Method for analyzing sulfur metabolite after grape fumigation

Abstract

The invention discloses a method for analyzing sulfur metabolites after grape fumigation, and relates to the field of agricultural product postharvest preservation, the method comprises the following steps: preparing experimental samples, and carrying out grouping design to obtain grouped grape samples; performing fumigation treatment on the experimental groups in the grouped grape samples by using stable isotope labeled sulfur dioxide gas to obtain fumigated grape samples; sampling and pre-treating the fumigated grape sample at a preset time point to obtain a sample solution at a series of time points; analyzing the sample solution at the series of time points by using a liquid chromatography-mass spectrometry technology to obtain mass spectrometry data of stable isotope information; and mass spectrum data based on the stable isotope information. According to the method, the cognitive leap from static content detection to dynamic process tracking is realized; safety evaluation is carried out through the identified key sulfur metabolites which are directly caused by fumigation and stably exist, so that the accuracy and perspectiveness of an evaluation result are improved.

Description

Technical Field

[0001] This invention relates to the field of postharvest preservation of agricultural products, and in particular to an analytical method for sulfur metabolites after grape fumigation. Background Technology

[0002] In the field of postharvest preservation of agricultural products, sulfur dioxide fumigation is a common technique for extending the storage and shelf life of grapes. The safety assessment of this technique has long relied on strict monitoring of the residual amount of free sulfur dioxide or sulfite in the product. Related detection methods, such as titration and ion chromatography, have formed mature standard operating procedures and have been incorporated into the food safety regulations of various countries. With the advancement of analytical technology, liquid chromatography-mass spectrometry has been applied to the research of sulfur-fumigated agricultural products, enabling more precise qualitative and quantitative analysis of sulfur-containing compounds in the fruit.

[0003] Although existing analytical techniques can identify changes in the static content of metabolites after fumigation treatment, they still face challenges in accurately distinguishing whether these changes are due to the direct binding and transformation of exogenous sulfur dioxide or to endogenous metabolic fluctuations in the fruit's own stress response. The accuracy and specificity of the conclusions of existing methods in supporting the safety assessment of sulfur-fumigated grape products need to be further improved. There is an urgent need for an analytical method that can clearly isolate and track the metabolic pathways of exogenous sulfur from a complex metabolic background. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides an analytical method for sulfur metabolites after grape fumigation, which solves the core problem that existing technologies cannot clearly distinguish and track the specific metabolic pathways of exogenous sulfur dioxide in grapes.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a method for analyzing sulfur metabolites after grape fumigation, comprising: preparing experimental samples and designing groupings to obtain grouped grape samples;

[0008] The experimental group of grape samples in the grouped samples was fumigated with stable isotope-labeled sulfur dioxide gas to obtain fumigated grape samples.

[0009] The fumigated grape samples were sampled and pretreated at preset time points to obtain sample solutions at a series of time points.

[0010] The sample solutions at the series of time points were analyzed using liquid chromatography-mass spectrometry to obtain mass spectrometry data with stable isotope information.

[0011] Based on the mass spectrometry data of the stable isotope information, the stable isotope labeling rate of sulfur metabolites is calculated, and a dynamic metabolic flow model of the sulfur metabolites is constructed.

[0012] Based on the dynamic metabolic flow model, key sulfur metabolites induced by fumigation were identified, and the safety of grape products was assessed based on these key sulfur metabolites.

[0013] As a preferred embodiment of the analytical method for sulfur metabolites after grape fumigation according to the present invention, the method includes the following steps: preparing experimental samples and designing groupings to obtain grouped grape samples:

[0014] Select fresh table grapes of similar variety, maturity, and size, free from mechanical damage and disease, to obtain fresh table grapes to be processed;

[0015] Gently wash the surface of the fresh table grapes with deionized water and let them air dry naturally to obtain clean fresh table grapes.

[0016] Fresh, clean table grapes were randomly divided into an experimental group and a control group to obtain grape samples for each group.

[0017] As a preferred embodiment of the analytical method for sulfur metabolites after grape fumigation according to the present invention, the method includes: fumigating the experimental group of the grouped grape samples with stable isotope-labeled sulfur dioxide gas to obtain fumigated grape samples, comprising the following steps:

[0018] The experimental group of grape samples was placed in a closed fumigation chamber with a controlled environment to obtain the experimental group grape samples placed in the closed fumigation chamber.

[0019] Grape samples from the experimental group were obtained by introducing a precisely controlled concentration of stable isotope-labeled sulfur dioxide gas into a sealed fumigation chamber.

[0020] Grape samples from the experimental group were treated under preset fumigation conditions in an environment of stable isotope-labeled sulfur dioxide gas to obtain fumigated grape samples.

[0021] As a preferred embodiment of the analytical method for sulfur metabolites after grape fumigation according to the present invention, the method includes the following steps: sampling and pretreatment of the fumigated grape samples at preset time points to obtain sample solutions at a series of time points:

[0022] At a series of preset time points after fumigation, sub-samples were collected from the fumigated grape samples to obtain grape sub-samples at different time points. Liquid nitrogen was used to rapidly freeze the grape sub-samples at different time points to obtain frozen grape sub-samples.

[0023] Frozen grape seed samples were ground into fine powder under low temperature conditions to obtain grape tissue powder. A certain amount of grape tissue powder was weighed and added to a pre-cooled extraction solvent for ultrasonic-assisted extraction to obtain crude extract.

[0024] The crude extract is centrifuged, and the supernatant is collected to obtain a clear extract. The clear extract is then purified by passing it through an activated solid-phase extraction column to obtain a purified eluent.

[0025] The purified eluent was dried and concentrated with nitrogen, and then redissolved with the initial mobile phase by liquid chromatography to obtain sample solutions at a series of time points.

