Method for screening non-sulfur marker of fresh bamboo shoot and detection method based on VB1 and its metabolites

Abstract

This invention discloses a method for screening sulfur-free biomarkers in fresh bamboo shoots and a detection method based on VB1 and its metabolites, belonging to the field of food preservation technology. The method selects sulfur (S), thiol, adenosine, VB1, and its metabolite 4-methyl-5-(beta-hydroxyethyl)thiazole as sulfur-free biomarkers for fresh bamboo shoots. The detection of sulfur-fumigated foods based on VB1 and its metabolites includes: 1. Determination of VB1 content; the VB1 content in sulfur-fumigated bamboo shoots is lower than that in fresh bamboo shoots; 2. Determination of 4-methyl-5-(beta-hydroxyethyl)thiazole content; sulfur-fumigated bamboo shoots contain 4-methyl-5-(beta-hydroxyethyl)thiazole. Through the above methods, this invention can identify whether bamboo shoots have been sulfur-fumigated by determining the content of VB1 and 4-methyl-5-(beta-hydroxyethyl)thiazole in bamboo shoots.

Description

Technical Field

[0001] This invention relates to the field of food preservation technology, specifically to a method for screening sulfur-free biomarkers in preserved bamboo shoots and a method for detecting VB1 and its metabolites. Background Technology

[0002] Bamboo shoots are an important food ingredient in my country, rich in nutrients and widely enjoyed. However, after harvesting, the quality of bamboo shoots declines rapidly, rendering them inedible within a short period, which severely restricts the development of the bamboo shoot industry. Therefore, extensive research has been conducted on bamboo shoot preservation to extend their shelf life.

[0003] Currently, bamboo shoot preservation methods include physical preservation, biological coating preservation, and chemical treatment preservation.

[0004] Sulfur fumigation is a traditional and commonly used method for preserving bamboo shoots, offering advantages such as simplicity and effectiveness. However, it is explicitly prohibited because the fumigation process generates large amounts of harmful substances that endanger human health. Despite this, the lack of effective alternatives means that some individuals and unscrupulous merchants continue to use sulfur fumigation to preserve bamboo shoots. Although strict regulations exist to prevent sulfur-fumigated bamboo shoots from entering the market and to ensure food safety, actual inspections primarily rely on measuring the SO2 content to determine if the bamboo shoots have undergone sulfur fumigation. However, in actual production, sulfur-fumigated bamboo shoots are often treated with rinsing, soaking, and sun-drying to remove sulfur. This significantly reduces the SO2 content, leading to false negatives in testing and allowing large quantities of sulfur-fumigated bamboo shoots to enter the market, seriously endangering public food safety.

[0005] Therefore, finding a detection method that can accurately identify whether bamboo shoots have been sulfur-fumigated even after desulfurization is of great significance to people's food safety.

[0006] Based on this, the present invention designs a method for screening sulfur-free biomarkers in fresh bamboo shoots and a detection method based on VB1 and its metabolites to solve the above problems. Summary of the Invention

[0007] In view of the above-mentioned shortcomings of the existing technology, the present invention provides a method for screening sulfur-free biomarkers in fresh bamboo shoots and a detection method based on VB1 and its metabolites.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for screening sulfur-free biomarkers in fresh bamboo shoots includes the following steps:

[0010] I. Pre-treatment of bamboo shoots;

[0011] Bamboo shoots of similar shape and size were divided into a control group and a sulfur-fumigated group for pretreatment.

[0012] II. Perform high-resolution time-of-flight mass spectrometry detection;

[0013] III. Analysis of small molecule compounds in bamboo shoots before and after sulfur fumigation;

[0014] IV. Analysis of differential metabolites in bamboo shoots before and after sulfur fumigation; including PCA analysis and OPLS-DA analysis;

[0015] V. Screening for significantly different metabolites;

[0016] VI. Sulfate, thiol, adenosine, vitamin B1 and its metabolite 4-methyl-5-(beta-hydroxyethyl)thiazole were selected as sulfur-free markers for fresh bamboo shoots.

[0017] Furthermore, the chromatographic conditions are as follows:

[0018] Chromatographic column: C18 column;

[0019] Column temperature: 40℃; Flow rate: 0.4 mL / min;

[0020] Injection volume: 2 μL;

[0021] In positive ion mode, the mobile phase composition is: A: water + 25 mM ammonium acetate + 0.5% formic acid; B: methanol.

[0022] In negative ion mode, the mobile phase composition is A: water, B: methanol.

[0023] Furthermore, the mass spectrometry conditions are as follows:

[0024] Ion source: electrospray ionization source; spray gas: 60 psi, auxiliary heating gas: 60 psi; curtain gas: 30 psi; temperature: 600℃; ionization pressure: ±5500V; primary scan range: 60-1000 Da; secondary scan range: 25-1000 Da; secondary mass spectrometry was obtained using IDA in high sensitivity mode, with declustering voltage: ±60V and collision energy: 35±15eV.

[0025] Furthermore, one or a combination of two of the following steps may be used:

[0026] I. Determination of VB1 content: The VB1 content in sulfur-fumigated bamboo shoots is lower than that in fresh bamboo shoots.

[0027] II. Determination of 4-methyl-5-(beta-hydroxyethyl)thiazole content; Sulfur-fumigated bamboo shoots contain 4-methyl-5-(beta-hydroxyethyl)thiazole.

[0028] Furthermore, the determination of VB1 content includes the following steps:

[0029] (1) Weigh bamboo shoot powder, add it to HCl solution and shake well; acid hydrolysis; cooling; adjust pH with sodium acetate solution; add protease-amylase mixture for enzymatic hydrolysis, and then cool to room temperature;

[0030] (2) Derivatization treatment: Transfer the supernatant to alkaline potassium ferricyanide solution, vortex and mix well, add n-butanol, vortex and let stand, take the upper layer solution and filter it through an organic microporous membrane, and then perform the determination on the instrument; take another standard solution and derivatize it simultaneously with the test solution;

[0031] (3) Calculation of results

[0032] VB1 content is calculated using the following formula:

[0033]

[0034] Where X: VB1 content in the sample; c: VB1 concentration; V: final volume of the test solution; f: dilution factor of the test solution before derivatization; M: mass of the sample.

