Method for rapidly analyzing trace insoluble protein particles in Baijiu

Abstract

The invention mainly relates to the technical field of microscopic infrared spectroscopy. The invention provides a method for rapidly analyzing trace insoluble protein particles in white spirit, and aims to rapidly identify and analyze insoluble protein particles with low content and small particle size in white spirit. The method mainly comprises the following steps: carrying out negative pressure suction filtration on white spirit through a silicon film, then naturally air-drying the white spirit, placing a slide on a placement table of a microscopic infrared spectrometer, adjusting and optimizing detection parameters, and focusing and splicing an image of the whole filter film; adding liquid nitrogen into the microscopic infrared spectrometer, and carrying out spot scanning, line scanning and surface scanning on the distributed visible foreign matters to collect spectral data; and deducting the background of the collected spectral data to obtain a spectral result, comparing the spectral result with a standard spectrum or a self-established spectrum library, and judging the source of the insoluble protein particles in the wine sample in combination with a sample source.

Description

Technical Field

[0001] The invention mainly relates to the technical field of microscopic infrared spectroscopy, and in particular to a method for rapidly analyzing trace insoluble protein particles in liquor. Background Art

[0002] Protein is an important ingredient in the raw materials for brewing liquor and plays a key role in a series of complex processes such as fermentation. Its content, type and changes will have a profound impact on the final taste, flavor and quality of the liquor. Therefore, accurate identification of protein in liquor has important industry significance.

[0003] In the field of food ingredient detection, protein detection technology has already had a certain development foundation. Traditional commonly used methods include Kjeldahl nitrogen determination method (detection limit 0.2-1 mg protein content), biuret method (detection limit 1-20 mg protein content), Coomassie brilliant blue method (detection limit minimum 1 μg protein content), ultraviolet absorption (detection limit 50-100 μg protein content), BCA method (detection range 20-2000 μg / ml) mass spectrometry (up to fg level). In summary, Kjeldahl nitrogen determination method and biuret method have low sensitivity and high requirements for protein content. Although Coomassie brilliant blue method and BCA method have low detection limits, the influence of liquor matrix is ​​relatively large. Mass spectrometry has high sensitivity and high specificity. It is a powerful tool for protein identification and analysis, but it is expensive and has high technical requirements for experimental personnel. In addition, traditional technology for protein detection is based on soluble protein, which presents a dissolved state and detects the protein content.

[0004] Protein substances in liquor usually come from brewing raw materials. During the distillation process of liquor, some macromolecular substances in the brewing raw materials will be dragged into the liquor with the distillation of liquor to form insoluble particles. These particles are characterized by "small quantity and small size". Most of the particle size distribution of protein particles in liquor is below 200μm. There are many difficulties in using conventional analysis methods, mainly because the precipitation amount of protein particles is relatively small, and the enrichment and separation of particles are difficult. In addition, the particles are prone to change during enrichment and separation, and are not easy to pick out. Summary of the invention

[0005] Technical Problems to be Solved by the Invention

[0006] The invention provides a method for rapidly analyzing trace insoluble protein particles in liquor, aiming to solve the problems that the detection methods for insoluble protein particles in liquor in the prior art are not efficient and accurate, and have high requirements on the technical ability of the detection personnel.

[0007] The technical solution adopted by the present invention to solve the above technical problems

[0008] A method for rapidly analyzing trace insoluble protein particles in liquor, comprising:

[0009] Step S1: filtering the liquor through a filter membrane under negative pressure and then placing it in a culture dish to air dry naturally;

[0010] Step S2: placing the air-dried filter membrane on a slide, placing the slide on the stage of the microscopic infrared spectrometer, adjusting the aperture illumination of the microscopic infrared spectrometer, and focusing and splicing the image of the entire filter membrane;

[0011] Step S3: adding liquid nitrogen to the micro-infrared spectrometer, collecting background data from the blank area of ​​the entire filter membrane image spliced ​​in step S2, and then collecting spectral data by performing point scanning, line scanning, and surface scanning according to the visible foreign matter distributed on the filter membrane;

[0012] Step S4: The microscopic infrared spectrometer automatically deducts the background from the spectral data collected in step S3 to obtain the spectral detection results, compares them with standard spectra of different types of proteins, determines the type of protein particles according to the protein absorption bands, and determines the source of the protein particles according to the type of protein particles.