[0026] As a preferred embodiment of the analytical method for sulfur metabolites after grape fumigation according to the present invention, the method includes the following steps: analyzing the sample solution at the series of time points using liquid chromatography-mass spectrometry to obtain mass spectrometry data with stable isotope information.

[0027] Sample solutions at a series of time points were injected into an ultra-high performance liquid chromatograph for separation, and the eluent after separation was obtained.

[0028] The separated eluent was directly introduced into a high-resolution mass spectrometer for ionization and mass analysis to obtain mass spectrometry data with stable isotope information.

[0029] As a preferred embodiment of the analytical method for sulfur metabolites after grape fumigation according to the present invention, the method includes the following steps: calculating the stable isotope labeling rate of sulfur metabolites based on the mass spectrometry data of the stable isotope information, and constructing a dynamic metabolic flux model of the sulfur metabolites:

[0030] The isotopic peak area data of sulfur metabolites are obtained by extracting the 32s background peak area and the 34s labeled peak area of ​​each sulfur metabolite from the mass spectrometry data of stable isotope information.

[0031] Based on the isotopic peak area data of sulfur metabolites, the percentage of the 34s labeled peak area of ​​each sulfur metabolite to the total peak area at different time points is calculated to obtain the stable isotopic labeling rate of sulfur metabolites.

[0032] By utilizing the curves of stable isotope labeling rates of sulfur metabolites over time, a dynamic metabolic flux model of sulfur metabolites is constructed using a kinetic fitting algorithm.

[0033] As a preferred embodiment of the analytical method for sulfur metabolites after grape fumigation according to the present invention, the identification of key sulfur metabolites induced by fumigation based on the dynamic metabolic flux model includes the following steps:

[0034] The trend of stable isotope labeling rate of each sulfur metabolite over time was analyzed in the dynamic metabolic flux model of sulfur metabolites to obtain the labeling dynamic characteristics of sulfur metabolites.

[0035] Based on the labeling dynamic characteristics of sulfur metabolites, sulfur metabolites with high stable isotope labeling rates and persistent existence in the later stages of storage and transportation were screened to obtain a list of candidate key sulfur metabolites.

[0036] By comparing the list of candidate key sulfur metabolites with data from unfumigated control group samples, it was confirmed that the sulfur metabolites in the list were present in extremely low amounts in the control group, thus identifying key sulfur metabolites induced by fumigation.

[0037] As a preferred embodiment of the analytical method for sulfur metabolites after grape fumigation according to the present invention, the safety assessment of grape products based on the key sulfur metabolites includes the following steps:

[0038] To determine the content of key sulfur metabolites induced by fumigation in grape products and obtain content data of key sulfur metabolites;

[0039] By comparing the content data of key sulfur metabolites with known toxicological reference values, the potential risk level of key sulfur metabolites can be obtained.

[0040] Based on the potential risk levels of key sulfur metabolites, a safety assessment report for grape products based on key sulfur metabolites is generated.

[0041] In a second aspect, the present invention provides a computer device including a memory and a processor, wherein the memory stores a computer program, wherein when the computer program is executed by the processor, it implements any step of the method for analyzing sulfur metabolites after grape fumigation as described in the first aspect of the present invention.

[0042] Thirdly, the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein: when the computer program is executed by a processor, it implements any step of the method for analyzing sulfur metabolites after grape fumigation as described in the first aspect of the present invention.

[0043] The beneficial effects of this invention are as follows: By using sulfur dioxide labeled with stable isotopes for fumigation treatment, a unique isotopic fingerprint is provided for metabolite tracing, enabling effective differentiation between sulfur metabolites induced by fumigation and the endogenous background of fruits. This solves the core problem that existing technologies cannot clearly define causal relationships. By constructing a dynamic metabolic flow model, the migration paths and conversion rates of sulfur atoms among different metabolites are quantitatively analyzed, revealing the dynamic laws of sulfur metabolism and achieving a cognitive leap from static content detection to dynamic process tracking. By identifying key sulfur metabolites that are directly caused by fumigation and exist stably, safety assessments are conducted, improving the accuracy and foresight of the assessment results and providing reliable technical support for the formulation of scientific safety standards. Attached Figure Description

[0044] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 A flowchart of the analytical method for sulfur metabolites after grape fumigation.

[0046] Figure 2 This is a schematic diagram of sample collection at a series of time points. Detailed Implementation

[0047] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0048] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0049] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0050] Reference Figures 1-2 As one embodiment of the present invention, this embodiment provides a method for analyzing sulfur metabolites after grape fumigation, comprising the following steps:

[0051] S1. Prepare experimental samples and design groupings to obtain grouped grape samples.

[0052] S1.1 Select fresh table grapes of similar variety, maturity, and size, free from mechanical damage and disease, to obtain fresh table grapes to be processed.

[0053] Furthermore, visual observation and manual sorting are conducted based on clear sensory and physical standards to ensure that the selected fresh table grapes are highly homogeneous in terms of genetic background, physiological stage, and basic morphology. Selecting fresh table grapes of the same variety is to control the potential impact of genetic factors on metabolome composition; ensuring that the ripeness and size are basically consistent is to ensure that all fresh table grapes are at similar physiological metabolic levels and have similar specific surface areas, thus producing comparable responses to fumigation treatment; and excluding fresh table grapes with mechanical damage and diseases is to prevent local abnormal metabolic activity or microbial contamination from interfering with the overall sulfur metabolite analysis.

[0054] S1.2. Gently wash the surface of the fresh table grapes to be treated with deionized water and let them air dry naturally to obtain fresh table grapes with a clean surface.