[0035] Furthermore, step (3) specifically involves: weighing thiamine hydrochloride and preparing standard solutions of 1 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 25 mg / L and 50 mg / L with HCl solution, and then measuring them on an instrument.

[0036] The conditions for the liquid chromatography instrument are:

[0037] Column: Agilent C18, 150mm × 4.6mm, 5μm;

[0038] Mobile phase: 0.01 mol / L sodium acetate solution - methanol;

[0039] Fluorescence detector: excitation wavelength 375nm, emission wavelength 435nm;

[0040] Flow rate: 0.8 mL / min;

[0041] Injection volume: 20 μL; Column temperature: 35℃.

[0042] Furthermore, the determination of the 4-methyl-5-(beta-hydroxyethyl)thiazole content includes the following steps:

[0043] 1) Add bamboo shoot powder to a pre-cooled methanol solution, vortex mix, centrifuge, and collect the supernatant for analysis.

[0044] 2) Preparation of standard solutions;

[0045] 3) Quantitative analysis and calculation methods

[0046] The content of 4-methyl-5-(beta-hydroxyethyl)thiazole in the sample was calculated using a standard curve.

[0047] Furthermore, step 2) specifically involves preparing a standard stock solution of 4-methyl-5-(beta-hydroxyethyl)thiazole standard using methanol solution, and then successively diluting it to prepare standard solutions of 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 25 mg / mL and 50 mg / mL for instrumental analysis.

[0048] Chromatographic conditions:

[0049] Column: Agilent C18 column;

[0050] Column temperature: 30℃; Flow rate: 0.8 mL / min; Mobile phase: water-methanol;

[0051] Injection volume: 10 μL; Autosampler temperature: 4℃; Detection wavelength: 249 nm.

[0052] Application of the method of detecting sulfur-fumigated food based on VB1 and its metabolites as sulfur-free markers in fresh bamboo shoots.

[0053] Application of the method of detecting sulfur-fumigated food based on VB1 and its metabolites as sulfur-free markers in fresh bamboo shoots.

[0054] Beneficial effects

[0055] This invention determined the VB1 content in bamboo shoots of different treatment groups using HPLC fluorescence spectrometry, revealing a significant difference in VB1 content before and after sulfur fumigation. Further verification and determination of its secondary metabolites were then conducted. A detection method for 4-methyl-5-(beta-hydroxyethyl)thiazole was established, and the linearity and stability of this method were verified. After analyzing the samples, 4-methyl-5-(beta-hydroxyethyl)thiazole was detected in the sulfur-fumigated samples, but not in fresh bamboo shoots. Therefore, the presence of sulfur fumigation in bamboo shoots can be determined by measuring the content of VB1 or 4-methyl-5-(beta-hydroxyethyl)thiazole.

[0056] This invention can also combine the content of VB1 and 4-methyl-5-(beta-hydroxyethyl)thiazole to comprehensively determine whether bamboo shoots have been fumigated with sulfur. Attached Figure Description

[0057] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0058] Figure 1 Volcano diagrams in positive (a) and negative (b) ion modes;

[0059] Figure 2 PCA score graphs are shown for positive (a) and negative (b) ion modes;

[0060] Figure 3 The images show the OPLS-DA score diagrams and displacement test results under positive and negative ion modes; (a and b are the OPLS-DA score diagrams and displacement test results under positive ion mode; c and d are the OPLS-DA score diagrams and displacement test results under negative ion mode).

[0061] Figure 4 Heatmap of differential metabolites; (a and b are positive and negative ion modes, respectively);

[0062] Figure 5 The curve shows the change in SO2 content;

[0063] Figure 6 The curve shows the change in thiol content;

[0064] Figure 7 Chromatograms of adenosine standard (a) and sulfur-fumigated bamboo shoot sample (b);

[0065] Figure 8 This is the standard curve for adenosine;

[0066] Figure 9 Chromatograms of VB1 standard (a), bamboo shoots (b), and sulfur-fumigated bamboo shoots (c);

[0067] Figure 10 Chromatograms of the standard and sample are shown; (a, b, c, and d are VB1 standard, sulfur-fumigated VB1 standard, 4-methyl-5-(beta-hydroxyethyl)thiazole, and (4-amino-2-methylpyrimidin-5-yl)methanesulfonic acid, respectively).

[0068] Figure 11 Chromatograms of 4-methyl-5-(beta-hydroxyethyl)thiazole standard (a), sulfur-fumigated bamboo shoots (b), and bamboo shoots (c);

[0069] Figure 12The standard curve for 4-methyl-5-(beta-hydroxyethyl)thiazole. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0071] The present invention will be further described below with reference to embodiments.

[0072] Example 1: Effects of Sulfur Fumigation on the Chemical Composition of Bamboo Shoots

[0073] Currently, no studies have investigated the effects of sulfur fumigation on the chemical composition of bamboo shoots. Therefore, this invention uses UPLC-TOF-MS to analyze the changes in the types of compounds in bamboo shoots before and after sulfur fumigation, and through multivariate statistical analysis, identifies differentially expressed metabolites that show significant changes before and after sulfur fumigation, laying the foundation for subsequent screening of sulfur fumigation biomarkers.

[0074] I. Experimental Methods

[0075] 3.1 Experimental Grouping

[0076] After peeling bamboo shoots of similar shape and size, divide them into two groups of 20 each.

