Auxiliary detection method and system for enzyme activity of low-temperature cooked meat products regulated by ultra-high pressure treatment

Abstract

The application discloses an auxiliary detection method and system for regulating enzyme activity of low-temperature cooked meat products by ultra-high pressure treatment, and relates to the technical field of enzyme activity detection. The method comprises the following steps: shearing and separating a meat block and an isotonic extraction solution to obtain sample extraction solution; based on an inhibition background calibration table, obtaining inhibition background intensity and a de-inhibition target window; adding an inert buffer to the sample extraction solution to obtain an in-situ reaction environment; adding a de-inhibition reference buffer to the sample extraction solution according to the de-inhibition target window to obtain a de-inhibition reference reaction environment; adding a proteolytic indicator probe, a lipid hydrolysis indicator probe and an additional internal standard enzyme reaction pair to the two reaction environments respectively to obtain a reaction time sequence; and outputting endogenous enzyme contribution ratio, microorganism-derived enzyme contribution ratio and a dominant source discrimination conclusion through a preset source splitting mapping rule. The application can still give traceable source splitting results under the condition that the formula inhibition background exists, and avoids misjudgment caused by total enzyme activity or single substrate reaction.

Description

Technical Field

[0001] This invention relates to the field of enzyme activity detection technology, and more specifically, to an auxiliary detection method and system for regulating enzyme activity in low-temperature cooked meat products under ultra-high pressure treatment. Background Technology

[0002] In the processing of chilled cooked meat products, ultra-high pressure non-thermal processing technology is often used to extend shelf life and maintain the product's tenderness, juiciness, and other quality characteristics. The core applications of this technology in actual production include microbial control of raw meat, regulation of endogenous enzyme activity, and the entire process of cold storage preservation of finished products. Ultra-high pressure processing can effectively reduce the number of viable bacteria in the product while regulating the rate of enzymatic reactions, thereby ensuring the product's quality stability during cold storage. In this application scenario, product quality deterioration mainly stems from the catalytic action of two types of enzymes: endogenous enzymes naturally present in the meat product and enzymes produced by microbial metabolism. Changes in the activity of these two types of enzymes directly affect the texture, flavor, and safety of the product during cold storage. Therefore, accurately identifying the source of the enzymes is a prerequisite for precise product quality control.

[0003] The core technology of ultra-high pressure (UHPP) processing of chilled cooked meat products lies in altering the structure of microbial cells and the conformation of enzyme molecules using high pressure. This reduces the number of viable bacteria by disrupting the integrity of the microbial cell membrane and regulates the catalytic activity of enzyme molecules by affecting their spatial structure. During microbial control, UHPP not only inhibits microbial reproduction but may also cause some microbial cells to lyse, releasing intracellular enzymes into the extracellular space. Simultaneously, extracellular enzymes secreted by microorganisms during growth and metabolism remain in the product system. Even after the number of viable bacteria decreases, these microbial-derived enzymes can continue to exert their catalytic effects under refrigeration conditions. To distinguish the contribution of endogenous enzymes from microbial-derived enzymes, existing technologies typically employ methods such as detecting total enzyme activity, analyzing the catalytic reaction characteristics of specific substrates, or detecting the content of volatile substances and degradation products generated by enzymatic reactions. The core logic is based on the differences in catalytic characteristics of enzymes from different sources, using characteristic detection indicators to differentiate the contributions of the two types of enzymes. Meanwhile, low-temperature cooked meat product formulas usually contain a variety of functional ingredients to achieve purposes such as preservation, texture improvement and flavor enhancement. These ingredients interact with enzymes in the system to form a specific reaction background environment.

[0004] However, in the actual processing and storage of chilled cooked meat products, the aforementioned enzyme source identification techniques are prone to failure. This problem does not stem from a lack of microbial enzymes, but rather from the combined effects of the unique components of the chilled cooked meat product system and the ultra-high pressure treatment. Components added to the chilled cooked meat product formulation, such as salts, nitrites, reducing agents, and phosphates, create strong inhibitory and metal ion chelating environments within the system. These environments inhibit enzymes from various sources. After ultra-high pressure treatment, intracellular enzymes released from microbial cell lysis, along with previously remaining extracellular enzymes and the meat product's own endogenous enzymes, all exhibit similar activity inhibition characteristics under this inhibitory background. The enzyme profile features that could originally be distinguished based on differences in enzyme catalytic properties are flattened by this inhibitory background, making existing identification methods based on total activity detection, specific substrate reaction analysis, or degradation product detection ineffective in distinguishing the dominant source of enzyme activity. Existing technologies lack effective means to circumvent background interference. They cannot directly eliminate the masking effect of the inhibition environment on enzyme spectrum differences, nor can they achieve accurate separation of enzyme sources by explicitly estimating the intensity of the inhibition background, thus leading to the failure of enzyme source identification.

[0005] Because it's difficult to accurately distinguish the dominant source of enzyme activity, changes in activity dominated by microbial enzymes are easily misinterpreted as insufficient regulation of endogenous enzyme activity during production. This leads to blind adjustments to ultra-high pressure processing parameters, such as increasing peak pressure or extending holding time. Such actions not only damage the product's texture and juiciness but may also increase energy consumption and costs. Conversely, misinterpreting changes in activity dominated by endogenous enzymes as a contribution from microbial enzymes may result in incorrect adjustments to refrigeration conditions or the addition of excessive preservatives. This not only fails to address the root cause of product quality deterioration but may also lead to quality defects such as softened texture and abnormal flavor during storage, shortening shelf life. Such misinterpretations also result in poor batch-to-batch quality stability, increasing production losses.

[0006] In view of this, the present invention proposes an auxiliary detection method and system for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment to solve the above problems. Summary of the Invention

[0007] To overcome the aforementioned deficiencies of the prior art and achieve the above objectives, the present invention provides the following technical solution: an auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment, comprising:

[0008] The meat chunks and isotonic extract were sheared and separated to obtain the sample extract.

[0009] Inhibition background characterization was obtained from the sample extract; based on the inhibition background characterization, the inhibition background intensity and the target window for de-inhibition were obtained by referring to the pre-established inhibition background calibration table.

[0010] An inert buffer was added to the sample extract to obtain an in-situ reaction environment. An anti-inhibition reference buffer was added to the sample extract according to the anti-inhibition target window to obtain an anti-inhibition reference reaction environment. Protein hydrolysis indicator probes, lipid hydrolysis indicator probes, and external internal standard enzyme reaction pairs were added to the two reaction environments respectively to obtain the reaction time series. The reaction time series were subtracted to obtain the inhibition relief increment curve.

[0011] Nucleic acid is extracted from the sample extract to obtain microbial lysis and release markers. The inhibition background intensity, inhibition relief increment curve, and microbial lysis and release markers are input into the preset source splitting mapping rule, and the endogenous enzyme contribution ratio, the microbial source enzyme contribution ratio, and the dominant source discrimination conclusion are output.

[0012] Furthermore, the method for shearing and separating the meat chunks from the isotonic extract to obtain the sample extract includes:

[0013] Meat pieces from the same package, along with meat pieces from the central area of ​​the package, are taken from the area closest to the packaging's adhesive surface and from the area of ​​the central meat piece, and combined as the source of the meat piece.

[0014] The isotonic extract was prepared according to the isotonic principle of matching the ionic environment of the low-temperature cooked meat products, and was placed in a low-temperature environment beforehand. The volume of the isotonic extract was recorded.

[0015] The meat chunks were placed in a sealed container and isotonic extract was added according to the volume of isotonic extract. The meat chunks and isotonic extract in the sealed container were mixed and sheared to obtain a uniform slurry.

[0016] The homogeneous slurry was allowed to stand at low temperature and then subjected to solid-liquid initial separation by fixed-size filtration to obtain filtrate.

[0017] The filtrate was placed in a centrifuge container and centrifuged at low speed. The supernatant was collected without disturbing the sedimentation layer and was defined as the sample extract.

[0018] Furthermore, methods for obtaining background suppression characterization from sample extracts include:

[0019] The sample extract was allowed to stand until the temperature stabilized within the same temperature range and then gently mixed. The sample extract was then dispensed into multiple equal-volume portions according to a predetermined volume, and the dispensing volume, dispensing temperature and dispensing time were recorded.

[0020] Based on the sample extract aliquots, conductivity data was collected by conductivity measurement, pH data was collected by pH measurement, oxidation-reduction potential data was collected by oxidation-reduction potential measurement, nitrite content data was collected by nitrite colorimetric measurement, and available divalent metal ion level data was collected by complexable metal ion indicator reaction.

[0021] Conductivity data, pH data, redox potential data, nitrite content data, and available divalent metal ion level data are compiled to form a background suppression characterization, which is then linked to the dispensing volume, dispensing temperature, and dispensing time.

[0022] Furthermore, methods for obtaining suppressed background intensity and desuppressed target window include:

[0023] The conductivity data, pH data, redox potential data, nitrite content data, and available divalent metal ion level data in the suppression background characterization were compared as a whole.

[0024] For each calibration entry in the background suppression calibration table, the matching deviation is calculated. The matching deviation is synthesized from the normalized results of conductivity data deviation, pH data deviation, redox potential data deviation, nitrite content data deviation, and available divalent metal ion level data deviation within the allowable deviation range.