[0055] Furthermore, to remove external contaminants such as dust, pesticide residues, natural wax layer, and environmental microorganisms from the surface of fresh table grapes, while avoiding damage to the grape skin structure, gentle rinsing with deionized water can effectively dissolve and wash away water-soluble contaminants and minimize the impact of ions in tap water on the microenvironment of the fresh table grapes. The subsequent natural drying process allows the moisture on the grape skin to evaporate gently, avoiding physical damage to the natural protective layer of the grape skin caused by rapid drying or wiping, thus maintaining its integrity and permeability.

[0056] S1.3. Fresh, clean table grapes were randomly divided into experimental and control groups to obtain grape samples for each group.

[0057] Furthermore, the fresh, clean table grapes treated in the aforementioned steps were randomly assigned to the experimental and control groups. Randomization means that each bunch or each grape has an equal chance of being assigned to either the experimental or control group. This assignment does not depend on any subjective judgment or known order. Randomization can evenly distribute subtle differences that are difficult to detect or control, such as small physiological fluctuations between individuals, to the experimental and control groups, thereby probabilistically ensuring that the two groups are statistically comparable in all characteristics before fumigation treatment.

[0058] Specifically, this grouping strategy ensures that any systematic differences observed between the experimental and control groups in subsequent analyses can be statistically more readily inferred to be due to the experimental factor of fumigation treatment rather than grouping bias, thus obtaining statistically comparable initial state grape samples for subsequent fumigation treatment and control groups as baseline references.

[0059] S2. Using stable isotope-labeled sulfur dioxide gas, the experimental group of the grouped grape samples was fumigated to obtain fumigated grape samples.

[0060] S2.1 Place the experimental group of grape samples in a controlled environment in a closed fumigation chamber to obtain the experimental group grape samples placed in the closed fumigation chamber.

[0061] Furthermore, the experimental group of grape samples was transferred from an open or fluctuating external environment to a sealed space where all key parameters could be precisely controlled. The grape samples were carefully placed in the sealed fumigation chamber, ensuring that the gas flow path could evenly cover the sample surface. The fundamental necessity of using a sealed fumigation chamber is to effectively isolate the interference of external air, prevent the stable isotope-labeled sulfur dioxide gas from being diluted or reacting uncontrollably with other substances in the environment, thereby ensuring the purity and stability of the fumigation atmosphere. The controllable environment means that the sealed fumigation chamber has the ability to regulate temperature and relative humidity. Precise temperature control can maintain the grape samples in a specific physiological metabolic state and affect gas molecular dynamics, while precise humidity control is related to the opening and closing state of the grape skin stomata and the gas adsorption and permeation efficiency, which is crucial for obtaining consistent and repeatable fumigation results.

[0062] S2.2. Introduce a stable isotope-labeled sulfur dioxide gas with a precisely controlled concentration into a sealed fumigation chamber to obtain experimental grape samples in a stable isotope-labeled sulfur dioxide gas environment.

[0063] Furthermore, relying on professional gas mixing and flow control devices, pre-prepared stable isotope-labeled sulfur dioxide gas is introduced into the sealed fumigation chamber at a controllable flow rate and duration. Through real-time monitoring or theoretical calculation, the gas concentration in the chamber is ensured to reach the preset target. The stable isotope-labeled sulfur dioxide gas, such as sulfur-34-labeled sulfur dioxide, differs in quality from sulfur-32, which is more abundant in nature. This quality difference allows metabolites derived from fumigants to be clearly distinguished from the endogenous background sulfur metabolites of grapes in subsequent high-resolution mass spectrometry analysis through their unique mass spectrometric signals.

[0064] Specifically, precise control of gas concentration simulates the fumigation conditions in actual preservation processes and ensures consistency in fumigation intensity. This avoids the situation where excessively high concentrations can mask normal metabolic pathways due to plant toxicity or insufficient concentrations can result in undetectable induction signals. By successfully introducing a traceable tracer into the biological system, a decisive prerequisite for achieving unambiguous metabolic flow analysis is created. The sealed fumigation chamber is filled with sulfur dioxide gas of known concentration and stable isotope labeling, exposing the experimental group grape samples to a specific fumigation atmosphere, thus obtaining experimental group grape samples in a stable isotope labeling sulfur dioxide gas environment.

[0065] S2.3. Treat the experimental group grape samples in a stable isotope-labeled sulfur dioxide gas environment under preset fumigation conditions to obtain fumigated grape samples.

[0066] Furthermore, the experimental group of grape samples in a stable isotope-labeled sulfur dioxide gas environment were continuously exposed for a necessary period of time under set environmental parameters to complete the gas penetration and absorption and the initial biochemical reaction. The preset fumigation conditions were a comprehensive set of parameters, including at least the fumigation duration and the guaranteed constant temperature and humidity. The fumigation duration directly affected the flux of stable isotope-labeled sulfur dioxide gas into grape tissue and the depth of its participation in metabolic transformation.

[0067] Specifically, maintaining stable environmental parameters in the sealed fumigation chamber throughout the entire process is crucial to ensure that all samples experience the exact same stress intensity and duration, thereby producing comparable metabolic responses. Sufficient fumigation time allows stable isotope-labeled sulfur dioxide gas to penetrate the fruit through the epidermis or be absorbed through stomata, where it combines with endogenous components such as sugars and aldehydes to form initial stable isotope-labeled sulfur-containing derivatives. These derivatives will be the starting point for subsequent tracking of the entire metabolic pathway. By controlling the key variable of time, sufficient and reproducible interactions can occur between stable isotope-labeled sulfur dioxide gas and the grape's biochemical system, thereby generating initial metabolites with clearly defined sources, necessary for subsequent dynamic metabolic analysis. The experimental group of grape samples completed the predetermined fumigation treatment, and their bodies already contained metabolites derived from stable isotope-labeled sulfur dioxide, thus obtaining fumigated grape samples.

[0068] S3. At preset time points, sample and pre-treat the fumigated grape samples to obtain sample solutions at a series of time points.