[0077] Control group: freeze-dried for 48 hours, then ground into powder, passed through a 60-mesh sieve, and stored at -20℃;

[0078] Sulfur-fumigated group: Fumigate overnight with sulfur at a ratio of 1:40 to bamboo shoots, then freeze-dry for 48 hours, remove, grind into powder, pass through a 60-mesh sieve, and store at -20℃.

[0079] 3.2 Sample preparation

[0080] Take 20.0 mg of sample and add 1 mL of pre-cooled 70% methanol solution. Vortex mix, sonicate at low temperature for 30 min, centrifuge at 10000 rpm and 4℃ for 15 min, and take the supernatant and pass it through a 0.22 μm organic membrane for analysis.

[0081] 3.3 Chromatographic conditions

[0082] Instrument analysis platform: UPLC-MS

[0083] Chromatographic column: C18 column

[0084] Column temperature: 40℃; Flow rate: 0.4 mL / min;

[0085] Injection volume: 2 μL;

[0086] In positive ion mode, the mobile phase composition is: A: water + 25 mM ammonium acetate + 0.5% formic acid; B: methanol.

[0087] In negative ion mode, the mobile phase composition is A: water, B: methanol;

[0088] The gradient elution procedure for the mobile phase is shown in Table 1.

[0089] Table 1 Mobile phase conditions for liquid chromatography

[0090]

[0091] 3.4 Mass Spectrometry Conditions

[0092] The first and second-order spectra of the samples were acquired using an AB Triple TOF 6600 mass spectrometer.

[0093] Ion source: Electrospray ionization (ESI); Spray gas (Gas1): 60 psi, auxiliary heating gas (GS2): 60 psi; Curtain gas (CUR): 30 psi; Temperature (TEM): 600℃; Ionization pressure (ISVF): ±5500V (positive and negative modes); Primary scan range: 60-1000 Da; Secondary scan range: 25-1000 Da; Secondary mass spectrometry was obtained using IDA in high-sensitivity mode; Declustering voltage (DP): ±60V (positive and negative modes); Collision energy (CE): 35±15eV.

[0094] 3.5 Data Analysis

[0095] The raw data were converted to .mzXML format using ProteoWizard, and then peak alignment, retention time correction, and peak area extraction were performed using MSDAIL software. The data extracted by MSDAIL were first subjected to metabolite structure identification and data preprocessing, then experimental data quality assessment, and finally data analysis.

[0096] II. Experimental Results

[0097] 4.1 Changes in small molecule compounds in bamboo shoots before and after sulfur fumigation

[0098] After the processed samples were analyzed, the chromatographic peaks were normalized and standardized. By regressing the original data, relevant mass spectrometry information was extracted from the mass spectra, including the precise mass-to-charge ratio (m / z), retention time, and secondary fragmentation spectrum. The results were then matched with a database to identify the structures of metabolites in the biological samples, and the identification results were verified and confirmed.

[0099] To further understand the changes in small molecule compounds in bamboo shoots before and after sulfur fumigation, data processing was performed, and the types and quantities of compounds identified before and after sulfur fumigation in positive and negative ion modes are shown in Table 2. It can be observed that there are significant differences in the compounds in bamboo shoots before and after sulfur fumigation. After sulfur fumigation, the quantities of compounds such as Benzenoids, Organic acids and derivatives, Lipids and lipid-like molecules increased to varying degrees, while the quantities of compounds such as Alkaloids and derivatives, Organic oheterocyclic compounds, Lignans, Neolignans and related compounds decreased. Metabolites with FC>1.5 or FC<0.67 and p value<0.05 in both positive and negative ion modes were selected for differential analysis, and the results are as follows: Figure 1 As shown, the content of many compounds in bamboo shoots changed significantly before and after sulfur fumigation. This indicates that sulfur fumigation has a significant impact on the quantity and content of compounds in bamboo shoots.

[0100] Table 2. Types and quantities of compounds under positive and negative ion modes.

[0101]

[0102]

[0103] In addition, a large number of new compounds are generated during the sulfur fumigation process of bamboo shoots. Statistical analysis revealed that 75 new compounds are generated under positive ion mode and 58 new compounds are generated under negative ion mode. The specific types are shown in Table 3.

[0104] Table 3. Types of newly generated compounds under positive and negative ion modes.

[0105]

[0106] Based on the precise mass number and MS / MS secondary mass spectra measured by UPLC-Q-TOF, and compared with the theoretical precise mass and MS / MS secondary mass spectra of fragment ions in the metabolomics database, a total of 9 sulfur-containing compounds were identified in the newly generated compounds, including 3 in positive ion mode and 6 in negative ion mode. Their names and structural formulas are shown in Table 4.

[0107] Table 4. Newly generated sulfur-containing compounds under positive and negative ion modes.

[0108]

[0109]

[0110] 4.2 Differential metabolites in bamboo shoots before and after sulfur fumigation

[0111] (1) PCA Analysis

[0112] Principal component analysis was performed on metabolites from the control group and the sulfur fumigation group, such as... Figure 2 As shown, the mass spectrometry data of the two groups of samples under both positive and negative ion states exhibit high discriminative power, indicating significant differences in the compound levels of bamboo shoots before and after sulfur fumigation. This provides a possibility for screening out potential differential metabolites.

[0113] (2) OPLS-DA analysis

[0114] Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) is a modified version of PLS-DA that filters out noise irrelevant to classification information, improving the model's analytical power and effectiveness. For example... Figure 3 a and 3c show that the OPLS-DA model can completely distinguish between the two groups of samples under both positive and negative ion modes, indicating that the compounds in bamboo shoots before and after sulfur fumigation are significantly different overall. To prove that the differences found in the OPLS-DA model are due to the true differences in compounds rather than overfitting, the model was cross-validated, such as... Figure 3 b, 3d, it can be seen that as the permutation retention gradually decreases, the R-value of the stochastic model decreases. 2 and Q 2 The values ​​all gradually decreased, indicating that the original model did not exhibit overfitting and had high reliability.