[0025] The calibration entry with the smallest matching deviation and a matching deviation not exceeding 1 is determined as the calibration entry, and the suppression background intensity and the desuppression target window are read. The desuppression target window limits the target range of conductivity data, redox potential data and available divalent metal ion level data.

[0026] Furthermore, methods for obtaining the inhibition release increment curve include:

[0027] The sample extract was divided into two equal-volume portions according to the dispensing volume, and the dispensing temperature and dispensing time were uniform.

[0028] An inert buffer solution was added to the extract of the first group of samples to obtain an in-situ reaction environment;

[0029] According to the target window for de-inhibition, the de-inhibition reference buffer was added to the extract of the second group of samples in portions to obtain the de-inhibition reference reaction environment. The in-situ reaction environment and the de-inhibition reference reaction environment had the same dilution factor.

[0030] Protein hydrolysis indicator probes, lipid hydrolysis indicator probes, and external internal standard enzyme reaction pairs were added to the in-situ reaction environment and the de-inhibition reference reaction environment, respectively, and signal readings were collected at fixed time intervals to obtain protein hydrolysis reaction time series, lipid hydrolysis reaction time series, and internal standard reaction time series, respectively.

[0031] Matrix background subtraction was performed on the reaction time series, and in the same direction subtraction was performed based on the internal standard reaction time series to obtain the inhibition relief increment curve.

[0032] Furthermore, methods for matrix background subtraction of reaction time series include:

[0033] External internal standard enzyme reaction pairs were added to the in situ reaction environment and the de-inhibition reference reaction environment, and readings were collected at fixed time intervals to obtain the in situ internal standard reaction time series and the de-inhibition reference internal standard reaction time series.

[0034] Based on the matrix background readings at the acquisition time points, matrix background subtraction was performed on the protein hydrolysis reaction time series and lipid hydrolysis reaction time series to obtain the in situ protein hydrolysis subtracted reaction time series, the de-inhibition reference protein hydrolysis subtracted reaction time series, the in situ lipid hydrolysis subtracted reaction time series, and the de-inhibition reference lipid hydrolysis subtracted reaction time series.

[0035] Furthermore, methods for performing co-directional subtraction based on internal standard reaction time series include:

[0036] The readout path drift was calculated based on the in situ internal standard reaction time series and the de-inhibition reference internal standard reaction time series, and the de-inhibition reference protein hydrolysis subtraction reaction time series and the de-inhibition reference lipid hydrolysis subtraction reaction time series were subtracted in the same direction.

[0037] At the time point of acquisition, the difference between the in situ protein hydrolysis subtracted reaction time series and the de-inhibition reference protein hydrolysis subtracted reaction time series was adjusted to obtain the protein hydrolysis inhibition relief increment curve. The difference between the in situ lipid hydrolysis subtracted reaction time series and the de-inhibition reference lipid hydrolysis subtracted reaction time series was adjusted to obtain the lipid hydrolysis inhibition relief increment curve.

[0038] Furthermore, the conclusions regarding the contribution ratio of endogenous enzymes, the contribution ratio of microbial enzymes, and the determination of the dominant source include:

[0039] Based on the aliquot volume, aliquots of the same volume standard were taken from the sample extract for nucleic acid extraction, and the sampling volume, sampling temperature and sampling time were recorded. The records were then correlated with the background suppression intensity and inhibition relief increment curves for the same batch.

[0040] The sample extracts used for nucleic acid extraction were aliquoted and centrifuged at low speed, and the supernatant was collected.

[0041] Nucleic acid extraction was performed on the supernatant to obtain a nucleic acid extract.

[0042] Microbial-specific nucleic acid copy numbers were obtained by conducting quantitative nucleic acid amplification reactions based on nucleic acid extracts; microbial-specific nucleic acid copy numbers were defined as microbial lysis and release markers and were batch-linked with inhibition background intensity and inhibition release increment curves;

[0043] The suppression background intensity, suppression release increment curve, and microbial lysis release markers are input into the source splitting mapping rule, and the contribution ratio of endogenous enzymes, the contribution ratio of microbial enzymes, and the dominant source discrimination conclusion are output.

[0044] Furthermore, the source splitting mapping rules include:

[0045] Based on the expansion of the inhibition background calibration table, source splitting mapping rules were established and the correspondence between inhibition background intensity, anti-inhibition target window, protein hydrolysis inhibition relief increment curve, lipid hydrolysis inhibition relief increment curve, microbial lysis release markers and endogenous enzyme contribution ratio, microbial source enzyme contribution ratio, and dominant source discrimination conclusion was recorded.

[0046] The suppression environment range is divided according to the suppression background intensity, and the conductivity data, redox potential data and available divalent metal ion level data are used as background constraints by limiting the desuppression target window.

[0047] The incremental curves of protein hydrolysis inhibition relief and lipid hydrolysis inhibition relief were extracted as curve feature inputs. Combined with microbial lysis release markers, the feasible range of the contribution ratio of microbial enzymes was determined, and the contribution ratios of endogenous enzymes, microbial enzymes, and dominant sources were given.

[0048] Furthermore, the correspondence between the input and output of the source splitting mapping rule includes:

[0049] The suppression environment interval is determined based on the suppression background intensity, and the corresponding relationship entries consistent with the desuppression target window are called within the suppression environment interval;

[0050] Based on the target window for desuppression, the target range is defined for conductivity data, redox potential data, and available divalent metal ion level data, and the incremental curve of desuppression relief is correlated with the target range.

[0051] Based on the curve characteristics of the proteolysis inhibition release increment curve and the lipid hydrolysis inhibition release increment curve, the distribution pattern of potential reaction increments along the proteolysis main line and the lipid hydrolysis main line is determined.

[0052] The feasible range of the contribution ratio of microbial enzymes is defined based on the markers of microbial lysis and release, and the remaining part is attributed to the contribution ratio of endogenous enzymes.

[0053] The dominant source is determined based on the contribution ratio of endogenous enzymes and the contribution ratio of enzymes from microorganisms.

[0054] An auxiliary detection system for regulating enzyme activity in low-temperature cooked meat products using ultra-high pressure processing includes:

[0055] The sample extraction module is used to shear and separate the meat chunks from the isotonic extract to obtain the sample extract.

[0056] The calibration control module is used to collect the inhibition background characterization from the sample extract; based on the inhibition background characterization, the inhibition background intensity and the de-inhibition target window are obtained by comparing with the pre-established inhibition background calibration table.

[0057] The sequence subtraction module is used to add inert buffer to the sample extract to obtain an in situ reaction environment, and to add a de-inhibition reference buffer to the sample extract according to the de-inhibition target window to obtain a de-inhibition reference reaction environment. Protein hydrolysis indicator probes, lipid hydrolysis indicator probes and external internal standard enzyme reaction pairs are added to the two reaction environments respectively to obtain the reaction time series. The reaction time series is subtracted to obtain the inhibition relief increment curve.

[0058] The proportion discrimination module is used to extract nucleic acids from the sample extract to obtain microbial lysis and release markers. The inhibition background intensity, inhibition relief increment curve, and microbial lysis and release markers are input into the preset source splitting mapping rules, and the output endogenous enzyme contribution ratio, microbial source enzyme contribution ratio, and dominant source discrimination conclusion are output.

[0059] Compared with existing technologies, the technical effects and advantages of the auxiliary detection method and system for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment of the present invention are as follows:

[0060] This invention uses sample extracts as the unified detection object. First, conductivity, pH, redox potential, nitrite content, and available divalent metal ion levels are collected to form an inhibition background characterization. The inhibition background intensity and de-inhibition target window are obtained by referring to an inhibition background calibration table. Under the constraints of the inhibition background intensity and de-inhibition target window, an in-situ reaction environment and a de-inhibition reference reaction environment are constructed. Protein hydrolysis indicator probes, lipid hydrolysis indicator probes, and external internal standard enzyme reaction pairs are introduced into the two reaction environments to obtain the reaction time series. Inhibition relief increment curves are formed by matrix background subtraction and internal standard subtraction. Nucleic acid extraction and quantitative nucleic acid amplification reactions are performed on the sample extracts to obtain microbial lysis and release markers. The inhibition background intensity, inhibition relief increment curves, and microbial lysis and release markers are input into a source splitting mapping rule, outputting the contribution ratio of endogenous enzymes, the contribution ratio of microbial source enzymes, and the dominant source discrimination conclusion.

[0061] This invention unifies the expression of the inhibition environment and the metal ion chelation environment as the inhibition background intensity and the de-inhibition target window, and deducts the influence of the treatment isolation matrix background and readout path. Under the condition of the presence of the formulation inhibition background, it can still give traceable source separation results. This solves the problem of the failure of source identification caused by the convergence of the activities of two types of enzymes and the masking of characteristics under the inhibition background formed by components such as salt, nitrite, reducing agent, and phosphate. It avoids misjudgment caused by total enzyme activity or single substrate reaction, and provides a directly referable discrimination basis for quality control during cold storage. Attached Figure Description

[0062] Figure 1This is a schematic diagram of an auxiliary detection system for regulating enzyme activity in low-temperature cooked meat products under ultra-high pressure treatment, according to an embodiment of the present invention.

[0063] Figure 2 This is a flowchart of an auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products under ultra-high pressure treatment, according to an embodiment of the present invention.

