Method for determining environmental persistent free radicals in barbecued food

Abstract

The invention discloses a method for determining environmental persistent free radicals in barbecued food, which comprises the following steps of: mincing the barbecued food by using a food processor, and drying at the temperature of 60 DEG C below zero for 24 hours by using a vacuum freeze-drying machine so as to retain the environmental persistent free radicals in a sample. And then, grinding the freeze-dried sample into powder by adopting an agate mortar, and storing at-20 DEG C under a dark condition. The method comprises the following steps: weighing a certain amount of sample, putting the sample into a quartz tube, packaging, and measuring spectral data through an electron paramagnetic resonance spectrometer (EPR); ePR instrument parameters including microwave power, resonant frequency, modulation frequency and the like are set to obtain high-quality spectral data. The spectral intensity (DI / N) of the sample is derived by calculating a double integral (DI) intensity and a normalization constant (N). And calculating the mass normalized spin concentration (spin / g) of the environmental persistent free radicals in the barbecued food. According to the method, the environmental persistent free radicals in the barbecued food can be accurately measured, and a scientific basis is provided for evaluating the safety and potential health risk of the barbecued food.

Description

Technical Field

[0001] The invention relates to the technical field of food safety and health assessment, and in particular to a method for accurately measuring generation and accumulation of environmental persistent free radicals in grilled food by using EPR detection technology. Background Art

[0002] Barbecue food is loved by consumers for its unique flavor and taste. According to the latest market data, by 2023, the scale of China's barbecue market has exceeded 300 billion yuan, reaching 322.4 billion yuan, a year-on-year increase of 27.1%. It is expected that by 2024, the market size will grow further and exceed 350 billion yuan. Although barbecue food is popular, harmful substances such as polycyclic aromatic hydrocarbons (PAHs) and environmentally persistent free radicals (EPFRs) generated during its thermal processing have not received enough attention.

[0003] Environmental persistent free radicals are a new type of environmental pollutants that can enter the human body through diet. Studies have shown that environmental persistent free radicals can induce the production of reactive oxygen species (ROS), triggering oxidative stress reactions, which may lead to pulmonary and cardiovascular dysfunction, and react with biological molecules to cause cell membrane instability and DNA oxidative damage, thus posing potential hazards to human health. Therefore, the detection of environmental persistent free radicals in barbecued food is of great significance for food safety and health assessment.

[0004] Electron paramagnetic resonance spectroscopy (EPR) is the main method for detecting environmental persistent free radicals. EPR can provide quantitative and qualitative information about environmental persistent free radicals by detecting the magnetic resonance signal of unpaired electrons. However, the accuracy of EPR depends on precise sample preparation, instrument calibration and optimization of measurement parameters. Currently, EPR technology has been widely used to detect environmental persistent free radicals in air, soil, water and biochar, but research on the detection of environmental persistent free radicals in barbecued foods has not yet been carried out. Summary of the invention

[0005] The present invention aims at the defects of the prior art and provides a method for determining environmental persistent free radicals in grilled food. By improving and applying EPR technology, the method helps to evaluate the food safety and potential health impact of grilled food.

[0006] In order to achieve the above invention object, the technical solution adopted by the present invention is as follows:

[0007] A method for determining environmental persistent free radicals in grilled food, characterized by comprising the following steps:

[0008] S1: The barbecue food purchased from the barbecue restaurant was minced in a food processor to ensure that the food was evenly dispersed. The minced food sample was placed in a vacuum freeze dryer until it was completely dry.

[0009] S2: Grind the freeze-dried barbecue food samples into powder using an agate mortar, put the ground samples into bottles, and store them in the dark at -20°C until the measurement;

[0010] S3: Weigh the grilled food sample and place it in a quartz tube, seal the bottom of the quartz tube with solid glue, and place the packaged quartz tube into the paramagnetic tube of the electron paramagnetic resonance spectrometer to prepare for measurement;

[0011] S4: Set the parameters of the electron paramagnetic resonance spectrometer, perform EPR spectroscopy measurements, and obtain preliminary data on environmental persistent free radicals.