[0115] Based on the OPLS-DA model criteria of VIP>1 and P value<0.05, a total of 85 differentially expressed metabolites were screened, including 30 in the positive ion mode and 55 in the negative ion mode. The hierarchical cluster analysis results of the significantly differentially expressed metabolites (VIP>1, P value<0.05) are as follows: Figure 4 As shown in the figure. Based on the changes in the content of these key metabolites, the samples from the control group and the sulfur-fumigated group could be clustered into completely different categories, indicating that these small molecule compounds have potential application value in distinguishing bamboo shoot samples from the sulfur-fumigated group and the control group.

[0116] (3) Screening of potential differential substances in sulfur-fumigated bamboo shoots

[0117] Thirty candidate differential metabolites obtained in the positive ion mode and 55 candidate differential metabolites obtained in the negative ion mode were excluded, and the reliability of each differential metabolite was verified. Finally, the 10 compounds with the most significant content differences in both the positive and negative ion modes were selected, as shown in Tables 5 and 6.

[0118] Table 5 Possible differential metabolites under positive ion mode

[0119]

[0120] Table 6. Possible differential metabolites under negative ion mode

[0121]

[0122]

[0123] III. Summary

[0124] Bamboo shoot samples were pretreated before and after sulfur fumigation and then analyzed by high-resolution time-of-flight mass spectrometry (HFS). The instrument stability was confirmed during the experiment by comparing the total ion chromatograms (TIC) of the QC samples, principal component analysis of the overall samples, and the variance of the QC sample data. Analysis of small molecule compounds in bamboo shoots before and after sulfur fumigation revealed 527 and 453 metabolites in positive ion mode, respectively, and 403 and 431 metabolites in negative ion mode, respectively. After sulfur fumigation, 75 new compounds were generated in positive ion mode, including 3 sulfur-containing compounds, while 68 new compounds were generated in negative ion mode, including 6 sulfur-containing compounds. Analysis showed that sulfur fumigation significantly affected the quantity and content of metabolites in bamboo shoots. Statistical analysis of the metabolites established a stable OPLS-DA analytical model. Based on the criteria of VIP>1 and P value<0.05, 30 and 55 candidate differential metabolites were screened in positive and negative ion modes, respectively. Further analysis revealed 10 significantly differential metabolites in each of the positive and negative ion modes. This lays the foundation for the subsequent screening of biomarkers for sulfur-fumigated bamboo shoots.

[0125] Example 2: Screening and Validation of Sulfur Fumigation Markers in Bamboo Shoots

[0126] This study investigated the changes in the content of sulfur, thiol, adenosine, vitamin B1, and their decomposition products in bamboo shoots after sulfur fumigation and desulfurization treatment by soaking in water, to determine their feasibility in identifying sulfur-fumigated bamboo shoots. The aim was to find a rapid and accurate detection method for sulfur-fumigated bamboo shoots.

[0127] I. Experimental Methods

[0128] 5.1 Determination of SO2 content

[0129] (1) Sample preparation

[0130] Control group: Fresh bamboo shoots were peeled, crushed, and mixed evenly;

[0131] Sulfur fumigation group: After peeling the bamboo shoots, fumigate them overnight with sulfur at a weight ratio of 1:40. After taking them out, crush and mix them evenly.

[0132] (2) Sample processing

[0133] Accurately weigh 10.00 g of the sample, add 100 mL of formaldehyde buffer solution, shake and soak for 2 h, then filter. Take 2.00 mL of the filtrate into a 25 mL stoppered test tube, add 8 mL of formaldehyde buffer solution, 0.5 mL of 3 g / L aminosulfonic acid ammonium solution, 0.5 mL of 1.5 mol / L NaOH solution, and 1.0 mL of 0.5 g / L pararosaniline hydrochloride solution in sequence, shake well, let stand for 20 min, and then measure the absorbance at 579 nm.

[0134] (3) Standard solution

[0135] Accurately measure 0.1 mL, 0.25 mL, 0.50 mL, 1.00 mL, 2.00 mL, 4.00 mL, and 8.00 mL of SO2 standard solution (equivalent to 1.0 μg, 2.5 μg, 5.0 μg, 10.0 μg, 20.0 μg, 40.0 μg, and 80.0 μg SO2), process them using the method described above, and then perform the determination. Plot a standard curve using SO2 concentration versus absorbance.

[0136] (4) Dynamic changes in SO2 content

[0137] Sulfur-fumigated bamboo shoots were soaked in clean water, and the SO2 content was determined using the method described above at 0h, 6h, 12h, 18h, 24h, 36h, and 48h. The water was changed every 6 hours.

[0138] 5.2 Determination of Sulfur Content

[0139] (1) Sample preparation

[0140] After peeling bamboo shoots of similar shape and size, divide them into 4 groups of 20 each.

[0141] Control group: freeze-dried for 48 hours, then crushed using a high-speed pulverizer, passed through a 60-mesh sieve, and stored at -20℃;

[0142] Control group: Bamboo shoots were peeled and soaked in water for 48 hours, with the water changed every 6 hours. They were then freeze-dried for 48 hours, removed and crushed using a high-speed grinder, passed through a 60-mesh sieve, and stored at -20℃.

[0143] Sulfur-fumigated group: Fumigate overnight with sulfur at a ratio of 1:40 to bamboo shoots, freeze-dry for 48 hours, take out and crush with a high-speed pulverizer, pass through a 60-mesh sieve, and store at -20℃.

[0144] Sulfur fumigation and soaking group: After fumigating overnight with sulfur at a ratio of 1:40 to bamboo shoots, soak in clean water for 48 hours, changing the water every 6 hours, freeze-dry for 48 hours, take out and crush with a high-speed pulverizer, pass through a 60-mesh sieve, and store at -20℃.