[0013] Furthermore, in step S1, the filter membrane is a silicon membrane, the pore size of the silicon membrane is 1 μm, and the size is 1 cm×1 cm; at the same time, the negative pressure of the filtration is controlled so that the filtration speed is not higher than 5 ml / min.

[0014] Furthermore, in step S2, the magnification of the microscope of the microscopic infrared spectrometer is 40 to 150 times, and the focusing conditions are: upper illumination is 90 to 100, aperture illumination is 0, and lower illumination is 45 to 55; the width and height of the aperture are both 150 um, and the angle is 0°.

[0015] Furthermore, the amount of liquid nitrogen added in step S3 is about 500-800 ml, the acquisition mode of the microscopic infrared device is: transmission mode, the detector is: MCT detector, and the spectral range of the detector is 4000 cm -1 -675cm -1 The acquisition method is: point scanning, line scanning, and surface scanning in sequence. The number of scans for collecting the single beam spectrum of each particle is 16, and the single scan time is not less than 3 seconds.

[0016] Furthermore, the diameter of the protein microparticles is 30um-300um.

[0017] Beneficial effects of the present invention

[0018] The present invention provides a method for rapidly analyzing trace insoluble protein particles in liquor, which can obtain infrared spectrum results of low-content and small-size proteins in liquor, and the content detection can reach nanogram (ng) level, which is much lower than conventional detection methods. At the same time, the method is not affected by the type and shape of protein foreign particles. The spectral image of the detected insoluble protein particles is compared with the standard spectrum or a self-built spectral library, and the source of the insoluble protein particles in the wine sample is determined in combination with the source of the sample, which can provide strong support for the identification and traceability of the components of protein foreign particles in liquor, and then improve the production process such as distillation point, blending and filtration point. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a scanning electron microscope image of the blank area of ​​the silicon film;

[0020] Figure 2 This is a mosaic diagram of protein particles in liquor in Example 1;

[0021] Figure 3 This is a distribution diagram of foreign particles intercepted by the silicon membrane after the liquor is filtered in Example 1;

[0022] Figure 4 This is a mosaic image of the protein extracted from the liquor lees in Example 2 under a microscope infrared microscope;

[0023] Figure 5 This is a comparison of the infrared spectra of protein particles in white wine and protein extracted from lees. DETAILED DESCRIPTION

[0024] Step S1: Select the filtration volume according to the content of visible foreign matter, filter the liquor containing protein particles through the filter membrane under negative pressure, and place it in a culture dish to air dry naturally; the size of the filter membrane is 1 cm×1 cm, and the pore size is 1 um.

[0025] Step S2: Place the air-dried filter membrane on the slide, and use the joystick configured by the Nicoleti N10 Fourier transform micro-infrared spectrometer to move the sample on the stage to the observation area of ​​the objective lens; adjust the aperture illumination, focus and stitch the entire silicon membrane image. The stage is the Nicoleti N10 automatic stage; the silicon membrane, the adapter slide, and the Nicoleti N10 Fourier transform micro-infrared spectrometer are all products of Thermo Fisher Scientific, USA; adjust the illumination parameters to upper illumination: 100, aperture illumination: 0, lower illumination: 50; the width and height of the aperture are both 150um, and the angle is 0°.

[0026] Step S3: Add liquid nitrogen to the microscopic infrared spectrometer, collect background data from the blank area of ​​the entire filter membrane image spliced ​​in step S2, and then collect spectral data by point scanning, line scanning, and surface scanning according to the visible foreign matter distributed on the filter membrane; the scanning time for each particle is 3 seconds, the number of scans is 16 times, the collection mode is transmission mode, and the MCT detector is used.

[0027] Step S4: The microscopic infrared device automatically deducts the background from the spectral data collected in step S3 to obtain the spectral detection result, compares it with the standard spectra of different types of proteins, determines the type of protein foreign matter based on the protein absorption band, and determines the source of the protein particles based on the type of protein foreign matter.