[0069] S3.1 At a series of preset time points after fumigation, sub-samples are collected from the fumigated grape samples to obtain grape sub-samples at different time points. Liquid nitrogen is used to rapidly freeze the grape sub-samples at different time points to obtain frozen grape sub-samples.

[0070] Furthermore, to obtain snapshots of the metabolic state of fumigated grape samples at different stages of storage and transportation, a series of representative preset time points were selected for sampling after fumigation to cover the key periods of metabolite generation, transformation, and degradation, thereby constructing a complete time series profile. When collecting subsamples from fumigated grape samples, it is necessary to ensure that each sampling is representative to reduce the impact of individual differences on the time series data. Immediately after collection, the grape subsamples at different time points were rapidly frozen using liquid nitrogen. This process of instantly freezing the tissue at the extremely low temperature of liquid nitrogen can quench enzyme activity in the sample to the maximum extent and effectively prevent any biochemical reactions from continuing after sampling, thereby completely fixing the metabolite profile at the moment of sampling.

[0071] Specifically, rapid freezing can truly preserve the metabolic state of the sample at a specific time point, avoiding metabolite degradation or transformation caused by slow freezing or processing delays. This ensures that subsequent analysis results can accurately reflect the true dynamics of metabolic evolution after fumigation, resulting in a series of frozen grape seed samples collected at different time points and with their instantaneous metabolic states fixed.

[0072] S3.2. Grind the frozen grape seed sample into a fine powder under low temperature conditions to obtain grape tissue powder. Weigh a certain amount of grape tissue powder, add pre-cooled extraction solvent, and perform ultrasonic-assisted extraction to obtain crude extract.

[0073] Furthermore, operating under low-temperature conditions prevents the heat generated during grinding from causing metabolite degradation or alteration. Frozen grape seed samples are typically ground into fine powder using pre-cooled grinding equipment in a liquid nitrogen environment. Obtaining uniform grape tissue powder greatly increases the contact surface area during subsequent extraction, thereby improving extraction efficiency. Accurately weighing a certain amount of grape tissue powder ensures comparability between samples and between samples at different time points, enabling quantitative or semi-quantitative analysis. Adding pre-cooled extraction solvent for ultrasonic-assisted extraction utilizes the solvent's solubility for target metabolites, as well as the cavitation effect and mechanical action of ultrasound, to promote the rapid diffusion and dissolution of metabolites from cell debris into the solvent. The pre-cooled solvent and environment help maintain the chemical stability of metabolites.

[0074] Specifically, ultrasound-assisted extraction enables rapid and efficient extraction while minimizing the risk of decomposition of thermally unstable metabolites in a low-temperature environment. Metabolites are transferred from grape tissue powder to a solvent to form a crude extract containing the target analyte.

[0075] S3.3 Centrifuge the crude extract, collect the supernatant to obtain a clear extract, and purify the clear extract by passing it through an activated solid-phase extraction column to obtain a purified eluent.

[0076] Furthermore, the extract is purified to remove interfering substances and ensure the accuracy and stability of subsequent instrumental analysis. Centrifugation of the crude extract uses the centrifugal force field to precipitate insoluble tissue residues, thereby obtaining a clear extract by taking the supernatant. This step initially removes particulate impurities. After obtaining the clear extract, it is purified by passing it through an activated solid-phase extraction column. The activation process restores the activity of the adsorbent in the solid-phase extraction column and creates a suitable retention environment. When the clear extract passes through, the target metabolites are selectively retained according to the interaction between their polar functional groups and the adsorbent, while strongly polar or non-polar interfering substances such as sugars, pigments, and organic acids are eluted and removed by the eluent. Finally, an appropriate elution solvent is used to elute the target metabolites from the solid-phase extraction column.

[0077] Specifically, solid-phase extraction purification can specifically or selectively enrich target analytes and effectively remove complex matrix components, significantly reducing matrix inhibition effects and background noise during subsequent liquid chromatography-mass spectrometry analysis, improving detection sensitivity and chromatographic peak shape, and yielding a purified eluent with a large number of interfering substances removed.

[0078] S3.4. The purified eluent is dried and concentrated by nitrogen gas, and then reconstituted with the initial mobile phase of liquid chromatography to obtain sample solutions at a series of time points.

[0079] Furthermore, nitrogen purging and concentration of the purified eluent utilizes inert nitrogen as a purge gas under gentle heating conditions to accelerate the evaporation of volatile solvents, thereby enriching the target metabolites at the bottom of the sample tube, concentrating the solution, and improving detection sensitivity. Reconstitution with the initial mobile phase of liquid chromatography after concentration ensures that the reconstituted sample solution matches the initial elution conditions of liquid chromatography in terms of solvent strength and pH, thus avoiding peak broadening or retention time shifts due to solvent effects and guaranteeing the reproducibility and quality of chromatographic separation.

[0080] Specifically, by concentrating and enriching the target analyte, the method's ability to detect low-abundance metabolites was improved. On the other hand, by using a solvent that matches the chromatographic starting conditions for reconstitution, the chromatographic separation process was ensured to be in the optimal state from the beginning after injection, resulting in sharp and symmetrical chromatographic peaks. Sample solutions at a series of time points that perfectly matched the instrument analysis conditions were prepared.

[0081] S4. Using liquid chromatography-mass spectrometry (LC-MS) to analyze sample solutions at a series of time points, mass spectrometry data of stable isotope information are obtained.

[0082] S4.1. Inject the sample solutions at a series of time points into an ultra-high performance liquid chromatograph for separation to obtain the separated eluent.