[0145] (2) Sample processing

[0146] Accurately weigh 0.1000 g of the above sample and place it in a digestion vessel. Add 5 mL of nitric acid (GR grade) and pre-digest at 120 °C for 30 min. Then transfer the sample to a microwave digester and digest according to the procedure in Table 7. After digestion, place the digestion vessel on a 135 °C hot plate to remove approximately 0.5 mL of acid. Transfer the digestion solution and bring the volume to 10 mL with ultrapure water. Perform a blank test simultaneously.

[0147] Table 7 Microwave Digestion Procedure

[0148]

[0149] (3) Preparation of standard solutions

[0150] The 1 mg / mL S standard solution was diluted to 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L and 80 mg / L.

[0151] (4) Instrument operating conditions

[0152] The instrument parameters were optimized using a tuning fluid, and the ICP-MS was tuned to its optimal working environment.

[0153] Test conditions: Carrier gas flow rate: 0.80 L / min; Peristaltic pump sampling speed: 0.2 r / s; Auxiliary gas flow rate: 0.40 L / min; Peristaltic pump stabilization time: 30 s; Single measurement: 3 times.

[0154] 5.3 Determination of total thiol content

[0155] (1) Sample preparation

[0156] After sulfur fumigation, bamboo shoots were soaked in clean water for 0h, 6h, 12h, 18h, 24h, 36h, 48h, 72h, and 96h. The bamboo shoots were then removed, dried, chopped, and juiced. The juice was collected, centrifuged, and the supernatant was used to determine the total sulfhydryl content. The water was changed every 6 hours.

[0157] (2) Preparation of standard solutions

[0158] Accurately weigh 10.00 mg of reduced glutathione, add 1.3 mL of distilled water to prepare a 25 μmol / mL standard stock solution, and then dilute it sequentially to prepare standard working solutions of 2.5 μmol / mL, 1 μmol / mL, 0.5 μmol / mL, 0.25 μmol / mL, 0.125 μmol / mL, 0.0625 μmol / mL, 0.03125 μmol / mL and 0.015625 μmol / mL.

[0159] (3) Sample determination

[0160] Perform the assay according to the kit's operating instructions.

[0161] 5.4 Determination of adenosine content

[0162] (1) Sample preparation

[0163] Same as "5.2".

[0164] (2) Sample processing

[0165] Same as "3.2".

[0166] (2) Preparation of standard solutions

[0167] Weigh 0.02 g of adenosine and prepare standard solutions with concentrations of 0.5 mg / L, 1 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 25 mg / L and 50 mg / L using 70% methanol solution. Filter the solutions through a 0.22 μm organic membrane and store at 4 °C.

[0168] (3) Chromatographic conditions

[0169] Instrument analysis platform: Shimadzu LC-20 high performance liquid chromatograph;

[0170] Column: Agilent C18 (150mm × 4.6mm, 3.5μm);

[0171] Column temperature: 30℃; Flow rate: 0.8 mL / min;

[0172] Mobile phase: water-methanol;

[0173] Injection volume: 10 μL; Autosampler temperature: 4℃;

[0174] The mobile phase gradient elution procedure is shown in Table 8.

[0175] Table 8 Gradient elution methods for liquid chromatography

[0176]

[0177] (4) Quantitative methods for adenosine in samples

[0178] The adenosine content in the sample was calculated using the adenosine standard curve.

[0179] (5) Methodological validation

[0180] ①Standard Curve

[0181] The above-prepared standard solutions were tested, and a standard curve was established with concentration on the x-axis and peak area on the y-axis.

[0182] ② Limit of detection and limit of quantitation of the method

[0183] The concentration of the target compound adenosine was determined, the baseline noise value was measured, and the limit of detection (LOD) of the method was determined by the compound concentration at three times the signal-to-noise ratio, and the limit of quantitation (LOQ) of the method was determined by the compound concentration at ten times the signal-to-noise ratio.

[0184] ③ Method recovery rate

[0185] High, medium, and low concentration standards were added to the sample extract for spike recovery experiments.

[0186] 5.5 Determination of VB1 content

[0187] (1) Sample preparation

[0188] Same as "5.2".

[0189] (2) Sample processing

[0190] Accurately weigh 5.00 g of bamboo shoot powder into a 100 mL Erlenmeyer flask, add 60 mL of 0.1 mol / L HCl solution, shake well, seal with a rubber stopper, and acid hydrolyze at 121 °C for 20 min. Remove and cool, adjust the pH to 4.0 with 2.0 mol / L sodium acetate solution, add 3 mL of protease-amylase mixture (1.27 g amylase and 1.76 g papain, add water to make up to 50 mL), and enzymatically hydrolyze at 37 °C for 16 h. Cool to room temperature, transfer to a 100 mL volumetric flask and add water to make up to 50 mL.

[0191] (3) Derivative processing

[0192] Transfer 2.0 mL of the supernatant, add 1.0 mL of alkaline potassium ferricyanide solution, vortex to mix, then add 2.0 mL of n-butanol, vortex for 1.5 min, let stand for 10 min, and then filter the upper layer through a 0.45 μm organic microporous membrane for analysis. Separately, take a standard solution and perform derivatization simultaneously with the test solution.

[0193] (4) Preparation of standard solutions

[0194] Weigh 0.02 g of thiamine hydrochloride and prepare standard solutions of 1 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 25 mg / L and 50 mg / L with 0.1 mol / L HCl solution. Filter the solutions through a 0.22 μm organic membrane and perform the determination on an instrument.

[0195] (5) Conditions for liquid chromatography instruments

[0196] Instrument analysis platform: Shimadzu LC-20;

[0197] Column: Agilent C18 (150mm × 4.6mm, 5μm);

[0198] Mobile phase: 0.01 mol / L sodium acetate solution - methanol (80:20);

[0199] Fluorescence detector: excitation wavelength 375nm, emission wavelength 435nm;

[0200] Flow rate: 0.8 mL / min;

[0201] Injection volume: 20 μL; Column temperature: 35℃.