[0064] Figure 3 This is a flowchart illustrating the method for obtaining suppressed background intensity and desuppressed target window according to an embodiment of the present invention. Detailed Implementation

[0065] The technical solutions of the embodiments of the present invention will be described in detail, clearly, and completely below with reference to the accompanying drawings. It should be particularly noted that the specific embodiments described below are only for better illustrating and explaining the technical solutions of the present invention, and are intended to enable those skilled in the art to better understand and implement the present invention, and should not be construed as limiting the scope of protection of the present invention. Without departing from the spirit and substance of the present invention, those skilled in the art can modify, adjust, or make equivalent substitutions based on the content disclosed in the present invention, and these should all be considered within the scope of protection of the present invention.

[0066] Example 1:

[0067] Please see Figure 1 As shown in the figure, this embodiment discloses an auxiliary detection system for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment, including a sample extraction module, a calibration control module, a sequence subtraction module, and a ratio discrimination module. Each module is connected by wired and / or wireless means to realize data transmission.

[0068] The sample extraction module is used to shear and separate the meat chunks from the isotonic extract to obtain the sample extract.

[0069] The specific implementation method for shearing and separating the meat chunks from the isotonic extract to obtain the sample extract is as follows.

[0070] Meat pieces were extracted from the same packaging, and the sampling locations were kept consistent. During sampling, meat pieces were extracted from the area near the packaging's adhesive surface and from the center area according to fixed sampling rules. These rules included selecting three sampling points axially evenly within a 3mm to 8mm thickness range inside the adhesive surface, with each sampling point yielding a 5g meat piece to form the meat piece near the packaging's adhesive surface. Simultaneously, two sampling points axially evenly selected within a 5mm radius around the center of the meat piece, with each sampling point yielding a 7g meat piece to form the meat piece in the center area. The meat pieces near the packaging's adhesive surface and the meat piece in the center area were then combined as the source meat for this processing. During the extraction process, the sampling location and corresponding meat mass of each sampling point were recorded simultaneously. The total mass of the source meat was controlled to 29g, and the mass deviation of each sampling point was controlled within 1g to reduce the impact of local component deviations caused by water accumulation on subsequent sample extraction. By limiting the source of meat to a combination of the area near the packaging and the center of the meat, and by quantifying and controlling the sampling conditions of each batch through the number of sampling points, the range of sampling locations, and the quality of sampling, it is possible to simultaneously cover the areas susceptible to water accumulation and the internal areas at the sample extract level, thereby reducing the spatial deviation of the sample extract in the distribution of inhibitory components and soluble enzymes.

[0071] An isotonic extract solution was prepared and pre-cooled to create an extraction environment consistent with the meat processing conditions. The isotonic extract solution was prepared according to the isotonic principle, matching the ionic environment of low-temperature cooked meat products, and was pre-placed in a low-temperature environment until its temperature stabilized within the target range. Simultaneously, the volume and temperature of the isotonic extract solution were recorded to ensure that the volume ratio of meat to isotonic extract solution remained consistent with the temperature conditions during subsequent shear separation. The isotonic extract solution was chosen because it can inhibit abnormal cell structure rupture caused by osmotic pressure shocks during the shearing phase, preventing the large-scale release of non-target intracellular substances that alter the background composition of the sample extract. It also reduces rapid protein conformational changes caused by abrupt changes in ionic strength, thus providing a more reproducible sample basis for subsequent background suppression characterization. By pre-fixing and recording the volume and temperature of the isotonic extract solution, the ionic and temperature backgrounds of the sample extract solution can be stabilized during subsequent shear separation, reducing fluctuations in soluble enzyme release caused by variations in extraction conditions.

[0072] The meat chunks and isotonic extract were mixed and sheared in a sealed container to obtain a homogeneous slurry. After adding the meat chunks obtained in the previous step to the sealed container, isotonic extract was added according to the pre-recorded volume, maintaining a consistent space within the sealed container to reduce air bubble entrainment during the shearing process. Subsequently, the meat chunks and isotonic extract in the sealed container were mixed and sheared. During the mixing and shearing process, the shearing time and intensity were controlled to avoid unnecessary temperature rise, while ensuring that the meat chunks were fully broken down and in full contact with the isotonic extract to form a homogeneous slurry with a relatively uniform particle distribution. The reason for using a sealed container and mixing and shearing is that the sealed container can reduce the introduction of external contaminants and environmental redox fluctuations into the sample extract background. Simultaneously, mixing and shearing can rapidly transfer soluble enzymes and soluble inhibitory components from the meat chunks to the isotonic extract phase, and the repeatability of this transfer process is maintained by controlling the shearing conditions. By mixing and shearing meat chunks with isotonic extract in a sealed container, a homogeneous slurry with uniform composition can be obtained, thus providing a stable starting material for subsequent shearing and separation and avoiding inconsistent extraction efficiency of soluble components in the sample extract due to different degrees of meat chunk breakage.

[0073] Shear separation is performed on the homogeneous slurry to reduce particle disturbance in subsequent separation processes. After the homogeneous slurry obtained in the previous step is allowed to stand briefly at low temperature to release macroscopic bubbles, it undergoes initial solid-liquid separation using sizing filtration. The pore size of the sizing filter is selected to intercept large tissue fragments without excessively retaining soluble components. This ensures that soluble enzymes and soluble inhibitors enter the filtrate while reducing the separation load caused by tissue fragments entering subsequent separation stages. The filtration volume and time are recorded during sizing filtration to maintain consistent shear separation intensity across batches. Sizing filtration for initial solid-liquid separation rapidly removes large tissue fragments without altering the isotonic extract background, reducing sedimentation instability and supernatant turbidity risks during subsequent low-speed centrifugation, thereby improving the homogeneity of the sample extract.

[0074] The filtrate after sizing and filtration was centrifuged at low speed to obtain the supernatant, forming the sample extract. The filtrate was then placed in a centrifuge container for low-speed centrifugation. The logic for low-speed centrifugation was to prioritize the sedimentation of residual fine particles without forcibly settling soluble protein complexes, thus avoiding the loss of soluble enzymes and soluble inhibitory components along with the precipitate due to excessively strong centrifugation conditions. After low-speed centrifugation, the supernatant was collected without disturbing the sedimentation layer and defined as the sample extract. To ensure that subsequent characterization of inhibition background, parallel reaction acquisition, and collection of microbial lysis release markers were conducted on a consistent basis, the sample extract was aliquoted, and the aliquot volume, temperature, and time point were recorded to ensure consistent traceability of the sample extract used in different subsequent operations in terms of volume, temperature, and time point. By obtaining a clear supernatant through low-speed centrifugation and using it as the sample extract, particulate interference can be reduced while retaining soluble enzymes and soluble inhibitory components. Furthermore, by recording the dispensing volume, dispensing temperature, and dispensing time, the usage conditions of the sample extract can be stabilized, thus providing a consistent sample basis for subsequent characterization of inhibition background and acquisition of reaction time series using the sample extract.

[0075] The calibration control module is used to collect the inhibition background characterization from the sample extract; based on the inhibition background characterization, the inhibition background intensity and the de-inhibition target window are obtained by comparing with the pre-established inhibition background calibration table.

[0076] The specific implementation method for obtaining the inhibition background characterization from the sample extract and obtaining the inhibition background intensity and desuppression target window based on the inhibition background characterization and the pre-established inhibition background calibration table is as follows.

[0077] Using the sample extract obtained in the previous process as the sole sampling object, the sample extract was first subjected to a state consistency treatment before conducting background suppression characterization. Specifically, the sample extract was allowed to stand within the same temperature range until the temperature stabilized, and then gently mixed. During mixing, only shaking in the same direction and with the same amplitude was used to avoid introducing new air bubbles and turbidity fluctuations. Subsequently, the sample extract was dispensed into multiple equal-volume portions according to a predetermined volume, and the dispensing volume, dispensing temperature, and dispensing time were recorded to ensure that all subsequent measurements used sample extract portions with the same volume basis, and that the time intervals between measurements remained consistent. By uniformly controlling the temperature and volume basis of the sample extract, the background changes caused by temperature drift and sedimentation during the measurement period were reduced, ensuring that the subsequent background suppression characterization could accurately reflect the suppression environment and metal ion chelation environment of the sample extract.

[0078] Based on the sample extract portions after achieving consistency, conductivity data was collected through conductivity measurement and incorporated into the inhibition background characterization. During conductivity measurement, the measuring probe was pretreated in isotonic extract before being introduced into the sample extract to maintain consistency between the ionic background on the probe surface and the ionic background of the sample extract. The sample extract was kept static during measurement, and the measurement temperature and time points were recorded. Conductivity data were read at the same range and recorded at the same time. The conductivity data was used to characterize the ion strength-related background of the sample extract. This ion strength-related background directly corresponds to the inhibitory environment formed by salt in low-temperature cooked meat product formulations, and it has a continuous impact on protein conformational stability and non-specific inhibition tendency. Therefore, conductivity data is used as one of the fundamental data for inhibition background characterization. The introduction of conductivity data allows inhibition background characterization to cover ionic environmental factors that have a wide impact on the enzyme reaction process and vary greatly between formulations, thereby improving the transferability of inhibition background intensity.

