A fish meat flavor deterioration evaluation method based on a protein-lipid co-oxidation simulation system

Abstract

The application relates to the technical field of aquatic product quality evaluation, and discloses a fish meat flavor deterioration evaluation method based on a protein-lipid co-oxidation simulation system, which comprises the following steps: obtaining a real sturgeon meat cold storage sample, and separating myofibrillar protein and glycerophospholipid components in the sturgeon meat; adding the myofibrillar protein and the glycerophospholipid components into a buffer system to construct a co-oxidation simulation system; synchronously cold storing the simulation system and the real sturgeon meat sample, detecting key volatile off-flavor substances, lipid oxidation bridging indexes and protein oxidation indexes; performing correlation analysis on dynamic changes of the key off-flavor substances in the real sturgeon meat sample and the simulation system; and according to the correlation analysis result and the oxidation indexes, evaluating off-flavor formation risks driven by co-oxidation. The application excludes interference of a fish meat complex matrix, and realizes accurate tracing and quantitative evaluation of an off-flavor mechanism.

Description

Technical Field

[0001] This invention relates to the field of aquatic product quality evaluation technology, specifically to a method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system. Background Technology

[0002] Aquatic products are prone to quality deterioration during storage, with flavor degradation being a core indicator for evaluating freshness and quality. Fish meat contains a large amount of polyunsaturated fatty acids and highly structured myofibril proteins. In refrigerated environments, lipid and protein components are easily oxidized under the influence of endogenous factors, leading to degradation and the release of large amounts of volatile odor compounds. Accurately evaluating the formation patterns of odor compounds and elucidating the chemical driving mechanisms behind flavor degradation are fundamental to developing aquatic product preservation technologies and establishing processing and storage standards.

[0003] Current technologies for evaluating the deterioration of aquatic product flavor typically involve directly extracting real fish tissue from different storage stages as the basic testing sample. During the evaluation process, technicians use gas chromatography-mass spectrometry (GC-MS) to extract volatile odor compounds from the real fish samples and determine their concentrations. Simultaneously, they measure macroscopic physicochemical indicators such as total bacterial count, volatile basic nitrogen, pH value, and thiobarbituric acid reactants. Finally, the overall decline in the flavor quality of the fish is characterized by the trends in these macroscopic physicochemical indicators and volatile substances over time.

[0004] However, real fish tissue is a highly complex bioreaction matrix, where complex biochemical processes such as microbial metabolism, endogenous enzymatic decomposition, and spontaneous oxidation of various macromolecules occur simultaneously. Existing evaluation methods that directly use real fish tissue as the detection entity cannot eliminate the cross-interference between biological activities and tissue enzymes in the complex matrix at the physical and chemical levels. As a result, the off-odor products ultimately captured by the detection instrument are the mixed superposition of multiple interfering factors. It is difficult to isolate the true reaction trajectory of lipid and protein components from the complex biochemical reaction network. Consequently, existing technologies cannot accurately define the specific contribution of lipid and protein interactions to off-odor formation, and face the technical deficiency of being unable to accurately trace specific chemical driving mechanisms. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system, thus solving the problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system, comprising the following steps: taking sturgeon meat samples and obtaining real sturgeon meat samples at multiple refrigeration time points under refrigeration conditions; separating myofibrillar proteins from the sturgeon meat samples and obtaining glycerophospholipid components from the lipid components of the sturgeon meat samples; adding the myofibrillar proteins and glycerophospholipid components to a buffer system to construct a myofibrillar protein-glycerophospholipid co-oxidation simulation system; and treating the myofibrillar proteins under refrigeration conditions corresponding to those of real sturgeon meat samples. A protein-glycerophospholipid co-oxidation simulation system was established, and system samples were collected at multiple refrigeration time points. Key volatile odor substances in real sturgeon meat samples and system samples were detected, along with lipid oxidation bridging indices and protein oxidation indices in the system samples. Correlation analysis was performed on the dynamic changes of key volatile odor substances in real sturgeon meat samples and system samples to obtain the correlation analysis results. Based on the correlation analysis results, lipid oxidation bridging indices, and protein oxidation indices, the risk of odor formation driven by the co-oxidation of myofibrillar proteins and glycerophospholipid components in sturgeon meat samples during refrigeration was evaluated.