[0012] S5: EPR spectral data were recorded using Bruker WinEPR Acquisition software and further analyzed using Microsoft Office Excel to determine the g-factor values ​​of the grilled food samples;

[0013] S6: Double integrate the EPR spectrum and calculate the intensity DI of the spectrum.

[0014] The normalization constant N is calculated by the formula:

[0015]

[0016] Calculate the spectral intensity of grilled food samples using DI / N;

[0017] S7: Calculate the mass-normalized spin concentration of ambient persistent free radicals in the grilled food samples by the following formula:

[0018]

[0019] Wherein, m represents the mass of the grilled food sample in g.

[0020] S8: Obtain the analysis results based on the g-factor value, spectral intensity DI / N and mass-normalized spin concentration spin / g.

[0021] Furthermore, in step S1, the vacuum freeze-drying temperature is -60°C and the drying time is 24 hours.

[0022] Furthermore, in step S2, the sample is ground and stored in a brown bottle.

[0023] Furthermore, in step S3, 2-5 mg of the grilled food sample is weighed and placed in a quartz tube with an inner diameter of 4 mm.

[0024] Further, in step S4, the electron paramagnetic resonance spectrometer is set with the following parameters:

[0025] The microwave power is 10.22mW; the resonance frequency is 9.87-9.88GHz; the modulation frequency is 100kHz; the modulation amplitude is 2.00G; the sweep width is 100G; the sweep time is 83.97s; the conversion time is 82.00ms; the time constant is 40.96ms; the X-axis resolution is 1024points; the receiver gain is 1.00×10 4 ; The spectrum scanning number is single.

[0026] Compared with the prior art, the advantages of the present invention are:

[0027] 1. Accurately determine the existence and concentration of free radicals: Through electron paramagnetic resonance spectrometer (EPR) measurement, combined with double integral intensity calculation (DI / N), the concentration of environmental persistent free radicals in grilled food can be accurately determined, realizing the quantitative analysis of unpaired electrons (free radicals) in the sample.

[0028] 2. Efficient identification of free radical types: By calculating the g-factor value, different types of free radicals in grilled food can be effectively identified. The g-factor value reflects the electronic structure characteristics of free radicals and can help identify specific types of free radicals, thereby revealing the formation mechanism of free radicals during the grilling process.

[0029] 3. Integrity and stability of the sample processing process: The barbecue food samples were processed and stored by vacuum freeze drying and low-temperature light-proof storage to avoid the decomposition or reaction of free radicals, thereby ensuring the stability of the free radical structure and the accuracy of the data during the measurement process.

[0030] 4. High sensitivity and high resolution: The parameter settings of EPR spectrum have been optimized, with high sensitivity and high resolution, which can detect extremely small amounts of persistent free radicals in the environment. At the same time, high-quality measurement data can be obtained through a single scan of the spectrum, which improves the measurement efficiency.

[0031] 5. Simple data analysis: The present invention combines Bruker WinEPR Acquisition software and Microsoft Office Excel for data processing, which can quickly calculate the g-factor value and free radical concentration, and perform data normalization and visual analysis through Excel, which simplifies the post-experimental processing process and improves the convenience of data analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention is a flow chart of a method for determining environmental persistent free radicals in grilled food according to an embodiment of the present invention.

[0033] Figure 2 4 is a schematic diagram of the calculation results of the spectral intensity (DI / N) of the grilled food sample according to the embodiment of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples.

[0035] The present invention provides a method for determining environmental persistent free radicals in grilled food, the method comprising the following steps:

[0036] S1: mince the barbecue pork purchased from the barbecue shop with a food processor, put it into a vacuum freeze dryer, and vacuum freeze dry it at -60°C for 24 hours until it is completely dry.