[0202] (6) Calculation of results

[0203] VB1 content is calculated using the following formula:

[0204]

[0205] X: VB1 content in the sample (mg / 100g); c: VB1 concentration (mg / L); V: final volume of the test solution (mL); f: dilution factor of the test solution before derivatization; M: mass of the sample (g).

[0206] 5.6 Determination of VB1 metabolites

[0207] (1) Sample preparation

[0208] A VB1 solution of a certain concentration was placed in a 50 mL centrifuge tube and fumigated simultaneously with bamboo shoots. The fumigated VB1 solution was diluted to 50 mg / L. At the same time, standard solutions of VB1, 4-methyl-5-(beta-hydroxyethyl)thiazole, and (4-amino-2-methylpyrimidin-5-yl)methanesulfonic acid were prepared.

[0209] (2) Chromatographic conditions

[0210] Instrument analysis platform: Shimadzu LC-20;

[0211] Chromatographic column: Agilent C18 column (250mm × 4.6mm, 5μm);

[0212] Column temperature: 30℃; flow rate: 0.8 mL / min;

[0213] Mobile phase: water-methanol (60:40);

[0214] Injection volume: 10 μL; Autosampler temperature: 4℃.

[0215] 5.7 Determination of 4-methyl-5-(beta-hydroxyethyl)thiazole

[0216] (1) Sample preparation

[0217] Same as "5.2".

[0218] (2) Sample processing

[0219] Same as "3.2".

[0220] (3) Preparation of standard solutions

[0221] Prepare a 1 mg / mL standard stock solution of 4-methyl-5-(beta-hydroxyethyl)thiazole using 70% methanol solution. Then, dilute the stock solution sequentially to prepare standard solutions of 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 25 mg / mL and 50 mg / mL. Filter the solutions through a 0.22 μm organic membrane and perform analysis.

[0222] (4) Chromatographic conditions

[0223] Instrument analysis platform: Shimadzu LC-20;

[0224] Chromatographic column: Agilent C18 column (250mm × 4.6mm, 5μm);

[0225] Column temperature: 30℃; Flow rate: 0.8 mL / min; Mobile phase: water-methanol;

[0226] Injection volume: 10 μL; Autosampler temperature: 4℃; Detection wavelength: 249 nm;

[0227] The gradient elution procedure for the mobile phase is shown in Table 9.

[0228] Table 9 Mobile Phase Conditions for Liquid Chromatography

[0229]

[0230] (5) Quantitative analysis calculation methods

[0231] Same as "5.4".

[0232] (6) Methodological validation

[0233] Same as "5.4".

[0234] 5.8 Data Processing

[0235] All experiments were performed in triplicate. Data were processed using WPS Office, and all data are expressed as mean ± SD. Origin 2018 software was used for graphing. Differences between experimental groups were analyzed using Duncan's multiple comparison test in IBM SPSS Statistics 20 (P < 0.05).

[0236] II. Experimental Results

[0237] 6.1 Results of SO2 content determination

[0238] The results showed that no SO2 was detected in the blank group, while the SO2 content in the sulfur-fumigated group was at a high level (177.53±13.42 mg / kg), far exceeding the national standard (50 mg / kg). The results of the determination of SO2 in sulfur-fumigated bamboo shoots after soaking treatment are as follows: Figure 5 As shown, soaking bamboo shoots in water reduces SO2 content. After 16 hours of soaking, the SO2 content in the bamboo shoots reaches the national standard threshold; after 48 hours, SO2 is almost undetectable. This indicates that soaking sulfur-fumigated bamboo shoots in water affects test results, leading to sulfur-fumigated bamboo shoots entering the market.

[0239] 6.2 Results of S element content determination

[0240] The results are shown in Table 10. The results indicate that even after soaking in water and meeting the SO2 content standards, the sulfur content in sulfur-fumigated bamboo shoots was still significantly higher than that in fresh bamboo shoots. Therefore, determining the sulfur content in samples can provide a reference for identifying whether bamboo shoots have been sulfur-fumigated.

[0241] Table 10 S content in samples

[0242]

[0243] 6.3 Results of Total Sulfhydryl Content Determination

[0244] The results of the thiol content measured after the samples were soaked for different times are as follows: Figure 6 As shown, the sulfhydryl content of fresh bamboo shoots did not change significantly with increasing soaking time; however, the sulfhydryl concentration of sulfur-fumigated bamboo shoots remained at a high level. Although the content showed a clear decreasing trend with increasing soaking time, even after 4 days of soaking, the sulfhydryl content was still much higher than that of fresh bamboo shoots. Furthermore, after 4 days of soaking, the bamboo shoots gradually developed an off-odor. Additionally, in the dynamic changes of SO2 in sulfur-fumigated bamboo shoots, the SO2 content met national standards after 24 hours of soaking, but at this point, the sulfhydryl content showed a significant difference. Therefore, the sulfhydryl content can be measured to determine whether bamboo shoots have been sulfur-fumigated.

[0245] 6.4 Results of Adenosine Content Determination

[0246] (1) Exclusivity

[0247] The standard and sample were taken and analyzed on the instrument. The specific chromatogram is shown in [link to chromatogram]. Figure 7 The retention time of adenosine was 8.57 min, and the separation effect of each substance was also good.

[0248] (2) Linearity and Range

[0249] A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation and linear range are shown in Table 11. Figure 8 The linear range of adenosine was 0.5–50 mg / kg, with a LOD of 0.17 mg / kg and a LOQ of 0.5 mg / kg. Spiking recovery experiments were conducted at low, medium, and high concentration gradients. The recoveries were all greater than 90% under different matrices, and the intra-day CV and inter-day CV were both less than 10%, meeting the requirements for sample quantification. Specific results are shown in Table 12.