[0028] Example 1

[0029] A method for rapidly analyzing trace insoluble protein particles in liquor comprises the following steps:

[0030] (1) 100 ml of liquor sample containing visible foreign matter was selected and filtered through a silicon membrane under negative pressure, and then naturally dried in a clean culture dish. The silicon membrane had a size of 1 cm×1 cm and a pore size of 1 μm.

[0031] (2) Place the naturally dried silicon film on an appropriate slide and transfer it to the stage. Use the joystick configured on the Nicoleti N10 Fourier transform microscope infrared spectrometer to move the sample on the stage and position it in the observation area of ​​the objective lens. Adjust the aperture illumination, focus and stitch the entire silicon film image. Adjust the illumination parameters to upper illumination: 100, aperture illumination: 0, lower illumination: 50; the width and height of the aperture are both 150 um, and the angle is 0°.

[0032] (3) Use the point scanning function of the instrument to mark the visible foreign particles on the obtained silicon film mosaic image. After marking, perform infrared collection of the particles. According to the prompt, select a background position on the silicon film that is not covered by the sample for background testing to obtain the following information: Figure 1 The blank background is shown, and the instrument will collect infrared spectra according to the order of particle marking; the collection method is: point scanning, line scanning, surface scanning, the infrared spectrum time of each particle is 3 seconds, scanned 16 times, the collection mode is transmission mode, and the MCT detector.

[0033] (4) After the background test, the Fourier transform calculation showed that the number of foreign protein particles trapped in the filtered wine sample was relatively large after the spectral image was matched with different component libraries as shown in Table 1 . Figure 2 This is a mosaic image of foreign particles in liquor obtained under infrared microscopy. Figure 3This is a comparison chart of the spectrum of foreign particles in liquor and the spectrum of the standard spectrum library. By comparing with the standard spectrum library and consulting the data, it is concluded that the foreign particles may be alcohol-soluble protein, and the matching degree with the standard substance is 92.6%. The analysis is as follows: The infrared spectrum of alcohol-soluble protein is at 3300cm -1 The corresponding absorption bands are the stretching vibration absorption bands of protein NH. The characteristic absorption bands of alcohol-soluble protein in the infrared spectrum are mainly composed of three groups, ranging from 1600 to 1700 cm -1 、1530-1550cm -1 and 1260-1300cm -1 The characteristic absorption peaks around the amides are amide I (C=0), amide II (CN stretching vibration and NH deformation vibration), and amide III. Combining the above characteristic peaks and the brewing process, it is speculated that the foreign matter may be alcohol-soluble protein particles.

[0034] Table 1 Infrared qualitative statistical results of foreign particles intercepted by silicon membrane after liquor filtration

[0035]

[0036] Example 2

[0037] Extraction and infrared spectroscopy identification of alcohol-soluble proteins in wine lees:

[0038] (1) During the distillation process, some protein may be dragged into the liquor by the liquor lees. The protein in the liquor lees is extracted by the "alkali extraction and acid precipitation method" and the extracted protein is freeze-dried to remove moisture.

[0039] (2) The dried protein is pressed into a thin layer and placed on a suitable window. The instrument parameters are set the same as (2) and (3) in Example 1.

[0040] (4) After the background test, the spectrum is obtained through Fourier transform calculation. Figure 4 This is a mosaic image of protein extracted from liquor lees under infrared microscopy. Figure 5 The spectrum of protein particles in liquor is compared with the spectrum of protein extracted from lees. The results show that the spectrum extracted from lees is qualitatively protein, with a matching rate of 94.89% with the standard library. It contains characteristic peaks of protein at 1600-1700cm -1 、1530-1550cm -1 and 1260-1300cm -1There is absorption around them, and their corresponding protein absorption bands are amide I (C=0), amide II (CN stretching vibration and NH deformation vibration), and amide III. In view of the fact that distiller's grains are fermented from grains, koji medicines, and husks, which contain a large amount of protein, the abnormal components in the distiller's grains are extracted by "alkali extraction and acid precipitation method" to be proteins, which proves the accuracy of the analysis result that the abnormal components are analyzed as proteins by the method for rapid analysis of trace insoluble protein particles in liquor described in the present invention.