[0037] S2: The freeze-dried barbecue pork samples were ground using an agate mortar and then stored in brown bottles at -20 °C in the dark until determination;

[0038] S3: Weigh 2-5 mg of barbecued pork sample and place it in a quartz tube with an inner diameter of 4 mm, and seal the bottom of the quartz tube with solid glue. Place the quartz tube containing the sample into a paramagnetic tube dedicated to the electron paramagnetic resonance spectrometer, and then place the quartz tube into the electron paramagnetic resonance spectrometer for testing;

[0039] S4: Set the EPR measurement parameters as follows: microwave power is 10.22 mW; resonance frequency is 9.87-9.88 GHz; modulation frequency is 100 kHz; modulation amplitude is 2.00 G; scan width is 100 G; scan time is 83.97 s; conversion time is 82.00 ms; time constant is 40.96 ms; X-axis resolution is 1024 points; receiver gain is 1.00 × 10 4 ; The spectrum scanning number is single.

[0040] S5: The obtained environmental persistent free radical data were analyzed using Bruker WinEPR Acquisition and Microsoft Office Excel.

[0041] The g-factor values ​​(properties of an electron in a specific environment) of the barbecue pork samples were estimated by using Bruker WinEPRAcquisition and Microsoft Office Excel.

[0042] The sub-steps of S5 are as follows:

[0043] S51: Data acquisition and preliminary processing using Bruker WinEPR Acquisition

[0044] Data acquisition: When measuring with an electron paramagnetic resonance spectrometer (EPR), the Bruker WinEPR Acquisition software should be used to collect the sample's electron paramagnetic resonance (EPR) spectrum data. During this process, it should be ensured that the instrument has been calibrated and the acquisition parameters (such as microwave frequency, power, modulation frequency, etc.) are set reasonably to avoid oversaturation or excessive signal noise.

[0045] Spectrum display and preliminary analysis: In Bruker WinEPR Acquisition software, EPR spectra are displayed in graphical form. In the EPR spectrum, the positive or negative peaks of the signal correspond to the resonance response of the free radicals in the sample. At this time, the symmetry of the peaks can be checked by eye to confirm whether the signal intensity is high enough and the noise is low enough. It is recommended to perform background subtraction before peak detection to improve the accuracy of the signal.

[0046] Resonance peak location: Find the location of the resonance peak. The g factor is directly related to the location of the resonance peak. The resonance peak is usually displayed as the maximum or minimum value of the signal. The magnetic field (B) value corresponding to these peaks needs to be recorded. The software usually provides automatic peak recognition function, but manual proofreading is recommended to ensure the accuracy of peak detection, especially in spectra with large noise.

[0047] S52: Calculate g-factor value

[0048] The relationship between the g-factor value and the resonant magnetic field (B) and microwave frequency (ν) is determined by the following formula:

[0049]

[0050] Where h is Planck's constant (6.62607015×10^-34 J·s), ν is the microwave frequency (usually 9.87-9.88 GHz), and μ B is the Bohr magneton (9.274009994×10^-24J·T^-1), and B is the resonant magnetic field (in Tesla T).

[0051] Bruker WinEPR Acquisition software can automatically calculate the g-factor value. The steps are as follows:

[0052] Input microwave frequency: Enter the microwave frequency used in the measurement (for example, 9.87-9.88GHz).

[0053] Magnetic field corresponding to the peak position: The software will automatically identify and output the corresponding resonant magnetic field (B).

[0054] Calculate g-factor: The software uses the formula above to calculate the g-factor value based on the microwave frequency and the resonant magnetic field.

[0055] S53: Data import into Excel for further analysis

[0056] Export data: Export the EPR spectrum data (including resonance peak position, signal intensity and corresponding magnetic field value) obtained in the Bruker WinEPR Acquisition software into a table format, such as a CSV file.

[0057] Processing in Excel: After importing into Excel, further data analysis and visualization are performed, including:

[0058] Calculate the mean of g-factor for multiple samples: For the g-factor of multiple samples, Excel can calculate the mean, standard deviation and other indicators through formulas to analyze the trend of g-factor. If necessary, you can also filter and select data under specific samples or experimental conditions for further analysis.

[0059] Plot spectra using the chart function: After importing into Excel, you can use the chart function to plot the spectral data (magnetic field B value versus signal intensity) as a line graph. It is recommended to generate a separate spectrum for each sample in order to visually compare the resonance characteristics of different samples. With Excel's data charting tool, you can also overlay the spectra of multiple samples in the same graph to observe the differences in signal intensity and resonance peak positions.