[0007] To eliminate interference from various physicochemical factors in the complex matrix of real fish meat, a myofibrillar protein-glycerophospholipid co-oxidation simulation system was established as the reaction baseline. When adding the myofibrillar protein and glycerophospholipid components to the buffer system, the final concentration of myofibrillar protein was 5-20 mg / mL, and the final concentration of the glycerophospholipid component was 1-10 mg / mL. The buffer system was either phosphate buffer or Tris buffer, and the pH was set to 6.5-7.8. Refrigeration conditions included refrigeration temperature and continuous refrigeration time period; the refrigeration temperature ranged from 0-6 ℃, and the continuous refrigeration time period ranged from 0-10 days.

[0008] When identifying substances causing flavor degradation, key volatile off-odors include at least one of hexanal, 1-octen-3-ol, (E)-2-octenal, 3-methylbutanal, octanal, (Z)-4-heptenal, 1-hexanol, 2,3-pentanedione, and 1-penten-3-ol. When detecting key volatile off-odors in real sturgeon meat samples and system samples, specific detection instruments are used to obtain the measured concentrations of the key volatile off-odors. The measured concentration of the key volatile off-odor is divided by the odor threshold of the key volatile off-odor in water to obtain the odor activity value (OAV); compounds with an OAV value greater than 1 are considered key off-odors. Specific detection instruments include HS-SPME-GC-MS, SAFE-GC-MS, or GC-IMS. In the testing operation, SAFE-GC-MS was used to detect key volatile odor substances in real sturgeon meat samples, and HS-SPME-GC-MS was used to detect key volatile odor substances in the samples. The key volatile odor substances detected were hexanal, 1-octen-3-ol, (E)-2-octenal, and 3-methylbutanal.

[0009] To quantify the degree of co-oxidation reaction, lipid oxidation bridging indicators include at least one of 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, and hydroxyl radicals; protein oxidation indicators include at least one of total thiol content, carbonyl content, Schiff base content, α-helix content, tryptophan endogenous fluorescence intensity, Raman spectral characteristic peaks, and Fourier transform infrared spectral characteristic peaks.

[0010] When preparing the buffer system, baseline data were simultaneously established for both glycerophospholipid oxidation and myofibrillar protein oxidation. The myofibrillar protein-glycerophospholipid co-oxidation simulation system met at least two of the following test conditions: the formation rate of 4-hydroxy-2-hexenal was 10-15 times that of the glycerophospholipid oxidation system alone; the formation rate of 4-hydroxy-2-nonenal was 4-6 times that of the glycerophospholipid oxidation system alone; the formation rate of hydroxyl radicals was 3-4 times that of the glycerophospholipid oxidation system alone; the generation rate of Schiff bases was 2-2.5 times that of the myofibrillar protein oxidation system alone; the loss rate of total thiol content was 1.3-1.7 times that of the myofibrillar protein oxidation system alone; and the increase rate of carbonyl content was 4-5 times that of the myofibrillar protein oxidation system alone.

[0011] Correlation analysis includes at least one of Pearson correlation analysis, Spearman correlation analysis, partial least squares regression, and principal component analysis. When performing correlation analysis on the dynamic changes of key volatile odor substances in real sturgeon meat samples and system samples, if the correlation coefficient between the total amount of at least one key volatile odor substance, or at least two key volatile odor substances, in the system sample and the corresponding indicator in the real sturgeon meat sample reaches a preset threshold, then the myofibrillar protein-glycerophospholipid co-oxidation simulation system is deemed capable of characterizing the formation process of refrigerated odor in real sturgeon meat. The preset threshold is a correlation coefficient of not less than 0.70.

[0012] Based on correlation analysis results, lipid oxidation bridging indices, and protein oxidation indices, the co-oxidation off-odor index was used to evaluate the risk of off-odor formation in sturgeon meat samples during refrigeration, driven by the co-oxidation of myofibrillar proteins and glycerophospholipid components. The odor activity (OAV) values ​​of the lipid oxidation bridging indices, protein oxidation indices, and key volatile odor substances were standardized. The Z-score method was used to standardize the raw measurement data.