[0083] Furthermore, after injecting sample solutions from a series of time points into the ultra-high performance liquid chromatograph (UHPLC) via an autosampler, the sample solutions flow through the chromatographic column under high pressure. The column is filled with tiny stationary phase particles, and separation is achieved based on the difference in the partition coefficients of the target sulfur metabolites between the mobile and stationary phases. The hydrophilic interaction column is suitable for separating polar metabolites, allowing sulfur-containing compounds such as thioamino acids and organic acids to elute sequentially according to their polarity. By optimizing the gradient program of the mobile phase, the elution intensity can be controlled, allowing metabolites of different polarities to elute from the column at different retention times. Compared with traditional liquid chromatography, the UHPLC, which uses high pressure and small particle size column technology, has higher resolution and faster analysis speed. By separating the complex mixture of metabolites in the sample solutions from a series of time points according to their physicochemical properties, each metabolite enters the mass spectrometer as a single component as possible at different time points, thereby greatly reducing the matrix inhibition effect during ionization and avoiding signal overlap caused by different substances entering the mass spectrometer simultaneously. The mixed sample solution is separated into a series of relatively pure components that elute in chronological order, which is the separated eluent.

[0084] S4.2. The separated eluent is directly introduced into a high-resolution mass spectrometer for ionization and mass analysis to obtain mass spectrometry data with stable isotope information.

[0085] Furthermore, the separated eluent is directly introduced into the ion source of the high-resolution mass spectrometer via an interface. At the ion source, the eluent is atomized and ionized to form charged ions. Electrospray ionization is a mild ionization method suitable for polar and thermally unstable metabolites, producing stable molecular ion signals. The generated ions are introduced into a mass analyzer. High-resolution mass analyzers, such as time-of-flight or orbital traps, can measure the mass-to-charge ratio of each ion with extremely high mass accuracy. The key capability of a high-resolution mass spectrometer lies in its ability to clearly distinguish ions with similar mass numbers; for example, it can accurately distinguish between a metabolite composed of sulfur 32 and the same metabolite composed of sulfur 34 because of the difference of two mass units. The mass spectrometry data acquired through full scan mode not only contains the precise molecular weight information corresponding to each chromatographic peak but also the abundance distribution information of its isotopic peak types.

[0086] Specifically, the high-resolution mass spectrometer provides high quality, precision, and resolution, enabling unambiguous determination of the elemental composition of metabolites and precise measurement of the signal intensity of isotope labels such as sulfur-32 and sulfur-34. This provides a direct basis for subsequent calculation of isotope labeling rates, resulting in a complete dataset containing the precise mass number and isotope abundance information of each metabolite, i.e., mass spectrometry data with stable isotope information.

[0087] S5. Based on mass spectrometry data with stable isotope information, calculate the stable isotope labeling rate of sulfur metabolites and construct a dynamic metabolic flow model of sulfur metabolites.

[0088] S5.1 Extract the 32s background peak area and 34s labeled peak area corresponding to each sulfur metabolite from the mass spectrometry data of stable isotope information to obtain the isotope peak area data of sulfur metabolites.

[0089] Furthermore, when processing mass spectrometry data of stable isotope information, the target sulfur metabolite must first be identified based on the known precise mass number or retention time. Then, the chromatographic peak area of ​​the background form metabolite composed of 32 sulfur atoms and the chromatographic peak area of ​​the labeled form metabolite composed of 34 sulfur atoms are integrated on the extracted ion chromatogram. The mass precision provided by the high-resolution mass spectrometer ensures that the 32s background peak and the 34s labeled peak, which differ by two atomic mass units, can be accurately distinguished to avoid interference from neighboring ions.

[0090] Specifically, peak area is extracted instead of simply relying on peak height because peak area is proportional to ion current intensity and can more accurately reflect the relative content of metabolites in the sample. The qualitative identification information contained in the mass spectrometry data with stable isotope information is transformed into numerical isotope peak area data of sulfur metabolites that can be used for subsequent quantitative calculations, providing a direct and reliable input for the calculation of labeling rate. A quantitative dataset of the 32s background peak area and 34s labeled peak area of ​​each sulfur metabolite at different time points was obtained, namely the isotope peak area data of sulfur metabolites.

[0091] S5.2 Based on the isotopic peak area data of sulfur metabolites, calculate the percentage of the 34s labeled peak area of ​​each sulfur metabolite to the total peak area at different time points to obtain the stable isotopic labeling rate of sulfur metabolites.

[0092] Furthermore, the key computational steps transform the raw signal into a traceability indicator with clear biological significance. The calculation process, based on isotopic peak area data of sulfur metabolites, divides the 34s labeled peak area corresponding to each sulfur metabolite at each time point by the sum of its 34s labeled peak area and its 32s background peak area, and then multiplies by a percentage to obtain the stable isotopic labeling rate of that metabolite at that time point. This percentage value intuitively reflects the concentration of sulfur metabolite molecules in the molecular population at a specific time point due to exogenous fumigation agents. The proportion of derived molecules and the stable isotope labeling rate are ratio indicators that, to some extent, offset the changes in absolute signal intensity caused by small fluctuations in sample pretreatment or instrument injection volume, making data from different samples at different time points more comparable.

[0093] Specifically, the abstract mass spectrometry signal is transformed into a quantitative parameter that can directly measure the source of metabolites. The stable isotope labeling rate of sulfur metabolites clearly shows the degree of exogenous sulfur incorporation into a specific metabolite pool, providing core evidence for determining whether the metabolite was directly induced by fumigation. The source contribution quantification value of each sulfur metabolite at different time points is obtained, namely the stable isotope labeling rate of sulfur metabolites.

[0094] Specifically, the expression is:

[0095] ;

[0096] in, For time points The percentage of the target sulfur metabolite in the 34S labeled form. For time points The peak area of ​​the target sulfur metabolite in 34S labeled form detected by mass spectrometry. For time points The peak area in the 32s background form corresponding to the same target sulfur metabolite detected by mass spectrometry. For a point in time.