[0079] After collecting conductivity data, pH data was collected from the same batch of sample extracts and incorporated into the inhibition background characterization. During pH measurements, the probe depth in the sample extract was kept consistent to avoid reading fluctuations caused by minor layering between the upper and lower layers. After measurement, the measurement temperature and time point were recorded, and the pH data was linked to the aforementioned conductivity data using the same aliquot volume. The pH data was used to characterize the reaction background of the sample extract in the low-temperature cooked meat product formulation environment. This is because pH simultaneously affects the spatial structural stability of soluble enzymes and the form of soluble inhibitory components. Furthermore, pH has a synergistic effect with nitrite content and redox potential data; therefore, pH data is used as a key dimension in the inhibition background characterization. The combination of pH and conductivity data allows the inhibition background characterization to simultaneously cover the constraints of both ionic strength-related background and reaction background, thus providing a more stable matching basis for subsequently extracting the de-inhibition target window from the inhibition background calibration table.

[0080] After collecting pH data, redox potential (RP) data was collected from sample extracts aliquoted from the same batch and incorporated into the background suppression characterization. During RRP measurement, the sample extract containers were kept open at the same time, and readings were recorded within a fixed time window to avoid incomparability caused by exposure time differences. Simultaneously, the measurement temperature and time point were recorded, and the RRP data were correlated with conductivity and pH data within the same data set. RRP data characterizes the redox background formed by reducing agents and other components in the sample extract. This redox background directly corresponds to the reaction background used for flavor and preservation in low-temperature cooked meat product formulations, and it tends to inhibit the activity of both endogenous and microbial enzymes, easily flattening enzyme profiles. Therefore, it must be explicitly collected in the background suppression characterization. The inclusion of RRP data allows the background suppression characterization to cover the reaction background factors most likely to cause source identification failure, establishing a necessary prerequisite for reliable extraction of the subsequent background suppression intensity.

[0081] After collecting redox potential data, nitrite content data was collected by colorimetric measurement of nitrite content in samples from the same batch, and incorporated into the inhibition background characterization. During colorimetric measurement, a fixed volume of sample extract was taken and mixed with a fixed volume of colorimetric reagent. After mixing, a fixed reaction time was maintained before reading the data, and the reaction temperature, reaction time, and reading time were recorded to ensure consistent acquisition conditions for nitrite content data across different batches. After reading, the nitrite content data and redox potential data were bound together in the same data object to reflect the reaction background jointly constituted by both. Nitrite content data was chosen as part of the inhibition background characterization because nitrite in chilled cooked meat products participates in the preservation reaction background and couples with reducing agents, thus exhibiting a systematic inhibitory tendency on the performance of soluble enzymes, further flattening the differences between enzymes from different sources. The introduction of nitrite content data allows the inhibition background characterization to cover the most representative inhibitory components in the formulation, thereby improving the accuracy of matching the inhibition background intensity with the target window for de-inhibition.

[0082] After collecting nitrite content data, samples from the same batch were aliquoted, and available divalent metal ion levels were collected using a complexing metal ion indicator reaction, which was then incorporated into the inhibition background characterization. During the complexing metal ion indicator reaction, a fixed volume of sample extract was taken and a fixed volume of indicator reagent was added. After a fixed reaction time, the indicator signal was read, and the measurement temperature and time point were recorded. The results were converted into available divalent metal ion level data, which, along with conductivity, pH, redox potential, and nitrite content data, constituted the inhibition background characterization. Available divalent metal ion level data is used to indirectly characterize the metal ion chelation environment formed by components such as phosphate. This chelation environment reduces the level of available divalent metal ions, thereby altering the reaction process and stability of soluble enzymes. Furthermore, after ultra-high pressure treatment, these chelates are more likely to co-inhibit microbial enzymes released extracellularly, leading to source discrimination failure. The introduction of available divalent metal ion level data allows the inhibition background characterization to cover this crucial dimension of the metal ion chelation environment, providing a direct basis for subsequently determining the target window for de-inhibition.

[0083] After collecting data on conductivity, pH, redox potential, nitrite content, and available divalent metal ion levels, these data were aggregated within the same data object to form an inhibition background characterization. This characterization was then linked to the sample extract's dispensing volume, dispensing temperature, and dispensing time. During aggregation, the data names were kept consistent with the collection order to ensure the inhibition background characterization fully reflects the inhibition and metal ion chelation environments of the sample extract. Simultaneously, the measurement temperature and time point for each data point were recorded along with the corresponding data to ensure that subsequent matching based on the inhibition background calibration table could be performed within the same temperature range and time scale. By forming a structurally complete inhibition background characterization linked to the sample extract's dispensing information, it was ensured that the subsequent acquisition of inhibition background intensity and the target window for de-inhibition was based on sample extracts from the same batch and under the same conditions.

[0084] Please see Figure 3As shown, based on the suppression background characterization, the suppression background intensity and the desuppression target window are obtained by referring to a pre-established suppression background calibration table. The suppression background calibration table, when established, already records conductivity data, pH data, redox potential data, nitrite content data, and available divalent metal ion level data under multiple level combinations, along with the corresponding suppression background intensity and desuppression target window. Therefore, in the specific acquisition process, the conductivity data, pH data, redox potential data, nitrite content data, and available divalent metal ion level data in the suppression background characterization are compared as a whole, and the matching deviation is calculated for each calibration entry in the suppression background calibration table. The calculation of matching deviation adopts a unified approach: first, the deviations of conductivity data, pH data, redox potential data, nitrite content data, and available divalent metal ion level data are calculated separately, and then the five deviations are converted by ratio with the corresponding upper limit of allowable deviation to obtain the five normalized deviations; when a certain deviation is 0, the corresponding normalized deviation is recorded as 0; when a certain deviation is equal to the corresponding upper limit of allowable deviation, the corresponding normalized deviation is recorded as 1; when a certain deviation is between 0 and the corresponding upper limit of allowable deviation, the corresponding normalized deviation falls into the 0 to 1 range according to the same proportion. To ensure consistency in the criteria for determining matching deviation, only calibration items whose deviations all fall within their respective allowable ranges are considered as candidate items for calculating matching deviation. Specifically, the allowable deviation range for conductivity data is set to 0 to 0.2 mSiemens per centimeter, for pH data to 0 to 0.3, for redox potential data to 0 to 20 mV, for nitrite content data to 0 to 5 mg per kilogram, and for available divalent metal ion levels to 0 to 0.2 mmol per liter. After normalization, the five normalized deviations are weighted and averaged to form the matching deviation, which is controlled within the range of 0 to 1. This ensures that the criterion of a matching deviation not exceeding 1 has a unified meaning and can be directly used to screen calibration items. In the weighted averaging, to ensure that the target window for de-inhibition is consistent with the pull-back constraint when adding the de-inhibition reference buffer to the sample extract, the conductivity data deviation, redox potential data deviation, and available divalent metal ion level data deviation are given larger weights, while the pH data deviation and nitrite content data deviation are given smaller weights. In one embodiment, the weights of the conductivity data deviation, redox potential data deviation, and available divalent metal ion level data deviation are all set to 0.25, and the weights of the pH data deviation and nitrite content data deviation are all set to 0.125, so that the dominant source of the matching deviation is consistent with the conductivity data, redox potential data, and available divalent metal ion level data at least defined by the target window for de-inhibition.

[0085] After obtaining the matching deviation of each calibration entry in the suppression background calibration table, the calibration entry with the smallest matching deviation (not exceeding 1) is identified as the closest to the suppression background characterization. The suppression background intensity and the desuppression target window are then directly read from this calibration entry. When multiple calibration entries have the same or similar matching deviation, to ensure consistent background constraints for entering the desuppression target window, the synthesized results of conductivity data deviation, redox potential data deviation, and available divalent metal ion level data deviation are further compared, and the calibration entry with the smaller synthesized result is preferentially selected. The desuppression target window at least defines the target range for conductivity data, redox potential data, and available divalent metal ion level data, used to control the pullback amplitude of the reaction background when adding the desuppression reference buffer to the sample extract. By using the suppression background characterization as input and directly extracting the suppression background intensity and the desuppression target window from the suppression background calibration table, the suppression environment and metal ion chelation environment of the sample extract can be converted into executable target range constraints, providing a consistent control basis for the next step of constructing the in-situ reaction environment and the desuppression reference reaction environment.

[0086] The sequence subtraction module is used to add inert buffer to the sample extract to obtain an in situ reaction environment, and add a de-inhibition reference buffer to the sample extract according to the de-inhibition target window to obtain a de-inhibition reference reaction environment. Protein hydrolysis indicator probes, lipid hydrolysis indicator probes and external internal standard enzyme reaction pairs are added to the two reaction environments respectively to obtain the reaction time series. The reaction time series is subtracted to obtain the inhibition relief increment curve.

[0087] The specific implementation method is as follows: adding inert buffer to the sample extract to obtain an in situ reaction environment; adding de-inhibition reference buffer to the sample extract according to the de-inhibition target window to obtain a de-inhibition reference reaction environment; adding protein hydrolysis indicator probe, lipid hydrolysis indicator probe and external internal standard enzyme to the two reaction environments respectively to obtain the reaction time series; and subtracting the reaction time series to obtain the inhibition relief increment curve.