[0013] To calculate the co-oxidation odor index, the standardized contents of 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, hydroxyl radicals, carbonyl groups, Schiff bases, and the total odor activity (OAV) were obtained. These were then weighted and summed. The total thiol content after standardization (with the assigned weight) was subtracted from the weighted sum to obtain the final co-oxidation odor index. A higher co-oxidation odor index indicates a higher risk of odor formation driven by the co-oxidation of myofibrillar proteins and glycerophospholipids.

[0014] This invention provides a method for evaluating the deterioration of fish flavor based on a protein-lipid co-oxidation simulation system. It has the following beneficial effects:

[0015] 1. This invention effectively eliminates interference factors such as microbial metabolism and tissue enzymatic reactions in the complex matrix of real fish meat by constructing an in vitro co-oxidation simulation system of myofibrillar proteins and glycerophospholipids. The extracted core components are subjected to isothermal refrigeration in a controlled buffer environment, enabling independent isolation and characterization of specific chemically driven mechanisms. This solves the problem of tracing the source of off-odors caused by lipid-protein interactions in real biological tissues, which is difficult to address with existing technologies, thus improving the purity of the detection system and the accuracy of mechanism studies.

[0016] 2. This invention constructs a multidimensional evaluation network encompassing the evolution of odor expression end products and underlying molecular structures by jointly detecting key volatile odor substances, lipid oxidation bridging indicators, and protein oxidation indicators. It breaks away from the existing single-mode flavor detection that relies on macroscopic spoilage physicochemical parameters, integrating multi-source measurement data such as odor activity data, the generation of various active aldehydes, and the degree of protein cross-linking. This comprehensively covers the dynamic reaction chain of flavor deterioration, achieving a comprehensive qualitative analysis of the odor formation process at both the molecular and sensory levels.

[0017] 3. This invention establishes a comprehensive evaluation standard, the co-oxidation odor index, by standardizing multidimensional measurement indicators dimensionlessly and introducing a correlation verification mechanism. Based on the confirmation that the dynamic evolution of biomarkers in the in vitro simulation system shows a significant correlation with real samples, the measurement results of various positive promoting products and negative consuming substances are weighted and summed. This dimensionless standardization and weighted summation of multidimensional measurement indicators transforms discrete biochemical detection values ​​into quantitative parameters that intuitively reflect the risk of deterioration, providing a standardized tool for quality monitoring during the cold storage of aquatic products. Attached Figure Description

[0018] Figure 1 This is a flowchart of the evaluation method for the formation of off-flavors in sturgeon meat during refrigeration driven by co-oxidation, as presented in this invention.

[0019] Figure 2 This is a schematic diagram showing the correspondence between real sturgeon meat and key off-odor substances in the MP+GP simulation system of this invention, with the normalized content trend of (E)-2-octenal (A) and the correlation of key indicators (B).

[0020] Figure 3 This is a schematic diagram of the enhanced lipid oxidation bridging index of the MP+GP system of the present invention, showing HHE content (A), HNE content (B), and hydroxyl radical content (C).

[0021] Figure 4 This is a schematic diagram of the MP+GP system for enhancing protein oxidation response according to the present invention, showing the total thiol content (A), carbonyl content (B), and Schiff base content (C). Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Please see the appendix Figure 1 -Appendix Figure 4 This invention provides a method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system, comprising the following steps:

[0024] Step S1 involves obtaining sturgeon meat samples at multiple refrigerated time points under cold storage conditions. Refrigeration conditions include refrigeration temperature and a continuous refrigeration time period. The refrigeration temperature ranges from 0 to 6 ℃, and the continuous refrigeration time period ranges from 0 to 10 days. Multiple refrigeration time points refer to sampling nodes set within the continuous refrigeration time period. Obtaining real sturgeon meat samples provides the physical objects for subsequent collection of baseline data.