[0060] Data fitting: When processing peak data, Gaussian or Lorentzian function fitting methods can be used to improve the accuracy of peak location and thus the accuracy of g-factor calculation.

[0061] Statistical analysis: In addition to the mean and standard deviation, more in-depth statistical analysis such as linear regression and analysis of variance (ANOVA) can be performed to explore the impact of differences between samples on the g factor.

[0062] S6: Figure 2 As shown, the spectral intensity (DI / N) of the barbecued pork sample was obtained by dividing the double integral (DI) intensity of the EPR spectrum by the normalization constant N.

[0063] S7: N in S6 is calculated by formula (1):

[0064]

[0065] S8: The mass-normalized spin concentration of EPFRs in the barbecued pork sample, spin / g, can be calculated by formula (2).

[0066]

[0067] In formula (2), DI / N represents the spectral intensity of the barbecued pork sample; m represents the mass of the barbecued pork sample, and the mass unit is g.

[0068] Those skilled in the art will appreciate that the embodiments described herein are intended to help readers understand the implementation methods of the present invention, and should be understood that the protection scope of the present invention is not limited to such special statements and embodiments. Those skilled in the art can make various other specific variations and combinations that do not deviate from the essence of the present invention based on the technical revelations disclosed by the present invention, and these variations and combinations are still within the protection scope of the present invention.

Claims

1. A method for determining environmental persistent free radicals in grilled food, characterized in that: The following steps are involved: S1: The barbecue food purchased from the barbecue restaurant was minced using a food processor to ensure that the food was evenly distributed; the minced food sample was placed in a vacuum freeze dryer until completely dry; S2: Grind the freeze-dried barbecue food samples into powder using an agate mortar and pestle. Place the ground samples in bottles and store them in the dark at -20°C until measurement. S3: Weigh the barbecued food sample and place it in a quartz tube. Seal the bottom of the quartz tube with solid glue. Place the sealed quartz tube into the paramagnetic tube of the electron paramagnetic resonance spectrometer and prepare for measurement. S4: Set the parameters of the electron paramagnetic resonance spectrometer, perform EPR spectroscopy measurements, and obtain preliminary data on environmental persistent free radicals; S5: EPR spectral data were recorded using Bruker WinEPR Acquisition software and further analyzed using Microsoft Office Excel to determine the g-factor values ​​of the grilled food samples; S6: Double integrate the EPR spectrum and calculate the intensity DI of the spectrum; The normalization constant N is calculated by the formula: Calculate the spectral intensity of grilled food samples using DI / N; S7: Calculate the mass-normalized spin concentration of ambient persistent free radicals in the grilled food samples using the following formula: Wherein, m represents the mass of the grilled food sample, in g; S8: Obtain analysis results based on the g-factor value, spectral intensity DI / N, and mass-normalized spin concentration spin / g.

2. The method for determining environmental persistent free radicals in grilled food according to claim 1, wherein: In step S1, the vacuum freeze drying temperature is -60°C and the drying time is 24 hours.

3. The method for determining environmental persistent free radicals in grilled food according to claim 1, wherein: In step S2, the sample is ground and stored in a brown bottle.

4. The method for determining environmental persistent free radicals in grilled food according to claim 1, wherein: In step S3, 2-5 mg of a barbecued food sample is weighed and placed in a quartz tube with an inner diameter of 4 mm.

5. The method for determining environmental persistent free radicals in grilled food according to claim 1, wherein: In step S4, the electron paramagnetic resonance spectrometer is set to the following parameters: The microwave power is 10.22 mW; the resonant frequency is 9.87-9.88 GHz; the modulation frequency is 100 kHz; the modulation amplitude is 2.00 G; the sweep width is 100 G; the sweep time is 83.97 s; the conversion time is 82.00 ms; the time constant is 40.96 ms; the X-axis resolution is 1024 points; and the receiver gain is 1.00 × 10 4 ; The spectrum scan number is single.

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