[0088] The protein hydrolysis indicator probe uses a fluorescent substrate probe and acquires fluorescence signals using a microplate reading method. The working solution of the protein hydrolysis indicator probe is prepared at a fixed volume and the working solution temperature is recorded. The addition volume of the protein hydrolysis indicator probe in the in-situ reaction environment is consistent with that in the de-inhibition reference reaction environment. The final concentration of the protein hydrolysis indicator probe is controlled within the range of 5 μmol / L to 20 μmol / L to ensure that a resolvable time series change is formed within the set acquisition time. The fluorescence reading uses a channel setting with an excitation wavelength of 360 nm to 390 nm and an emission wavelength of 440 nm to 470 nm. Before each reading, the plate is gently oscillated for the same duration to mix and unify the concentration distribution in the well. The lipid hydrolysis indicator probe also uses a fluorescent substrate probe and acquires fluorescence signals using a microplate reading method. The working solution of the lipid hydrolysis indicator probe is prepared at a fixed volume and the working solution temperature is recorded. The addition volume of the lipid hydrolysis indicator probe in the in-situ reaction environment is consistent with that in the de-inhibition reference reaction environment. The final concentration of the lipid hydrolysis indicator probe is controlled in the range of 2 μmol / L to 10 μmol / L. The fluorescence reading of the lipid hydrolysis indicator probe uses a channel setting with an excitation wavelength of 480 nm to 510 nm and an emission wavelength of 510 nm to 540 nm. It is subjected to the same gentle oscillation mixing conditions and the same reading rhythm as the protein hydrolysis indicator probe, so that the reaction time series obtained by the protein hydrolysis indicator probe and the lipid hydrolysis indicator probe have the same time scale.

[0089] The external internal standard enzyme reaction pair consists of an external internal standard enzyme and an external internal standard enzyme substrate pair. The external internal standard enzyme reaction pair is independent of endogenous enzymes and microbial enzymes in the sample extract. The addition volume of the external internal standard enzyme reaction pair is consistent in the in-situ reaction environment and the de-inhibition reference reaction environment. The external internal standard enzyme substrate pair uses a fluorescent substrate that is compatible with the external internal standard enzyme and the fluorescence signal is acquired by microplate reading. The final concentration of the external internal standard enzyme substrate pair is controlled in the range of 5 μmol / L to 15 μmol / L. The fluorescence reading of the external internal standard enzyme reaction pair uses a fixed excitation wavelength and a fixed emission wavelength channel set that matches the external internal standard enzyme substrate pair. The readings are completed at the same acquisition time sequence as the readings of the protein hydrolysis indicator probe and the lipid hydrolysis indicator probe to ensure that the internal standard reaction time sequence can be used to sort out the readout path drift.

[0090] The reaction time series was acquired with the start time of the in-situ reaction environment and the start time of the de-inhibition reference reaction environment as their respective zero points. The sampling interval was controlled within the range of 30 to 120 seconds, and the total acquisition time was controlled within the range of 10 to 30 minutes. The reaction temperature was maintained within the range of 4 to 10 degrees Celsius throughout the process. At each acquisition time point, the fluorescence readings of the protein hydrolysis indicator probe, the lipid hydrolysis indicator probe, and the external internal standard enzyme reaction were recorded. Before each reading, the plate was gently shaken for the same duration to reduce the influence of well layering on the readings. To support subsequent matrix background subtraction, protein hydrolysis blank wells and lipid hydrolysis blank wells were set up in the in-situ reaction environment and the de-inhibition reference reaction environment, respectively. The protein hydrolysis blank wells and lipid hydrolysis blank wells did not contain the corresponding indicator probes but contained an equal volume of inert buffer. Background readings were collected at the same sampling interval and with the same total acquisition time, so that a correspondence between indicator probe readings and matrix background readings was established at each acquisition time point.

[0091] Using the sample extract obtained in the previous step as the source for this step, the sample extract is first divided into two equal-volume portions according to the dispensing volume, maintaining the same dispensing temperature and time point for both portions. Before dispensing, the sample extract is gently mixed in the same direction with a fixed amplitude to avoid introducing new air bubbles and turbidity fluctuations. After dispensing, both equal-volume portions are allowed to stand within the same temperature range until the temperature stabilizes to reduce the impact of temperature drift on the subsequent reaction time series. By dispensing the sample extract into equal volumes and unifying the dispensing temperature and time point, it is ensured that the in-situ reaction environment and the de-inhibition reference reaction environment are based on the same volume and temperature baseline, reducing the incomparability of reaction time series caused by differences in the state of the sample extract from the source.

[0092] Based on the de-inhibition target window obtained in the previous process, inert buffer and de-inhibition reference buffer were prepared, and their addition volumes were fixed. The inert buffer was prepared according to the principle of not reacting specifically with the sample extract and not providing metal ion compensation. The ionic background of the inert buffer was consistent with that of the isotonic extract, so that only volume dilution was introduced after adding the inert buffer without introducing new changes to the inhibitory environment. The de-inhibition reference buffer was prepared according to the principle of corresponding to the inhibitory environment and the metal ion chelation environment. The de-inhibition reference buffer contained at least a metal ion compensation component, an ion environment buffer component, and a redox buffer component. The initial conductivity, initial redox potential, and initial available divalent metal ion level of the de-inhibition reference buffer were recorded. By fixing the addition volumes of the inert buffer and the de-inhibition reference buffer and clarifying their differences in ionic background and compensation capabilities, it was possible to distinguish between the "same-direction change caused by volume dilution" and the "rebound of the inhibitory environment caused by the de-inhibition reference buffer" in subsequent subtraction processing.

[0093] Using the first set of equal-volume aliquots of the sample extract as the subject, inert buffer was added to the sample extract to establish an in-situ reaction environment. The inert buffer was added at the same rate and from a fixed location. After addition, the mixture was gently mixed until the liquid appearance was uniform. The time point at which mixing was completed was immediately recorded and defined as the start time point of the in-situ reaction environment. Subsequently, conductivity data, redox potential data, and available divalent metal ion levels were collected from and recorded in the in-situ reaction environment. The collected results, along with the aliquot volume, aliquot temperature, and aliquot time point, were used to record the background of the in-situ reaction environment. By establishing the in-situ reaction environment with inert buffer and simultaneously recording conductivity data, redox potential data, and available divalent metal ion levels, the overall characteristics of the original inhibitory environment and metal ion chelating environment of the sample extract can be preserved, providing a baseline reaction time series of "unresolved inhibition background" for subsequent incremental inhibition relief curves.

[0094] Using the second aliquot of the sample extract as an example, a reference buffer for inhibition was added to the sample extract according to the inhibition target window to form a reference reaction environment for inhibition. The reference buffer was added in multiple additions, with gentle mixing after each addition and the collection of conductivity, redox potential, and available divalent metal ion levels. The collected results were correlated with the target range defined by the inhibition target window. Addition was stopped once the collected results fell within the target range defined by the inhibition target window, and the cumulative added volume was recorded. The cumulative added volume and the aliquot volume together determined the final dilution factor, which was consistent with the dilution factor of the in-situ reaction environment. The time point at which mixing was completed was recorded and defined as the start time point of the reference reaction environment for inhibition. By controlling the amount of reference buffer added according to the inhibition target window and simultaneously collecting conductivity, redox potential, and available divalent metal ion levels, the inhibitory environment and metal ion chelation environment in the sample extract could be pulled back to the executable target range, while maintaining a dilution factor consistent with the in-situ reaction environment, avoiding misinterpreting dilution differences as inhibition relief.

[0095] Protein hydrolysis indicator probes were added to both the in-situ reaction environment and the de-inhibition reference reaction environment, and the protein hydrolysis reaction time series were obtained. Before adding the protein hydrolysis indicator probes, a working solution of the protein hydrolysis indicator probes was prepared at a fixed volume and the temperature of the working solution was recorded. During addition, the volume of protein hydrolysis indicator probes added to the in-situ reaction environment and the de-inhibition reference reaction environment was kept completely consistent. After addition, the same mixing action was used to start the reaction, and signal readings were collected at fixed time intervals based on their respective starting time points. The collection time points were recorded to form the protein hydrolysis reaction time series.

[0096] To ensure that the protein hydrolysis reaction time series simultaneously includes readings related to the enzyme action of the protein hydrolysis indicator probe and readings related to the matrix background, a paired well acquisition method was used to obtain both types of readings in each reaction environment, ensuring that the liquid source, liquid volume, mixing action, and acquisition time point were completely consistent for both types of wells. Specifically, protein hydrolysis probe reaction wells and protein hydrolysis blank wells were set up in the in-situ reaction environment, and similarly, protein hydrolysis probe reaction wells and protein hydrolysis blank wells were set up in the de-inhibition reference reaction environment. Working solution of the protein hydrolysis indicator probe was added to the protein hydrolysis probe reaction wells, and readings related to the enzyme action of the protein hydrolysis indicator probe were recorded in subsequent readings. In the protein hydrolysis blank wells, no working solution of the protein hydrolysis indicator probe was added, but an inert buffer of the same volume as the working solution was added, and readings related to the matrix background were recorded in subsequent readings. The liquid composition of the protein hydrolysis blank well is consistent with that of the protein hydrolysis probe reaction well. The only difference is that the protein hydrolysis blank well does not contain a protein hydrolysis indicator probe. This allows the readings of the protein hydrolysis blank well to characterize the background changes introduced by factors such as color, turbidity, effluent particles and container wall scattering under the same reaction environment, and to be recorded at the same acquisition time point as the protein hydrolysis probe reaction well.