[0025] Step S2 involves separating myofibrillar proteins from the sturgeon meat sample and obtaining glycerophospholipid components from the sturgeon meat lipid fraction. For the specific centrifugation and washing steps for separating myofibrillar proteins from sturgeon meat tissue, those skilled in the art can refer to standard protein extraction procedures. Separating myofibrillar proteins is a well-known technique in the art and will not be elaborated further in the patent document. For the specific solvent extraction and precipitation steps for obtaining glycerophospholipid components from the sturgeon meat lipid fraction, those skilled in the art can refer to conventional lipid purification methods. Obtaining glycerophospholipid components is a well-known technique in the art and will not be elaborated further in the patent document.

[0026] Step S3 involves adding myofibrillar protein and glycerophospholipid components to a buffer system to construct a myofibrillar protein-glycerophospholipid co-oxidation simulation system. The buffer system provides a constant pH liquid-phase reaction environment. Specific features of the buffer system include phosphate buffer and Tris buffer, with a pH range of 6.5-7.8. When adding myofibrillar protein and glycerophospholipid components to the buffer system, the final concentration of myofibrillar protein is 5-20 mg / mL, and the final concentration of the glycerophospholipid component is 1-10 mg / mL. This myofibrillar protein-glycerophospholipid co-oxidation simulation system eliminates microbial metabolic factors and tissue enzymatic reaction interferences present in real sturgeon meat samples.

[0027] Step S4 involves processing the myofibrillar protein-glycerophospholipid co-oxidation simulation system under refrigeration conditions corresponding to those of real sturgeon meat samples, and collecting system samples at multiple refrigeration time points. The corresponding refrigeration conditions refer to the exact same refrigeration temperature and continuous refrigeration time period as in Step S1. These multiple time points are synchronized with the sampling nodes in Step S1, constituting a synchronized reaction process between the real sturgeon meat samples and the co-oxidation simulation system.

[0028] Step S5 involves detecting key volatile odor compounds in real sturgeon meat samples and the co-oxidation simulation system, as well as lipid oxidation bridging indices and protein oxidation indices in the co-oxidation simulation system. Key volatile odor compounds are detected using specific instruments, and odor activity values ​​(OAVs) are calculated based on odor thresholds. Compounds with an OAV greater than 1 are considered key odor compounds. Detection instruments include HS-SPME-GC-MS, SAFE-GC-MS, or GC-IMS. Specific sub-characteristics of key volatile odor compounds include hexanal, 1-octen-3-ol, (E)-2-octenal, 3-methylbutanal, octanal, (Z)-4-heptenal, 1-hexanol, 2,3-pentanedione, and 1-penten-3-ol. Lipid oxidation bridging indices include 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, and hydroxyl radicals. Protein oxidation indices include total thiol content (see attached table). Figure 4 (A) Carbonyl content (attached) Figure 4 (B) Schiff base content (see appendix) Figure 4 The study included C content, α-helix content, tryptophan endogenous fluorescence intensity, Raman spectral characteristic peaks, and Fourier transform infrared spectral characteristic peaks.

[0029] Step S6 involves performing correlation analysis on the dynamic changes of key volatile odor substances in the real sturgeon meat sample and the co-oxidation simulation system. Dynamic changes refer to the quantitative evolution trajectory of the concentration or odor activity values ​​of the same type of key volatile odor substances at multiple time points. Specific sub-features of the correlation analysis include Pearson correlation analysis, Spearman correlation analysis, partial least squares regression, and principal component analysis.

[0030] Step S7: Based on the correlation analysis results, lipid oxidation bridging indices, and protein oxidation indices, evaluate the risk of off-odor formation driven by myofibrillar protein-glycerophospholipid co-oxidation during sturgeon meat refrigeration. The evaluation relies on the correlation analysis results to establish validity, and uses the quantitative measurements of lipid oxidation bridging indices and protein oxidation indices to determine the degree of co-oxidation reaction, outputting an off-odor formation risk assessment conclusion.

[0031] Regarding the technical features of separating myofibrillar protein from sturgeon meat and obtaining glycerophospholipid components from sturgeon meat lipid components in step S2 above, the following details the separation and extraction techniques of the core reaction components.