[0250] Table 11 Linearity and Range Test Results

[0251]

[0252] Table 12 Spiked recoveries and CV of adenosine

[0253]

[0254] (3) Detection results of actual samples

[0255] The solutions obtained after sample processing were analyzed and the data were recorded. The adenosine content in fresh bamboo shoots and sulfur-fumigated bamboo shoots was calculated, and the specific results are shown in Table 13. The results show that the adenosine content in bamboo shoots treated with sulfur fumigation and soaking was still much higher than that in fresh bamboo shoots. Therefore, using adenosine as a potential biomarker is feasible.

[0256] Table 13 Adenosine content in bamboo shoot samples

[0257]

[0258]

[0259] 6.5 Results of VB1 content determination

[0260] (1) Exclusivity

[0261] Take the standard and sample separately, and analyze them on the instrument. See the specific chromatograms below. Figure 9 The retention time of VB1 was 5.03 min, and the separation effect of each substance was also good.

[0262] (2) Linearity and Range

[0263] Table 14 shows the regression equation and linear range with concentration on the x-axis and peak area on the y-axis. The linear range of VB1 is 1–50 mg / kg, LOD is 0.33 mg / kg, and LOQ is 1.0 mg / kg.

[0264] Table 14 Linearity and Range of VB1 Standard Curve

[0265]

[0266] (3) Sample test results

[0267] Samples of fresh bamboo shoots, freshly soaked bamboo shoots, sulfur-fumigated bamboo shoots, and sulfur-fumigated soaked bamboo shoots were processed using various methods to prepare sample solutions, which were then analyzed using a mass spectrometry method. Data were recorded, and the VB1 content was calculated using a standard curve. The specific results are shown in Table 15. The results show that the VB1 content of bamboo shoots decreased significantly after sulfur fumigation. Furthermore, mass spectrometry analysis also revealed that the ionic strength of VB1 in sulfur-fumigated samples was significantly lower than that in fresh bamboo shoots. Therefore, it can be determined that sulfur fumigation destroys the VB1 structure in bamboo shoots and reduces its content; thus, it can be used as a method to identify whether bamboo shoots have been sulfur-fumigated.

[0268] Table 15. VB1 content in bamboo shoot samples

[0269]

[0270] 6.6 Determination of VB1 metabolites

[0271] The standard and sample were analyzed on the instrument, and the resulting chromatogram is shown below. Figure 10 As shown in the figure, the retention times of VB1, 4-methyl-5-(beta-hydroxyethyl)thiazole, and (4-amino-2-methylpyrimidin-5-yl)methanesulfonic acid were 3.67 min and 14.89 min, respectively. The two peaks of VB1 after sulfur fumigation appeared at 3.67 min and 14.89 min, respectively. Therefore, it can be determined that VB1 will produce 4-methyl-5-(beta-hydroxyethyl)thiazole after sulfur fumigation. Since the retention time of (4-amino-2-methylpyrimidin-5-yl)methanesulfonic acid is the same as that of VB1, it cannot be determined whether (4-amino-2-methylpyrimidin-5-yl)methanesulfonic acid was produced. Therefore, further testing is needed to determine whether 4-methyl-5-(beta-hydroxyethyl)thiazole is present in the sulfur-fumigated bamboo shoot samples.

[0272] 6.7 Results of 4-Methyl-5-(beta-hydroxyethyl)thiazole content determination

[0273] (1) Exclusivity

[0274] Take the standard and sample separately, and analyze them on the instrument. See the specific chromatograms below. Figure 11 The retention time of 4-methyl-5-(beta-hydroxyethyl)thiazole was 16.49, and the separation effect of each substance was also good.

[0275] (2) Linearity and Range

[0276] A standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The regression equation and linear range are shown in [reference needed]. Figure 12 And Table 16.

[0277] Table 16 Linearity and Range Test Results

[0278]

[0279] Spiking recovery experiments were conducted at low, medium, and high concentration gradients. The spiked recoveries were all greater than 90% under different matrices, and the intraday CV and interday CV were both less than 10%, which met the requirements for sample quantification. The specific results are shown in Table 17.

[0280] Table 17 Spiking recoveries and CV of 4-methyl-5-(beta-hydroxyethyl)thiazole

[0281]

[0282] (3) Sample test results

[0283] The treated samples were analyzed under the chromatographic conditions described above, and the data were recorded. The content of 4-methyl-5-(beta-hydroxyethyl)thiazole was calculated using a standard curve. The specific results are shown in Table 18. The data revealed that 4-methyl-5-(beta-hydroxyethyl)thiazole was not detected in the control group and the control soaking group, but it was detected in the sulfur-fumigated group and the sulfur-fumigated soaking group. Therefore, by determining whether 4-methyl-5-(beta-hydroxyethyl)thiazole is present in bamboo shoots, sulfur-fumigated bamboo shoots can be identified.

[0284] Table 18 Content of 4-methyl-5-(beta-hydroxyethyl)thiazole in bamboo shoot samples

[0285]

[0286] III. Summary

[0287] This chapter divides the experiment into four groups: a control group, a control soaking group, a sulfur fumigation group, and a sulfur fumigation soaking group. By measuring the content of sulfur, thiol groups, adenosine, vitamin B1, and 4-methyl-5-(beta-hydroxyethyl)thiazole in bamboo shoots of each group, the presence or absence of sulfur fumigation was determined. The following results were obtained:

[0288] (1) The changes in the sulfur content of bamboo shoots and sulfur-fumigated bamboo shoots before and after soaking in water were determined by ICP-MS. It was found that the sulfur content in the sulfur-fumigated and sulfur-soaked bamboo shoots was much higher than that in fresh bamboo shoots. Therefore, this method can provide a reference for identifying whether bamboo shoots have been fumigated with sulfur.

