume of CHCA matrix. After the matrix is ​​dried, matrix-assisted laser desorption ionization time-of-flight mass spectrometry is used for detection. Mass spectrometric signals with a mass-to-charge ratio in the range of 2000-20000 Daltons are collected in the linear positive ion mode. The type of unknown Daqu is identified based on the position of the maximum intensity characteristic peak of the mass spectrum.

[0073] The black, white and yellow koji of known Daqu types were tested according to Comparative Example 1. Specifically, the same Daqu was subjected to two mass spectrometry tests according to the above method. The mass spectra were as follows: Figures 13-18 As shown, Figures 13-15 These are the mass spectra of white, black, and yellow koji in the first test; Figures 16-18 The mass spectra of white koji, black koji and yellow koji in the second test are shown in Figure 2. Figures 13-18 It can be seen that the characteristic peaks of different Daqu are not obvious when they are not enriched with magnetic beads. The characteristic peaks interfere with each other, the difference in the intensity of the characteristic peaks is small, the classification boundaries of different types of Daqu are not clear, and different types of Daqu cannot be distinguished based on the position of the characteristic peaks.

[0074] Validation of Daqu identification method

[0075] Fifteen manually tested Daqu blocks of known Daqu types were tested according to the method of Example 1. The known Daqu were identified based on the position of the maximum intensity characteristic peak in the mass spectrum. The identification results are shown in Table 1.

[0076] Table 1 Daqu identification verification results

[0077] Number of test blocks (blocks) Accuracy (%) 15 93

[0078] This application enriches the microorganisms in Daqu, then performs a cracking treatment, uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect the cracked substances, and judges whether the detected Daqu belongs to black qu, yellow qu or white qu based on the position of the characteristic peak with the largest intensity in the mass spectrum. It can more quickly and accurately identify the type of Daqu, thereby improving the brewing quality of liquor. In the case of characteristic spectral signals, the classification boundaries between Daqu with different fermentation degrees are clearer. In the actual production process, the mixing ratio of different types of Daqu can be evaluated and adjusted more objectively and accurately, which is conducive to the optimization of the later brewing process.

[0079] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for identifying Daqu, characterized in that: The following steps are involved: (1) Weighing a Daqu sample, filtering the Daqu sample to obtain a filtrate; (2) measuring the filtrate, adding magnetic beads to the filtrate, and incubating to obtain an incubation solution; the magnetic beads are prepared by incubating magnetic particles modified with mannose lectin and streptavidin; the amount of the added magnetic beads is 15-25 μL; the average particle size of the magnetic beads is 250 nm to 500 nm; and the incubation time is greater than or equal to 30 min; (3) removing the magnetic beads from the incubation solution and performing a lysis treatment on the microorganisms on the magnetic beads to obtain a sample to be tested; the lysis treatment comprises: adding a formic acid solution to the magnetic beads for lysis to obtain a lysis solution, and then adding acetonitrile to the lysis solution; the volume of the formic acid solution added is 4 to 6 μL, and the lysis treatment time is greater than or equal to 5 minutes; (4) Detecting the sample to be tested by matrix-assisted laser desorption ionization time-of-flight mass spectrometry; identifying the type of Daqu according to the characteristic peaks in the mass spectrum of the sample to be tested; detecting the sample to be tested by matrix-assisted laser desorption ionization time-of-flight mass spectrometry includes: setting the matrix-assisted laser desorption ionization time-of-flight mass spectrometry to a linear positive ion mode, collecting mass spectrometry signals with a mass-to-charge ratio in the range of 2000-20000 Daltons; identifying the type of Daqu according to the characteristic peaks in the mass spectrum of the sample to be tested is: when the maximum intensity characteristic peak in the mass spectrum of the sample to be tested is near 4545 m / z, the Daqu is black qu; when the maximum intensity characteristic peak in the mass spectrum of the sample to be tested is near 4916 m / z, the Daqu is white qu; when the maximum intensity characteristic peak in the mass spectrum of the sample to be tested is near 4541 m / z, and the larger intensity characteristic peak is near 3840 m / z, the Daqu is yellow qu.

2. The Daqu identification method according to claim 1, wherein: In step (1), the filtering treatment of the Daqu sample includes: adding Tris-HCl buffer to the Daqu sample, mixing and then filtering.

3. The Daqu identification method according to claim 2, wherein: The mass of the Daqu sample is 1-3 g, and the volume of the added Tris-HCl buffer is 4-6 mL.

4. The Daqu identification method according to claim 2, wherein: The mass of the Daqu sample was 2 g, and the volume of the added Tris-HCl buffer was 5 mL.

5. The Daqu identification method according to claim 1, wherein: In step (2), adding magnetic beads to the filtrate includes: adding Tris-HCl buffer and magnetic beads to the filtrate.

6. The Daqu identification method according to claim 5, characterized in that: The volume of the filtrate is 150 μL to 250 μL, and the total volume of the Tris-HCl buffer and the filtrate is 1 mL.

7. The Daqu identification method according to claim 5, characterized in that: The volume of the filtrate was 200 μL.

8. The Daqu identification method according to claim 5, characterized in that: The amount of magnetic beads was 20 μL.

9. The Daqu identification method according to claim 5, characterized in that: The average particle size of the magnetic beads is 500 nm.

10. The Daqu identification method according to claim 1, characterized in that: In step (2), the incubation time is 30 minutes.

11. The Daqu identification method according to claim 1, characterized in that: In step (3), the volume of the added formic acid solution was 5 μL, and the lysis time was 5 min.

12. The Daqu identification method according to claim 1, characterized in that: In step (3), the volume of acetonitrile added to the lysate is 4-6 μL.

13. The Daqu identification method according to claim 1, characterized in that: In step (3), the volume of acetonitrile added to the lysate is 5 μL.

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