[0097] During data collection, signal readings were synchronously acquired at the same time points for both the proteolysis probe reaction wells and the proteolysis blank wells in the in-situ reaction environment. Similarly, for the de-inhibition reference reaction environment, signal readings were synchronously acquired at the same time points for both the proteolysis probe reaction wells and the proteolysis blank wells. This ensured that each acquisition time point established a correspondence between the enzymatically related readings of the proteolysis indicator probe and the matrix background readings, thus constructing a proteolysis reaction time series. By obtaining the proteolysis reaction time series in both the in-situ and de-inhibition reference reaction environments with consistent addition volumes, mixing actions, and acquisition intervals, and by acquiring matrix background readings in paired well locations and recording them corresponding to the enzymatically related readings of the proteolysis indicator probe at the same acquisition time points, the background readings from the same batch and at the same time point could be used as the basis for subtraction in subsequent subtraction processing. This allowed for the time-series characterization of the proteolysis main line related to quality deterioration of low-temperature cooked meat products in both the in-situ and de-inhibition reference reaction environments.

[0098] Lipid hydrolysis indicator probes were added to both the in-situ reaction environment and the de-inhibition reference reaction environment, and lipid hydrolysis reaction time sequences were obtained. Before adding the lipid hydrolysis indicator probes, a working solution of the probes was prepared at a fixed volume, and the working solution temperature was recorded. The added volume of the lipid hydrolysis indicator probes was kept completely consistent in both reaction environments. After addition, the reaction was initiated using the same mixing action, and signal readings were collected at fixed time intervals based on their respective starting time points. The collection time points were recorded to form the lipid hydrolysis reaction time sequence. The lipid hydrolysis reaction time sequence also included readings related to the enzyme action of the lipid hydrolysis indicator probes and readings related to the matrix background, and both types of readings were recorded correspondingly at the same collection time point. By simultaneously obtaining lipid hydrolysis reaction time sequences in both reaction environments, the main lipid hydrolysis curve related to the quality deterioration of low-temperature cooked meat products can be covered in time series form, avoiding the deviation between the inhibition de-inhibition increment curve and the actual deterioration driver caused by relying solely on a single type of reaction time sequence.

[0099] External internal standard enzyme (ECS) reaction pairs were added to both the in-situ reaction environment and the de-inhibition reference reaction environment, and the internal standard reaction time series were obtained. The external ECS reaction pairs consisted of an external ECS enzyme and its standard substrate pair, and were independent of endogenous enzymes and microbial enzymes in the sample extract. The added volumes of the ECS reaction pairs were kept completely consistent in both reaction environments, and the addition order was fixed to match that of the protein hydrolysis indicator probes and lipid hydrolysis indicator probes to minimize the impact of differences in the addition order on the initial reaction state. After addition, readings were collected at fixed time intervals, and the time points were recorded to form the in-situ internal standard reaction time series and the de-inhibition reference internal standard reaction time series. By introducing external ECS reaction pairs into the two reaction environments and obtaining the internal standard reaction time series, a common reference for the reading path can be provided for subsequent subtraction processing, avoiding misinterpretation of reading fluctuations caused by ion background pullback as inhibition relief.

[0100] When subtracting the reaction time series to obtain the inhibition relief increment curve, matrix background subtraction was first performed on the protein hydrolysis reaction time series and the lipid hydrolysis reaction time series respectively. For each acquisition time point, the readings related to the matrix background were used as subtraction factors on the readings related to the enzyme action of the indicator probe at the same acquisition time point, resulting in in situ protein hydrolysis subtracted reaction time series, inhibition relief reference protein hydrolysis subtracted reaction time series, in situ lipid hydrolysis subtracted reaction time series, and inhibition relief reference lipid hydrolysis subtracted reaction time series. The subtraction order was kept consistent during the subtraction process, and the names of the readings used for subtraction and the corresponding acquisition time points were recorded. By performing matrix background subtraction first, the co-directional fluctuations caused by color, turbidity, and effluent can be separated from the reaction time series, reducing the interference of matrix background differences on the morphology of the inhibition relief increment curve.

[0101] After matrix background subtraction, readout path drift was generated based on the in-situ internal standard reaction time series and the de-inhibition reference internal standard reaction time series and included in the subtraction process. Specifically, early change trends, mid-term incremental trends, and late plateau trends were extracted from the in-situ internal standard reaction time series and the de-inhibition reference internal standard reaction time series at the same acquisition time point, and the differences between the two were compiled into readout path drift. Subsequently, when forming the de-inhibition reference protein hydrolysis subtraction reaction time series and the de-inhibition reference lipid hydrolysis subtraction reaction time series, the readout path drift was introduced for co-direction subtraction, thus obtaining the de-inhibition reference protein hydrolysis subtraction reaction time series and the de-inhibition reference lipid hydrolysis subtraction reaction time series after readout path drift subtraction. By adding an external internal standard enzyme reaction to generate readout path drift and performing co-direction subtraction on the de-inhibition reference reaction time series, the readout path changes caused by the de-inhibition reference buffer can be distinguished from the inhibition relief effect, reducing the risk of amplifying the inhibition relief increment curve due to readout path changes.

[0102] After obtaining the subtracted reaction time series, the time series differences between the in-situ and de-inhibition reference on the protein hydrolysis indicator probe and lipid hydrolysis indicator probe are extracted to form inhibition relief increment curves. Specifically, the difference between the in-situ protein hydrolysis subtracted reaction time series and the de-inhibition reference protein hydrolysis subtracted reaction time series after readout path drift subtraction is processed at the same acquisition time point to obtain the protein hydrolysis inhibition relief increment curve; the difference between the in-situ lipid hydrolysis subtracted reaction time series and the de-inhibition reference lipid hydrolysis subtracted reaction time series after readout path drift subtraction is processed at the same acquisition time point to obtain the lipid hydrolysis inhibition relief increment curve; the protein hydrolysis inhibition relief increment curve and the lipid hydrolysis inhibition relief increment curve are bound together with the inhibition background intensity and de-inhibition target window obtained in the previous process to form a data object that can be directly used for subsequent source splitting and mapping rules. By performing difference analysis on the in-situ and desuppression reference subtraction response time series at the same acquisition time point, the "potential response increment released after the suppression background is removed" can be expressed in the form of suppression removal increment curve, and a clear correspondence can be established with the suppression background intensity and the desuppression target window, providing continuous and traceable key data for the next step of input source splitting and mapping rules.

[0103] The proportion discrimination module is used to extract nucleic acids from the sample extract to obtain microbial lysis and release markers. The inhibition background intensity, inhibition relief increment curve, and microbial lysis and release markers are input into the preset source splitting mapping rules, and the output endogenous enzyme contribution ratio, microbial source enzyme contribution ratio, and dominant source discrimination conclusion are output.

[0104] Nucleic acid extraction was performed on the sample extract to obtain microbial lysis and release markers. The specific implementation method is as follows: the suppression background intensity, the suppression relief increment curve, and the microbial lysis and release markers are input into the source splitting mapping rule, and the endogenous enzyme contribution ratio, the microbial source enzyme contribution ratio, and the dominant source discrimination conclusion are output.

[0105] Using the aforementioned sample extract as the sole source for this step, a portion of the same volume is taken from the sample extract for nucleic acid extraction, ensuring that the dispensing temperature and time point of this portion are consistent with the portion used to form the inhibition relief increment curve. Before sampling, the sample extract is gently mixed in the same direction and amplitude as in the previous steps to avoid sampling differences caused by sedimentation or air bubbles. Immediately after sampling, the sampling volume, sampling temperature, and sampling time point are recorded, and this record is correlated with the inhibition background intensity and inhibition relief increment curve for the same batch. This step ensures that the microbial lysis release markers, inhibition background intensity, and inhibition relief increment curve originate from the same batch of sample extract with consistent volume, reducing the drift in source identification conclusions caused by differences across dispensing portions.

[0106] The sample extract for nucleic acid extraction was aliquoted and centrifuged at low speed, and the supernatant was collected. The operating conditions for low-speed centrifugation were the same as those for obtaining the sample extract, prioritizing the sedimentation of residual fine particles without altering the soluble background of the sample extract. During supernatant collection, the sedimentation layer was kept undisturbed, and the collection volume was recorded to ensure a stable liquid phase composition and controllable turbidity for nucleic acid extraction, reducing interference from particles in subsequent nucleic acid extraction and quantitative nucleic acid amplification reactions. This step stabilizes the initial liquid phase conditions for nucleic acid extraction without altering the inhibitory and metal ion chelating environments of the sample extract, thereby improving the consistency of microbial lysis release marker collection.

[0107] Nucleic acid extraction was performed on the supernatant to obtain a nucleic acid extract. During the extraction process, the mixing, settling time, and low-speed centrifugation time were maintained consistent across all operations. After each low-speed centrifugation, only the supernatant was transferred, without introducing the sedimentation layer. The final volume of the nucleic acid extract and the time point at which it was obtained were recorded and linked to the aforementioned sampling volume, sampling temperature, and sampling time point to form a traceable data object. This step ensures the stable output of nucleic acid components from the sample extract in the form of a nucleic acid extract. The volume and time point recordings guarantee the traceability of the source of microbial lysis release markers and allow for a one-to-one correspondence with the inhibition background intensity and inhibition release increment curves.