[0289] (2) Using a total sulfhydryl assay kit, the changes in total sulfhydryl content in bamboo shoots and sulfur-fumigated bamboo shoots during soaking were determined. It was found that after soaking in water for 4 days, the total sulfhydryl content in sulfur-fumigated bamboo shoots was still much higher than that in fresh bamboo shoots, at which point the bamboo shoots began to emit a foul odor. Therefore, determining the sulfhydryl content in bamboo shoot juice is a rapid and effective method to identify whether bamboo shoots have been fumigated with sulfur.

[0290] (3) An HPLC method for the detection of adenosine was established, using adenosine as a potential biomarker for sulfur-fumigated bamboo shoots. The linearity and stability of the method were verified. Quantitative analysis showed that adenosine has high sensitivity and specificity in distinguishing whether bamboo shoots have been fumigated with sulfur.

[0291] (4) The content of VB1 in each treatment group of bamboo shoots was determined by HPLC fluorescence method, and a significant difference in VB1 content before and after sulfur fumigation was found. Therefore, its secondary metabolites were further verified and determined. A detection method for 4-methyl-5-(beta-hydroxyethyl)thiazole was established, and the linearity and stability of the method were verified. After testing the samples, it was found that 4-methyl-5-(beta-hydroxyethyl)thiazole was detected in the sulfur-fumigated samples, but not in fresh bamboo shoots. Therefore, the presence of sulfur fumigation in bamboo shoots can be identified by measuring the content of VB1 and 4-methyl-5-(beta-hydroxyethyl)thiazole. The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting sulfur-fumigated bamboo shoots based on vitamin B1 and its metabolites as sulfur-free markers in fresh bamboo shoots, characterized in that, Use one or a combination of two of the following steps: I. Determination of Vitamin B1 Content; The determination of vitamin B1 content can be used to identify whether bamboo shoots have been treated with sulfur. The vitamin B1 content in sulfur-treated bamboo shoots is lower than that in fresh bamboo shoots. The determination of vitamin B1 content includes the following steps: (1) Weigh bamboo shoot powder, add it to HCl solution and shake well; acid hydrolysis; cooling; adjust pH with sodium acetate solution; add protease-amylase mixture for enzymatic hydrolysis, and then cool to room temperature; (2) Derivatization treatment: Transfer the supernatant to alkaline potassium ferricyanide solution, vortex and mix, add n-butanol, vortex and let stand, take the upper layer solution and filter it through an organic microporous membrane, and then perform the determination on the instrument; take another standard solution and derivatize it simultaneously with the test solution; The conditions for the liquid chromatography instrument are as follows: Column: Agilent C18, 150 mm × 4.6 mm, 5 μm; Mobile phase: 0.01 mol / L sodium acetate solution - methanol, with a sodium acetate solution to methanol ratio of 80:20; Fluorescence detector: excitation wavelength 375 nm, emission wavelength 435 nm; Flow rate: 0.8 mL / min; Injection volume: 20 μL; Column temperature: 35℃; II. Determination of 4-methyl-5-(beta-hydroxyethyl)thiazole content; The determination of 4-methyl-5-(beta-hydroxyethyl)thiazole content in bamboo shoots can help identify whether bamboo shoots have been sulfur-fumigated. Sulfur-fumigated bamboo shoots contain 4-methyl-5-(beta-hydroxyethyl)thiazole, while fresh bamboo shoots do not. The determination of the 4-methyl-5-(beta-hydroxyethyl)thiazole content includes the following steps: 1) Add bamboo shoot powder to a pre-cooled methanol solution, vortex mix, centrifuge, and collect the supernatant for analysis. Chromatographic conditions: Column: Agilent C18 column; Column temperature: 30℃; Flow rate: 0.8 mL / min; Mobile phase: water-methanol; The mobile phase conditions for liquid chromatography are as follows: ； Injection volume: 10 μL; Autosampler temperature: 4℃; Detection wavelength: 249 nm.

2. The method for detecting sulfur-fumigated bamboo shoots based on vitamin B1 and its metabolites as sulfur-free markers in fresh bamboo shoots, as described in claim 1, is characterized in that... The determination of vitamin B1 content also includes the following steps: (3) Calculation of results Vitamin B1 content is calculated using the following formula: ; Where X: vitamin B1 content in the sample; c: vitamin B1 concentration; V: final volume of the test solution; f : Dilution factor of the test solution before derivatization; m: Mass of the sample.

3. The method for detecting sulfur-fumigated bamboo shoots based on vitamin B1 and its metabolites as sulfur-free markers in fresh bamboo shoots, as described in claim 2, is characterized in that... Step (3) specifically involves weighing thiamine hydrochloride and preparing standard solutions of 1 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 25 mg / L and 50 mg / L with HCl solution, and then measuring them on the instrument.

4. The method for detecting sulfur-fumigated bamboo shoots based on vitamin B1 and its metabolites as sulfur-free markers in fresh bamboo shoots, as described in claim 1, is characterized in that... The determination of the 4-methyl-5-(beta-hydroxyethyl)thiazole content also includes the following steps: 2) Preparation of standard solutions; 3) Quantitative analysis and calculation methods The content of 4-methyl-5-(beta-hydroxyethyl)thiazole in the sample was calculated using a standard curve.

5. The method for detecting sulfur-fumigated bamboo shoots based on vitamin B1 and its metabolites as sulfur-free markers in fresh bamboo shoots according to claim 4, characterized in that, Step 2) Specifically, prepare a standard stock solution of 4-methyl-5-(beta-hydroxyethyl)thiazole standard with methanol solution, and then dilute it sequentially to prepare standard solutions of 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 25 mg / mL and 50 mg / mL for instrumental analysis.

6. The application of the method for detecting sulfur-fumigated bamboo shoots based on vitamin B1 and its metabolites as sulfur-free markers in any one of claims 1 to 5 in the detection of sulfur-fumigated bamboo shoots.

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