[0032] Step S201: Prepare sturgeon meat samples. The sturgeon meat can be from hybrid sturgeon (female Kaluga sturgeon and male Amur sturgeon), or from other sturgeon sources. The sturgeon meat samples are divided; some sturgeon meat is used to extract myofibrillar protein, and some sturgeon meat is freeze-dried to prepare freeze-dried sturgeon meat. The freeze-dried sturgeon meat is used to extract glycerophospholipid components.

[0033] Step S202: Isolation of myofibrillar protein from sturgeon meat. The sturgeon meat was homogenized in pre-cooled KCl-Tris-maleic acid buffer. The homogenized mixture was centrifuged, the precipitate was collected, and repeatedly washed with KCl-Tris-maleic acid buffer. The washed precipitate was dissolved in buffer containing 0.6 mol / L KCl. The resulting mixture was centrifuged again, and the supernatant was collected; this supernatant is the isolated myofibrillar protein. The protein concentration of the myofibrillar protein was determined using the biuret method.

[0034] Step S203: Obtain the glycerophospholipid component from the sturgeon meat lipid fraction. Total lipids are extracted from freeze-dried sturgeon meat using a chloroform and methanol mixture. After solvent removal and evaporation separation of the mixture, pre-cooled acetone is added to the total lipids to precipitate the glycerophospholipid component. The precipitated glycerophospholipid component is then dried for later use. The glycerophospholipid component can be crude phospholipids or further enriched PC, PE, LPC, or a mixture thereof.

[0035] For the specific centrifugation parameters set during the sturgeon meat homogenization process and the evaporation operation method during the total lipid solvent removal process, those skilled in the art can refer to the conventional biochemical separation procedures in the laboratory. The setting of operation parameters is a well-known technology in the field and will not be elaborated in the patent document.

[0036] Regarding the technical features of constructing the myofibrillar protein-glycerophospholipid co-oxidation simulation system in steps S3 and S4 above, and processing and sampling under corresponding refrigeration conditions, the following details the construction and reaction control of the co-oxidation simulation system.

[0037] Step S301: Prepare a buffer system. The buffer system is used to provide a reaction environment with a constant pH. The buffer system is a phosphate buffer, Tris buffer, or an equivalent buffer system. The pH of the buffer system is set to 6.5-7.8. Preferably, the buffer system is a 1x phosphate buffer, and the pH of the 1x phosphate buffer is set to 7.0-7.4.

[0038] Step S302: Assemble the myofibrillar protein-glycerophospholipid co-oxidation simulation system. Myofibrillar protein and glycerophospholipid components isolated from sturgeon meat are added to a buffer system to construct the myofibrillar protein-glycerophospholipid co-oxidation simulation system. The final concentration of myofibrillar protein in the co-oxidation simulation system is 5-20 mg / mL, and the final concentration of the glycerophospholipid component is 1-10 mg / mL. Preferably, the final concentration of myofibrillar protein is 8-12 mg / mL, and the final concentration of the glycerophospholipid component is 4-6 mg / mL. Specifically, an MP+GP group simulation system is established in a 1-fold phosphate buffer solution, containing a final concentration of 5 mg / mL glycerophospholipid component and a final concentration of 10 mg / mL myofibrillar protein. To facilitate subsequent quantification of the oxidation rate changes of lipid oxidation bridging indicators and protein oxidation indicators, a GP group simulation system and an MP group simulation system are simultaneously established in the same 1-fold phosphate buffer solution. The GP group simulation system contains only a final concentration of 5 mg / mL glycerophospholipid component. The MP group simulation system contained only myofibrillar protein at a final concentration of 10 mg / mL.

[0039] Step S303: Process the co-oxidation simulation system and collect samples. The co-oxidation simulation system is processed under refrigeration conditions corresponding to those of real sturgeon meat samples. The refrigeration conditions are 0-6 ℃, and the refrigeration time is 0-10 days. The preferred refrigeration condition is 4 ℃. During the refrigeration process, each simulation system is continuously stirred. Samples are collected at multiple time points during the refrigeration process. These multiple time points include 0, 2, 4, 6, and 8 days. Specifically, each simulation system is continuously stirred and reacted at 4 ℃ for 8 days, and system samples are collected at the corresponding time points of 0, 2, 4, 6, and 8 days. The time points for collecting system samples are consistent with the refrigeration time points of the real sturgeon meat samples.