[0108] Nucleic acid amplification and quantification (QAQ) reactions were conducted using nucleic acid extraction solutions to obtain microbial-specific nucleic acid copy numbers. The QAQ used bacterial ribosomal gene-specific fragments as amplification targets. These fragments do not match the gene sequences of animal tissues, thus reducing interference from meat-derived nucleic acids in the readings. The sampling volume, reaction temperature, and reaction time remained consistent across different batches, and the start and reading times for each reaction were recorded. The readings were then compiled into microbial-specific nucleic acid copy numbers and bound to the volume of the nucleic acid extraction solution. This step allows for the preferential characterization of microbial-derived nucleic acid load levels in low-temperature cooked meat product systems, and expresses the impact of microbial cell lysis and release of intracellular components after ultra-high pressure processing with quantifiable data.

[0109] Microbial-specific nucleic acid copy number is defined as a biomarker for microbial lysis and release. This biomarker is then batch-specifically linked to the inhibition background intensity and inhibition release increment curves. The linking process includes at least the time-series difference between the proteolysis inhibition release increment curve and the lipid hydrolysis inhibition release increment curve, retaining the corresponding acquisition time points to ensure the biomarker and inhibition release increment curve correspond on the same time scale. Simultaneously, the biomarker is associated with sample batch information to ensure that subsequent outputs of endogenous enzyme contribution ratios, microbial-derived enzyme contribution ratios, and dominant source identification conclusions can be traced back to the same batch of sample extracts. This step transforms the nucleic acid extraction data into the key input required for source separation and ensures consistency with the inhibition background intensity and inhibition release increment curves across batches, volumes, and time points.

[0110] Prepare source splitting mapping rules and complete the matching and location of input data. Source splitting mapping rules are pre-established data objects. Source splitting mapping rules are extended from the inhibition background calibration table. Source splitting mapping rules record the correspondence between inhibition background intensity, anti-inhibition target window, protein hydrolysis inhibition relief increment curve, lipid hydrolysis inhibition relief increment curve, microbial lysis release markers and endogenous enzyme contribution ratio, microbial source enzyme contribution ratio, and dominant source discrimination conclusion. Among them, the suppression background intensity is used to limit the suppression environment interval to which the input data belongs; the de-suppression target window is used to limit the target interval of conductivity data, redox potential data, and available divalent metal ion level data and serve as background constraints; the protein hydrolysis inhibition de-suppression increment curve and the lipid hydrolysis inhibition de-suppression increment curve are used to characterize the distribution pattern of potential reaction increment under de-suppression conditions on the two main lines; and the microbial lysis release marker is used to characterize the loading context of microbial enzymes and limit the feasible range of the contribution ratio of microbial enzymes. Under the premise that the suppression environment interval and background constraints are consistent, the source splitting mapping rule maps the distribution pattern of potential reaction increment on the two main lines and the loading context of microbial lysis release markers to the contribution ratio of endogenous enzymes, the contribution ratio of microbial enzymes, and the dominant source discrimination conclusion. Matching and positioning determine the suppression environment interval with the suppression background intensity and use the conductivity data, redox potential data, and available divalent metal ion level data limited by the de-suppression target window as background constraints to ensure that the input data entering the same correspondence have a consistent suppression environment and metal ion chelation environment context. This step can classify input data under different formulations and different inhibition backgrounds into a comparable correspondence range, thereby avoiding source separation distortion caused by the inhibition background flattening effect.

[0111] The inhibition release increment curves are organized into curve feature inputs that can be used for source separation mapping rules. During this process, the reading names and acquisition time points of the aforementioned subtraction treatment remain unchanged. The increment value sequences of the proteolysis inhibition release increment curves and lipid hydrolysis inhibition release increment curves at each acquisition time point are extracted separately. The concentrated period of the release response and the consistency difference of the release response between the two main curves are retained as curve feature inputs. These curve feature inputs, along with microbial lysis and release markers, constitute the input set for the source separation mapping rules and form a batch-specific input combination with the inhibition background intensity. This step transforms the inhibition release increment curves from raw time series differences into a stable input set, ensuring the consistency of the source separation mapping rules across different batches.

[0112] The process involves inputting the inhibition background intensity, inhibition relief increment curve, and microbial lysis release markers into a source-separation mapping rule. The rule outputs the contribution ratios of endogenous enzymes, microbial enzymes, and the dominant source determination conclusion. Input is based on the inhibition environment interval corresponding to the inhibition background intensity, and within this interval, it retrieves the corresponding relationship entries consistent with the de-inhibition target window. Curve feature input determines the distribution pattern of potential reaction increments along the proteolysis and lipidolysis main lines. Microbial lysis release markers limit the feasible range of the contribution ratio of microbial enzymes, and the remaining portion is attributed to the endogenous enzyme contribution ratio. The source-separation mapping rule provides the endogenous enzyme contribution ratio, microbial enzyme contribution ratio, and dominant source determination conclusion within the corresponding relationship entries. This step enables the output of traceable endogenous enzyme contribution ratios, microbial enzyme contribution ratios, and dominant source determination conclusions under inhibition background flattening conditions, providing directly applicable determination results for the quality stability control of ultra-high pressure cooked meat products during refrigeration.

[0113] For example, in one data instance, the suppression background intensity is 0.8, the desuppression target window limits the conductivity data to 12 mSiemens per centimeter to 14 mSiemens per centimeter, the desuppression target window limits the redox potential data to -90 mV to -60 mV, and the desuppression target window limits the available divalent metal ion level data to 0.9 mmol / L to 1.2 mmol / L. The incremental value sequence of the protein hydrolysis inhibition desuppression release curve at the acquisition time points of 0 seconds, 60 seconds, 120 seconds, and 180 seconds is 0, 0.18, 0.36, and 0.44, respectively. The incremental value sequence of the lipid hydrolysis inhibition desuppression release curve at the acquisition time points of 0 seconds, 60 seconds, 120 seconds, and 180 seconds is 0, 0.06, 0.10, and 0.11, respectively. The concentrated period of the desuppression response falls between 60 seconds and 120 seconds. The incremental value of the protein hydrolysis inhibition desuppression release curve at each acquisition time point is greater than the incremental value of the lipid hydrolysis inhibition desuppression release curve at the corresponding acquisition time point. The biomarker for microbial lysis and release is the copy number of microbial-specific nucleic acids (MSCs), which is 2,500,000 copies per milliliter of nucleic acid extract. The source splitting and mapping rule selects corresponding entries within the inhibition environment interval corresponding to an inhibition background intensity of 0.8 that match the target window for de-inhibition. Based on the concentrated time period and consistency differences in the de-inhibition response, the potential reaction increment is determined to be distributed primarily along the proteolytic axis. The contribution ratio of microbial-derived enzymes is limited to the range of 0.55 to 0.85 based on the microbial lysis and release biomarker. The source splitting and mapping rule outputs a contribution ratio of 0.7 for microbial-derived enzymes and 0.3 for endogenous enzymes, and concludes that the dominant source is microbial-derived enzymes.

[0114] Example 2:

[0115] Please see Figure 2As shown, this embodiment provides an auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment, including:

[0116] The meat chunks and isotonic extract were sheared and separated to obtain the sample extract.

[0117] Inhibition background characterization was obtained from the sample extract; based on the inhibition background characterization, the inhibition background intensity and the target window for de-inhibition were obtained by referring to the pre-established inhibition background calibration table.

[0118] An inert buffer was added to the sample extract to obtain an in-situ reaction environment. An anti-inhibition reference buffer was added to the sample extract according to the anti-inhibition target window to obtain an anti-inhibition reference reaction environment. Protein hydrolysis indicator probes, lipid hydrolysis indicator probes, and external internal standard enzyme reaction pairs were added to the two reaction environments respectively to obtain the reaction time series. The reaction time series were subtracted to obtain the inhibition relief increment curve.

[0119] Nucleic acid is extracted from the sample extract to obtain microbial lysis and release markers. The inhibition background intensity, inhibition relief increment curve, and microbial lysis and release markers are input into the preset source splitting mapping rule, and the endogenous enzyme contribution ratio, the microbial source enzyme contribution ratio, and the dominant source discrimination conclusion are output.

[0120] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An auxiliary detection method for enzyme activity regulation in low-temperature cooked meat products under ultra-high pressure treatment, characterized in that, include: The meat chunks and isotonic extract were sheared and separated to obtain the sample extract. Background suppression characterization was obtained from the sample extract; Based on the suppression background characterization, the suppression background intensity and the desuppression target window are obtained by referring to the pre-established suppression background calibration table; An inert buffer was added to the sample extract to obtain an in-situ reaction environment; an anti-inhibition reference buffer was added to the sample extract according to the anti-inhibition target window to obtain an anti-inhibition reference reaction environment; protein hydrolysis indicator probes, lipid hydrolysis indicator probes, and external internal standard enzyme reaction pairs were added to the two reaction environments respectively to obtain the reaction time series; the reaction time series were subtracted to obtain the inhibition relief increment curve; Nucleic acid is extracted from the sample extract to obtain microbial lysis and release markers. The inhibition background intensity, inhibition relief increment curve, and microbial lysis and release markers are input into the preset source splitting mapping rule, and the endogenous enzyme contribution ratio, the microbial source enzyme contribution ratio, and the dominant source discrimination conclusion are output.

2. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 1, characterized in that, Methods for shearing and separating meat chunks from isotonic extracts to obtain sample extracts include: Meat pieces from the same package, along with meat pieces from the central area of ​​the package, are taken from the area closest to the packaging's adhesive surface and from the area of ​​the central meat piece, and combined as the source of the meat piece. The isotonic extract was prepared according to the isotonic principle of matching the ionic environment of the low-temperature cooked meat products, and was placed in a low-temperature environment beforehand. The volume of the isotonic extract was recorded. The meat chunks were placed in a sealed container, and isotonic extract was added according to the volume of isotonic extract. The meat chunks and isotonic extract in the sealed container were mixed and sheared to obtain a uniform slurry. The homogeneous slurry was allowed to stand at low temperature, and the filtrate was obtained by initial solid-liquid separation using a fixed-size filter. The filtrate was placed in a centrifuge container and centrifuged at low speed. The supernatant was collected without disturbing the sedimentation layer and was defined as the sample extract.

3. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 2, characterized in that, Methods for obtaining background suppression characterization from sample extracts include: The sample extract was allowed to stand until the temperature stabilized within the same temperature range and then gently mixed. The sample extract was then dispensed into multiple equal-volume portions according to a predetermined volume, and the dispensing volume, dispensing temperature and dispensing time were recorded. Based on the sample extract aliquots, conductivity data was collected by conductivity measurement, pH data was collected by pH measurement, oxidation-reduction potential data was collected by oxidation-reduction potential measurement, nitrite content data was collected by nitrite colorimetric measurement, and available divalent metal ion level data was collected by complexable metal ion indicator reaction. Conductivity data, pH data, redox potential data, nitrite content data, and available divalent metal ion level data are compiled to form a background suppression characterization, which is then linked to the dispensing volume, dispensing temperature, and dispensing time.

4. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 3, characterized in that, Methods for obtaining suppressed background intensity and desuppressed target window include: The conductivity data, pH data, redox potential data, nitrite content data, and available divalent metal ion level data in the suppression background characterization were compared as a whole. For each calibration entry in the background suppression calibration table, the matching deviation is calculated. The matching deviation is synthesized from the normalized results of conductivity data deviation, pH data deviation, redox potential data deviation, nitrite content data deviation, and available divalent metal ion level data deviation within the allowable deviation range. The calibration entry with the smallest matching deviation and a matching deviation not exceeding 1 is determined as the calibration entry, and the suppression background intensity and the desuppression target window are read. The desuppression target window limits the target range of conductivity data, redox potential data and available divalent metal ion level data.

5. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 3, characterized in that, Methods for obtaining the inhibition release increment curve include: The sample extract was divided into two equal-volume portions according to the dispensing volume, and the dispensing temperature and dispensing time were uniform. An inert buffer solution was added to the extract of the first group of samples to obtain an in-situ reaction environment; According to the target window for de-inhibition, the de-inhibition reference buffer was added to the extract of the second group of samples in portions to obtain the de-inhibition reference reaction environment. The in-situ reaction environment and the de-inhibition reference reaction environment had the same dilution factor. Protein hydrolysis indicator probes, lipid hydrolysis indicator probes, and external internal standard enzyme reaction pairs were added to the in-situ reaction environment and the de-inhibition reference reaction environment, respectively, and signal readings were collected at fixed time intervals to obtain protein hydrolysis reaction time series, lipid hydrolysis reaction time series, and internal standard reaction time series, respectively. Matrix background subtraction was performed on the reaction time series, and in the same direction subtraction was performed based on the internal standard reaction time series to obtain the inhibition relief increment curve.

6. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 5, characterized in that, Methods for matrix background subtraction of reaction time series include: External internal standard enzyme reaction pairs were added to the in situ reaction environment and the de-inhibition reference reaction environment, and readings were collected at fixed time intervals to obtain the in situ internal standard reaction time series and the de-inhibition reference internal standard reaction time series. Based on the matrix background readings at the acquisition time points, matrix background subtraction was performed on the protein hydrolysis reaction time series and lipid hydrolysis reaction time series to obtain the in situ protein hydrolysis subtracted reaction time series, the de-inhibition reference protein hydrolysis subtracted reaction time series, the in situ lipid hydrolysis subtracted reaction time series, and the de-inhibition reference lipid hydrolysis subtracted reaction time series.

7. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 6, characterized in that, Methods for in-direction subtraction based on internal standard reaction time series include: The readout path drift was calculated based on the in situ internal standard reaction time series and the de-inhibition reference internal standard reaction time series, and the de-inhibition reference protein hydrolysis subtraction reaction time series and the de-inhibition reference lipid hydrolysis subtraction reaction time series were subtracted in the same direction. At the time point of acquisition, the difference between the in situ protein hydrolysis subtracted reaction time series and the de-inhibition reference protein hydrolysis subtracted reaction time series was adjusted to obtain the protein hydrolysis inhibition relief increment curve. The difference between the in situ lipid hydrolysis subtracted reaction time series and the de-inhibition reference lipid hydrolysis subtracted reaction time series was adjusted to obtain the lipid hydrolysis inhibition relief increment curve.

8. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 3, characterized in that, The conclusions regarding the contribution ratio of endogenous enzymes, the contribution ratio of microbial enzymes, and the determination of the dominant source include: Based on the aliquot volume, aliquots of the same volume standard were taken from the sample extract for nucleic acid extraction, and the sampling volume, sampling temperature and sampling time were recorded. The records were then correlated with the background suppression intensity and inhibition relief increment curves for the same batch. The sample extracts used for nucleic acid extraction were aliquoted and centrifuged at low speed, and the supernatant was collected. Nucleic acid extraction was performed on the supernatant to obtain a nucleic acid extract. Microbial-specific nucleic acid copy numbers were obtained by conducting quantitative nucleic acid amplification reactions based on nucleic acid extracts; microbial-specific nucleic acid copy numbers were defined as microbial lysis and release markers and were batch-linked with inhibition background intensity and inhibition release increment curves; The suppression background intensity, suppression release increment curve, and microbial lysis release markers are input into the source splitting mapping rule, and the contribution ratio of endogenous enzymes, the contribution ratio of microbial enzymes, and the dominant source discrimination conclusion are output.

9. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 1, characterized in that, Source splitting mapping rules include: Based on the expansion of the inhibition background calibration table, source splitting mapping rules were established and the correspondence between inhibition background intensity, anti-inhibition target window, protein hydrolysis inhibition relief increment curve, lipid hydrolysis inhibition relief increment curve, microbial lysis release markers and endogenous enzyme contribution ratio, microbial source enzyme contribution ratio, and dominant source discrimination conclusion was recorded. The suppression environment range is divided according to the suppression background intensity, and the conductivity data, redox potential data and available divalent metal ion level data are used as background constraints by limiting the desuppression target window. The incremental curves of protein hydrolysis inhibition relief and lipid hydrolysis inhibition relief were extracted as curve feature inputs. Combined with microbial lysis release markers, the feasible range of the contribution ratio of microbial enzymes was determined, and the contribution ratios of endogenous enzymes, microbial enzymes, and dominant sources were given.

10. The auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment according to claim 9, characterized in that, The correspondence between the input and output of the source splitting mapping rule includes: The suppression environment interval is determined based on the suppression background intensity, and the corresponding relationship entries consistent with the desuppression target window are called within the suppression environment interval; Based on the target window for desuppression, the target range is defined for conductivity data, redox potential data, and available divalent metal ion level data, and the incremental curve of desuppression relief is correlated with the target range. Based on the curve characteristics of the proteolysis inhibition release increment curve and the lipid hydrolysis inhibition release increment curve, the distribution pattern of potential reaction increments along the proteolysis main line and the lipid hydrolysis main line is determined. The feasible range of the contribution ratio of microbial enzymes is defined based on the markers of microbial lysis and release, and the remaining part is attributed to the contribution ratio of endogenous enzymes. The dominant source is determined based on the contribution ratio of endogenous enzymes and the contribution ratio of enzymes from microorganisms.

11. An auxiliary detection system for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment, used to implement the auxiliary detection method for regulating enzyme activity in low-temperature cooked meat products by ultra-high pressure treatment as described in any one of claims 1-10, characterized in that, include: The sample extraction module is used to shear and separate the meat chunks from the isotonic extract to obtain the sample extract. The calibration control module is used to collect suppression background characterization data from the sample extract. Based on the suppression background characterization, the suppression background intensity and the desuppression target window are obtained by referring to the pre-established suppression background calibration table; The sequence subtraction module is used to add inert buffer to the sample extract to obtain an in situ reaction environment, and to add a de-inhibition reference buffer to the sample extract according to the de-inhibition target window to obtain a de-inhibition reference reaction environment. Protein hydrolysis indicator probes, lipid hydrolysis indicator probes and external internal standard enzyme reaction pairs are added to the two reaction environments respectively to obtain the reaction time series. The reaction time series is subtracted to obtain the inhibition relief increment curve. The proportion discrimination module is used to extract nucleic acids from the sample extract to obtain microbial lysis and release markers. The inhibition background intensity, inhibition relief increment curve, and microbial lysis and release markers are input into the preset source splitting mapping rules, and the output endogenous enzyme contribution ratio, microbial source enzyme contribution ratio, and dominant source discrimination conclusion are output.

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