[0097] S5.3. Using the curves of stable isotope labeling rates of sulfur metabolites over time, a dynamic metabolic flow model of sulfur metabolites is constructed using a kinetic fitting algorithm.

[0098] Furthermore, plotting the stable isotope labeling rate of each key sulfur metabolite relative to time yields a curve describing its labeling dynamics. This curve contains information about the generation and conversion rate of the metabolite. By selecting appropriate kinetic model equations, such as first- or second-order reaction kinetic equations describing the conversion of precursors into products, and using kinetic fitting algorithms such as nonlinear least squares, the experimental curve of the stable isotope labeling rate of sulfur metabolites changing with time is fitted and the model parameters are optimized to minimize the difference between the model calculation curve and the experimental data points.

[0099] Specifically, a successful fit means finding a mathematical expression that best describes the dynamics of the metabolite label, i.e., a dynamic metabolic flow model of sulfur metabolites. This model includes key kinetic parameters such as the generation rate constant and the conversion rate constant, going beyond static snapshot-style analysis. By constructing a dynamic metabolic flow model of sulfur metabolites, the flow direction and speed of sulfur atoms between different metabolites can be quantitatively revealed, thereby analyzing the dynamic structure and regulatory laws of the entire metabolic network. This yields a mathematical relationship that can quantitatively describe the generation and conversion kinetics of sulfur metabolites, i.e., a dynamic metabolic flow model of sulfur metabolites.

[0100] Specifically, the expression is:

[0101] ;

[0102] in, For time points Metabolites stable isotope labeling rate, Precursor The initial marking rate at time zero, For the precursor Transformation into metabolites The reaction rate constant, For the precursor The rate constant of the reaction that converts a substance into another metabolite. Metabolites The rate constant of its own further transformation and degradation. To describe metabolites The exponential term that decays naturally over time. To describe the precursor The exponential term that consumes time. It is a metabolite. It is a precursor.

[0103] S6. Based on the dynamic metabolic flow model, key sulfur metabolites induced by fumigation were identified.

[0104] S6.1 Analyze the trend of stable isotope labeling rate of each sulfur metabolite over time in the dynamic metabolic flux model of sulfur metabolites to obtain the labeling dynamic characteristics of sulfur metabolites.

[0105] Furthermore, the morphological and kinetic characteristics of the stable isotope labeling rate of each sulfur metabolite over time are identified. For example, the maintenance of the plateau period when the slope of the curve rises from the starting point to the peak and whether a downward trend appears in the later stage are observed. The labeling dynamic characteristics of sulfur metabolites may be characterized by rapid labeling, i.e., the labeling rate rises rapidly and reaches a high value, indicating that the metabolite is a direct primary product after fumigation; or it may be characterized by slow accumulation, i.e., the labeling rate rises slowly, indicating that it may be a secondary product formed through multiple transformations; or it may be characterized by an instantaneous peak, i.e., the labeling rate rises rapidly and then falls rapidly, indicating that the metabolite is an unstable intermediate.

[0106] Specifically, by analyzing the trend of stable isotope labeling rates of each sulfur metabolite over time in the dynamic metabolic flow model of sulfur metabolites, we can infer the relative position and function of each sulfur metabolite in the metabolic network, such as whether it is a precursor, intermediate, or final product. Transforming the abstract mathematical model output into an understandable biological behavioral description of the labeling dynamic characteristics of sulfur metabolites provides a qualitative basis for subsequent rule-based screening. Summarizing the unique labeling behavior pattern of each sulfur metabolite yields the labeling dynamic characteristics of sulfur metabolites.

[0107] S6.2 Based on the labeling dynamic characteristics of sulfur metabolites, sulfur metabolites with high stable isotope labeling rates and persistent presence in the later stages of storage and transportation are screened to obtain a list of candidate key sulfur metabolites.

[0108] Furthermore, these criteria can be applied to evaluate the labeling dynamics of sulfur metabolites. For example, an ideal candidate key sulfur metabolite may exhibit a rapid rise in labeling rate to a high plateau and remain near that plateau throughout the observation window. Screening based on the labeling dynamics of sulfur metabolites can effectively exclude metabolites with low and volatile labeling rates or those that only appear briefly in the early stages from the scope of focus. Utilizing dynamic data enables targeted initial screening to obtain a list of candidate key sulfur metabolites, greatly narrowing the target range that needs in-depth evaluation and improving analytical efficiency. A list of metabolites that meets the preset risk characteristics is thus obtained, i.e., the list of candidate key sulfur metabolites.

[0109] S6.3 Compare the list of candidate key sulfur metabolites with the data of the control group samples that were not fumigated to confirm that the sulfur metabolites in the list were present in very low amounts in the control group, and identify the key sulfur metabolites induced by fumigation.

[0110] Furthermore, the detection signals of each metabolite in the experimental group samples from the candidate key sulfur metabolite list were directly compared with the detection signals of the same metabolite in the unfumigated control group samples. The confirmation process required verifying that in the unfumigated control group samples, these candidate key sulfur metabolites were either completely undetectable or their signal intensity was much lower than that in the experimental group samples, typically several orders of magnitude lower. This comparison ensured that the appearance and accumulation of the finally identified key sulfur metabolites were indeed specifically induced by the experimental factor of fumigation treatment, rather than by non-specific factors such as variety differences, environmental fluctuations, or analytical errors. This provided the final evidence of causality. By setting up an unfumigated control group for comparison, the direct induced relationship between the metabolites and fumigation treatment could be confirmed unambiguously, thus transforming the candidate key sulfur metabolite list into a confirmed set of key sulfur metabolites induced by fumigation. This completed the transition from the candidate list to the confirmed list, i.e., the identification of key sulfur metabolites induced by fumigation.

[0111] S7. Conduct safety assessments of grape products based on key sulfur metabolites.