[0040] For pH adjustment during the preparation of buffer systems, those skilled in the art can use conventional acid-base titration instruments in conjunction with chemical reagents. Adjusting pH using conventional acid-base titration instruments in conjunction with chemical reagents is a well-known technique in the field and will not be elaborated further in the patent documents.

[0041] Regarding the technical characteristics of key volatile odor substances detected in real sturgeon meat samples and co-oxidation simulation systems in step S5 above, the details of key volatile odor substance detection are disclosed below.

[0042] Step S501 involves detecting key volatile odor substances in the real sturgeon meat sample and the co-oxidation simulation system. The key volatile odor substances are detected using HS-SPME-GC-MS, SAFE-GC-MS, GC-IMS, or a combination of HS-SPME-GC-MS, SAFE-GC-MS, and GC-IMS. In a preferred embodiment, SAFE-GC-MS is used to detect the real sturgeon meat sample, as shown in Table 1, and HS-SPME-GC-MS is used to detect the co-oxidation simulation system sample, as shown in Table 2.

[0043] Table 1. Changes in volatile compounds in real sturgeon meat

[0044]

[0045] Table 2. Changes in volatile compounds in the MP+GP simulation system.

[0046]

[0047] Step S502: Determine the specific types of key volatile odor substances. Key volatile odor substances include one or more of hexanal, 1-octen-3-ol, (E)-2-octenal, 3-methylbutyraldehyde, octaldehyde, (Z)-4-heptenal, 1-hexanol, 2,3-pentanedione, and 1-penten-3-ol. Preferably, hexanal, 1-octen-3-ol, (E)-2-octenal, and 3-methylbutyraldehyde are detected in real sturgeon meat samples and in a co-oxidation simulation system. Hexanal, 1-octen-3-ol, (E)-2-octenal, and 3-methylbutyraldehyde detected in real sturgeon meat samples can all be detected and identified in the co-oxidation simulation system.

[0048] Step S503: Calculate the Odor Activity Value (OAV) based on the odor threshold to determine the key odor substances. Obtain the measured concentration of the key volatile odor substances. Divide the measured concentration of the key volatile odor substances by the odor threshold of the key volatile odor substances in water to obtain the OAV value. The OAV value is calculated using the following formula:

[0049]

[0050] In the formula: Indicates the first Odor activity values ​​of key volatile odor substances; Indicates the first Measured concentrations of key volatile odor substances; Indicates the first Odor threshold of key volatile odor substances.

[0051] After calculating the odor activity value (OAV), compounds with an OAV value greater than 1 were identified as key odor substances. Specifically, (E)-2-octenal maintained an OAV value greater than 1 for 0-8 days in the co-oxidation simulation system, with its content increasing from 0.0035 μg / g to 0.0050 μg / g. The OAV value of 3-methylbutyraldehyde increased from 0.49 to 1.49, and exceeded 1 after 4 days. Based on the changes in OAV values, hexanal, 1-octen-3-ol, (E)-2-octenal, and 3-methylbutyraldehyde were identified as co-oxidation odor markers.

[0052] For the selection of chromatographic columns and the setting of mass spectrometry bombardment parameters during the operation of HS-SPME-GC-MS or SAFE-GC-MS, those skilled in the art can refer to the analysis manual of conventional chromatography-mass spectrometry instruments. Setting the operating parameters of the analytical instrument is a well-known technology in the field and will not be elaborated in the patent documents.

[0053] Regarding the technical characteristics of detecting lipid oxidation bridging indices and protein oxidation indices in the co-oxidation simulation system in step S5 above, the implementation details of quantitative analysis of lipid and protein multidimensional oxidation response indices are disclosed below.