[0041] Example 3

[0042] To further confirm that the foreign particles in liquor are proteins, the BCA protein analysis kit was used to identify the proteins in Examples 1 and 2. Principle of BCA protein quantitative analysis: The macromolecular structure of proteins, the number of peptide bonds, and the presence of four specific amino acids (cysteine, cystine, tryptophan, and tyrosine) produce color reactions with BCA. Based on bicinchoninic acid (BCA), the biuret reaction and the color development reaction are combined: the former is in an alkaline medium, and proteins can be Cu 2+ Reduction to Cu 1+ The latter is a colorimetric detection of Cu using a unique reagent containing bicinchoninic acid (BCA) 1+ It has the characteristics of high sensitivity and high selectivity. When the sample to be tested contains protein, it will react with the BCA detection working solution to produce a purple color-developing substance, and the total protein concentration is determined by colorimetry.

[0043] (1) The visible foreign matter in the liquor in Example 1 and the protein extracted from the lees in Example 2 were placed in a microplate for testing.

[0044] (2) According to the operating instructions of BCA protein quantitative analysis, prepare BCA assay working solution: mix 50 parts of BCA reagent A with 1 part of BCA reagent B (the ratio of reagent A to reagent B = 50:1), and record the prepared BCA reagent as BCA assay working solution.

[0045] (3) Take a small amount of BCA detection working solution and drop it into the microplate containing the protein particle samples obtained in Example 1 and Example 2, and shake it on an oscillator for 30 seconds to fully blend them.

[0046] (4) Seal the microplate with plastic wrap, incubate at 37°C for 30 min, and observe whether color develops.

[0047] (5) According to the color reaction results, the protein foreign particles in the liquor and the protein extracted from the lees reacted with the BCA test working solution, proving that the substances contained peptide bonds or amino acids, further indicating that the substances were protein substances.

Claims

1. A method for rapidly analyzing trace insoluble protein particles in liquor, characterized in that: include: Step S1: filtering the liquor through a filter membrane under negative pressure and placing it in a culture dish to air dry naturally; Step S2: placing the air-dried filter membrane on a slide, placing the slide on the stage of the microscopic infrared spectrometer, adjusting the aperture illumination of the microscopic infrared spectrometer, and focusing and splicing the image of the entire filter membrane; Step S3: adding liquid nitrogen to the micro-infrared spectrometer, collecting background data from the blank area of ​​the entire filter membrane image spliced ​​in step S2, and then collecting spectral data by performing point scanning, line scanning, and surface scanning according to the visible foreign matter distributed on the filter membrane; Step S4: The microscopic infrared spectrometer automatically deducts the background from the spectral data collected in step S3 to obtain the spectral detection results, compares them with standard spectra of different types of proteins, determines the type of protein particles according to the protein absorption bands, and determines the source of the protein particles according to the type of protein particles.

2. A method for rapid analysis of trace insoluble protein particles in liquor as claimed in claim 1, characterized in that: In step S1, the filter membrane is a silicon membrane with a pore size of 1 μm and a size of 1 cm×1 cm; at the same time, the negative pressure of the filtration is controlled so that the filtration speed is not higher than 5 ml / min.

3. A method for rapid analysis of trace insoluble protein particles in liquor as claimed in claim 1, characterized in that: In step S2, the magnification of the microscope of the microscopic infrared spectrometer is 40 to 150 times, and the focusing conditions are: upper illumination is 90 to 100, aperture illumination is 0, and lower illumination is 45 to 55; the width and height of the aperture are both 150 um, and the angle is 0°.

4. A method for rapid analysis of trace insoluble protein particles in liquor as claimed in claim 1, characterized in that: The amount of liquid nitrogen added in step S3 is about 500-800 ml. The acquisition mode of the microscopic infrared device is transmission mode, the detector is MCT detector, and the spectral range of the detector is 4000 cm -1 -675cm -1 The acquisition method is: point scanning, line scanning, and surface scanning in sequence. The number of scans for collecting the single beam spectrum of each particle is 16, and the single scan time is not less than 3 seconds.

5. A method for rapidly analyzing trace insoluble protein particles in liquor as described in any one of claims 1 to 4, characterized in that: The diameter of the protein particles is 30um-300um.

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