[0112] S7.1 Determine the content of key sulfur metabolites induced by fumigation in grape products and obtain content data of key sulfur metabolites;

[0113] Furthermore, using external or internal standard methods, a series of standard solutions of known concentrations are prepared using high-purity standards for each key sulfur metabolite induced by fumigation. After injection and analysis, a standard curve is established. This curve describes the quantitative relationship between the mass spectrometry response signal (e.g., peak area) and the metabolite concentration. By substituting the mass spectrometry signal intensity of the key sulfur metabolite in the grape product sample into this standard curve, its specific content in the sample can be calculated. To ensure accuracy, the entire quantitative process requires quality control, including using blank samples and spiked recovery samples to verify the accuracy and precision of the method.

[0114] Specifically, the presence of key sulfur metabolites was moved from a qualitative judgment of whether they were present or absent to a precise measurement of how much they were, providing a reliable numerical basis for subsequent risk comparisons and obtaining quantitative values ​​for each key sulfur metabolite in grape products, i.e., the content data of key sulfur metabolites.

[0115] S7.2 Compare the content data of key sulfur metabolites with known toxicological reference values ​​to obtain the potential risk level of key sulfur metabolites;

[0116] Furthermore, the concentration data of key sulfur metabolites are compared with these reference values ​​to calculate the ratio of the estimated exposure level of the key sulfur metabolite to the toxicological reference value, i.e., the risk quotient. Based on the magnitude of the risk quotient, different potential risk levels can be classified. For example, a risk quotient much less than 1 indicates that the potential risk is negligible, while a risk quotient close to or greater than 1 indicates that attention is required. This provides an objective, scientifically based framework that transforms the detected chemical concentrations into understandable risk levels, making the assessment results scientifically grounded and comparable. Based on the comparison results, a qualitative or semi-quantitative risk level is assigned to each key sulfur metabolite, thus obtaining the potential risk level of the key sulfur metabolite.

[0117] S7.3. Based on the potential risk levels of key sulfur metabolites, a safety assessment report for grape products based on key sulfur metabolites is generated.

[0118] Furthermore, the comprehensive process does not simply list the potential risk levels of individual key sulfur metabolites, but rather considers the combination of risk levels of all identified key sulfur metabolites, their respective concentration levels, and potential combined toxicological effects. The assessment report will systematically present the analytical results, including a list of the key sulfur metabolites detected, their respective concentration data, the corresponding toxicological reference values, and the calculated risk quotient or determined potential risk level.

[0119] Specifically, based on this comprehensive information, the report will draw an overall conclusion on the safety of grape products. For example, it may indicate that the key sulfur metabolites induced by fumigation in the product are generally at an acceptable risk level, or it may identify specific metabolites and their risks that require special attention. The report integrates the scattered risk assessment results of individual compounds into a comprehensive, structured, and clearly guiding professional document, providing direct and clear scientific support for the decision-making of regulatory authorities or production enterprises, and generating a formal document with clear conclusions, namely, a grape product safety assessment report based on key sulfur metabolites.

[0120] This embodiment also provides a computer device applicable to the analysis method of sulfur metabolites after grape fumigation, comprising: a memory and a processor; the memory is used to store computer-executable instructions, and the processor is used to execute the computer-executable instructions to implement the analysis method of sulfur metabolites after grape fumigation as proposed in the above embodiment.

[0121] The computer device can be a terminal, comprising a processor, memory, communication interface, display screen, and input devices connected via a system bus. The processor provides computing and control capabilities. The memory includes non-volatile storage media and internal memory. The non-volatile storage media stores the operating system and computer programs. The internal memory provides an environment for the operation of the operating system and computer programs stored in the non-volatile storage media. The communication interface is used for wired or wireless communication with external terminals; wireless communication can be achieved through Wi-Fi, carrier networks, NFC (Near Field Communication), or other technologies. The display screen can be an LCD screen or an e-ink screen. The input devices can be a touch layer covering the display screen, buttons, a trackball, or a touchpad on the computer device's casing, or an external keyboard, touchpad, or mouse.

[0122] This embodiment also provides a storage medium storing a computer program that, when executed by a processor, implements the analytical method for sulfur metabolites after grape fumigation as proposed in the above embodiments. The storage medium can be implemented by any type of volatile or non-volatile storage device or a combination thereof, such as Static Random Access Memory (SRAM), Electrically Erasable Programmable Read-Only Memory (EEPROM), Erasable Programmable Read Only Memory (EPROM), Programmable Red-Only Memory (PROM), Read-Only Memory (ROM), magnetic storage, flash memory, magnetic disk, or optical disk.

[0123] In summary, this invention provides a unique isotopic fingerprint for metabolite tracing by using sulfur dioxide labeled with stable isotopes for fumigation treatment. This enables effective differentiation between sulfur metabolites induced by fumigation and the endogenous background of the fruit, solving the core problem of existing technologies that cannot clearly define causal relationships. By constructing a dynamic metabolic flow model, the invention quantitatively analyzes the migration paths and conversion rates of sulfur atoms among different metabolites, revealing the dynamic laws of sulfur metabolism and achieving a cognitive leap from static content detection to dynamic process tracking. Furthermore, by identifying key sulfur metabolites that are directly caused by fumigation and are stably present, the invention conducts safety assessments, improving the accuracy and foresight of the assessment results and providing reliable technical support for the formulation of scientific safety standards.

[0124] It should be noted that 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for analyzing sulfur metabolites after grape fumigation, characterized in that: include, Prepare experimental samples and design groupings to obtain grouped grape samples; The experimental group of grape samples in the grouped samples was fumigated with stable isotope-labeled sulfur dioxide gas to obtain fumigated grape samples. The fumigated grape samples were sampled and pretreated at preset time points to obtain sample solutions at a series of time points. The sample solutions at the series of time points were analyzed using liquid chromatography-mass spectrometry to obtain mass spectrometry data with stable isotope information. Based on the mass spectrometry data of the stable isotope information, the stable isotope labeling rate of sulfur metabolites is calculated, and a dynamic metabolic flow model of the sulfur metabolites is constructed. Based on the dynamic metabolic flow model, key sulfur metabolites induced by fumigation were identified, and the safety of grape products was assessed based on these key sulfur metabolites.

2. The analytical method for sulfur metabolites after grape fumigation as described in claim 1, characterized in that: Prepare experimental samples and design groupings to obtain grouped grape samples, including the following steps: Select fresh table grapes of similar variety, maturity, and size, free from mechanical damage and disease, to obtain fresh table grapes to be processed; Gently wash the surface of the fresh table grapes with deionized water and let them air dry naturally to obtain clean fresh table grapes. Fresh, clean table grapes were randomly divided into an experimental group and a control group to obtain grape samples for each group.

3. The analytical method for sulfur metabolites after grape fumigation as described in claim 2, characterized in that: The experimental group of grape samples in the grouped samples was fumigated with stable isotope-labeled sulfur dioxide gas to obtain fumigated grape samples, including the following steps: The experimental group of grape samples was placed in a closed fumigation chamber with a controlled environment to obtain the experimental group grape samples placed in the closed fumigation chamber. Grape samples from the experimental group were obtained by introducing a precisely controlled concentration of stable isotope-labeled sulfur dioxide gas into a sealed fumigation chamber. Grape samples from the experimental group were treated under preset fumigation conditions in an environment of stable isotope-labeled sulfur dioxide gas to obtain fumigated grape samples.

4. The analytical method for sulfur metabolites after grape fumigation as described in claim 3, characterized in that: The fumigated grape samples are sampled and pretreated at preset time points to obtain sample solutions at a series of time points, including the following steps: At a series of preset time points after fumigation, sub-samples were collected from the fumigated grape samples to obtain grape sub-samples at different time points. Liquid nitrogen was used to rapidly freeze the grape sub-samples at different time points to obtain frozen grape sub-samples. Frozen grape seed samples were ground into fine powder under low temperature conditions to obtain grape tissue powder. A certain amount of grape tissue powder was weighed and added to a pre-cooled extraction solvent for ultrasonic-assisted extraction to obtain crude extract. The crude extract is centrifuged, and the supernatant is collected to obtain a clear extract. The clear extract is then purified by passing it through an activated solid-phase extraction column to obtain a purified eluent. The purified eluent was dried and concentrated with nitrogen, and then redissolved with the initial mobile phase by liquid chromatography to obtain sample solutions at a series of time points.

5. The analytical method for sulfur metabolites after grape fumigation as described in claim 4, characterized in that: The sample solutions at the aforementioned time points were analyzed using liquid chromatography-mass spectrometry (LC-MS) to obtain mass spectrometry data containing stable isotope information, including the following steps: Sample solutions at a series of time points were injected into an ultra-high performance liquid chromatograph for separation, and the eluent after separation was obtained. The separated eluent was directly introduced into a high-resolution mass spectrometer for ionization and mass analysis to obtain mass spectrometry data with stable isotope information.

6. The analytical method for sulfur metabolites after grape fumigation as described in claim 5, characterized in that: Based on the mass spectrometry data containing the stable isotope information, the stable isotope labeling rate of sulfur metabolites is calculated, and a dynamic metabolic flux model of the sulfur metabolites is constructed, including the following steps: Extract the ³²S background peak area and ³²S content of each sulfur metabolite from the mass spectrometry data containing stable isotope information. 4 S-labeled peak area, obtaining isotopic peak area data of sulfur metabolites; Based on isotopic peak area data of sulfur metabolites, the ³a of each sulfur metabolite at different time points was calculated. 4 The percentage of the S-labeled peak area to the total peak area is used to obtain the stable isotope labeling rate of sulfur metabolites. By utilizing the curves of stable isotope labeling rates of sulfur metabolites over time, a dynamic metabolic flux model of sulfur metabolites is constructed using a kinetic fitting algorithm.

7. The analytical method for sulfur metabolites after grape fumigation as described in claim 6, characterized in that: Based on the dynamic metabolic flow model, key sulfur metabolites induced by fumigation were identified, including the following steps: The trend of stable isotope labeling rate of each sulfur metabolite over time was analyzed in the dynamic metabolic flux model of sulfur metabolites to obtain the labeling dynamic characteristics of sulfur metabolites. Based on the labeling dynamic characteristics of sulfur metabolites, sulfur metabolites with high stable isotope labeling rates and persistent existence in the later stages of storage and transportation were screened to obtain a list of candidate key sulfur metabolites. By comparing the list of candidate key sulfur metabolites with data from unfumigated control group samples, it was confirmed that the sulfur metabolites in the list were present in extremely low amounts in the control group, thus identifying key sulfur metabolites induced by fumigation.

8. The analytical method for sulfur metabolites after grape fumigation as described in claim 7, characterized in that, The safety assessment of grape products based on the aforementioned key sulfur metabolites includes the following steps: To determine the content of key sulfur metabolites induced by fumigation in grape products and obtain content data of key sulfur metabolites; By comparing the content data of key sulfur metabolites with known toxicological reference values, the potential risk level of key sulfur metabolites can be obtained. Based on the potential risk levels of key sulfur metabolites, a safety assessment report for grape products based on key sulfur metabolites is generated.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, characterized in that: When the processor executes the computer program, it implements the steps of the method for analyzing sulfur metabolites after grape fumigation as described in any one of claims 1 to 8.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by the processor, it implements the steps of the method for analyzing sulfur metabolites after grape fumigation as described in any one of claims 1 to 8.

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