[0054] Before detecting lipid oxidation bridging indices, the consumption of fatty acid precursors in the system was measured. The changes in fatty acid precursor consumption in the simulated system of co-oxidation of real sturgeon meat and myofibrillar protein-glycerophospholipids are shown in the table below:

[0055] Table 3. Changes in fatty acid precursors in real sturgeon meat and the MP+GP simulation system.

[0056]

[0057] Step S504: Detect lipid oxidation bridging indices in the co-oxidation simulation system and the control system. Lipid oxidation bridging indices include one or more of 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, and hydroxyl radicals. The contents of 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal were detected using derivatization HPLC. The level of hydroxyl radicals was detected using DMPO spin trapping combined with electron spin resonance technology.

[0058] The myofibrillar protein-glycerophospholipid co-oxidation simulation system significantly enhanced lipid oxidation bridging indices, including HHE content (see appendix). Figure 3 (A), HNE content (attached) Figure 3 (B) Hydroxyl radical content (see attached) Figure 3 The evolution trend of (C) is shown in the appendix. Figure 3 As shown in the table below, the specific results of the changes in lipid oxidation bridging indices for each system during the 0-8 day refrigeration period are as follows:

[0059] Table 4 Changes in lipid oxidation bridging indices

[0060]

[0061] In the co-oxidation simulation system, the formation rate of 4-hydroxy-2-hexenal was 10-15 times that of the glycerophospholipid oxidation system alone. Specifically, during a 0-8 day refrigeration period, the concentration of 4-hydroxy-2-hexenal in the glycerophospholipid oxidation system alone increased from 0.0823 μg / mL to 0.1693 μg / mL, while in the co-oxidation simulation system it increased from 0.1748 μg / mL to 1.3108 μg / mL, representing a formation rate 13.13 times that of the glycerophospholipid oxidation system alone. The formation rate of 4-hydroxy-2-nonenal was 4-6 times that of the glycerophospholipid oxidation system alone. Specifically, in the glycerophospholipid oxidation system alone, the concentration of 4-hydroxy-2-nonenal increased from 0.8921 μg / mL to 2.5132 μg / mL, while in the co-oxidation simulation system, it increased from 0.8998 μg / mL to 8.7528 μg / mL. The formation rate of 4-hydroxy-2-nonenal was 4.56 times that of the glycerophospholipid oxidation system alone. The formation rate of hydroxyl radicals was 3-4 times that of the glycerophospholipid oxidation system alone, or the hydroxyl radical level at the reaction endpoint was 2-3 times that of the glycerophospholipid oxidation system alone. Specifically, the hydroxyl radical concentration in the glycerophospholipid oxidation system alone increased from 0.96 to 2.07, while in the co-oxidation simulation system it increased from 1.35 × 10⁻⁶. 17 Spins / g increased to 5.09×10 17 The hydroxyl radical formation rate of the co-oxidation simulation system was 3.35 times that of the glycerophospholipid oxidation system alone, and the hydroxyl radical level of the co-oxidation simulation system was 2.46 times that of the glycerophospholipid oxidation system alone after 8 days of refrigeration.

[0062] Step S505 involves detecting protein oxidation indicators in the co-oxidation simulation system and the control system. Protein oxidation indicators include one or more of the following: total thiol content, carbonyl content, Schiff base content, α-helix content, tryptophan intrinsic fluorescence intensity, Raman spectral characteristic peaks, and Fourier transform infrared spectral characteristic peaks. Total thiol and carbonyl content were determined using a detection kit, and Schiff base content was detected using fluorescence spectroscopy.

[0063] The myofibrillar protein-glycerophospholipid co-oxidation mimicry system simultaneously enhanced the protein oxidation response, and its evolution trend is shown in the attached figure. Figure 4 As shown in the table below, the specific results of the changes in protein oxidation parameters for each system during the 0-8 day refrigeration period are as follows:

[0064] Table 5 Changes in protein oxidation indicators

[0065]

[0066] In the co-oxidation simulation system, the Schiff base formation rate was 2-2.5 times that of the myofibrillar protein oxidation system alone. Specifically, during a refrigeration period of 0-8 days, the Schiff base concentration in the myofibrillar protein oxidation system increased from 62.28 to 102.77, while that in the co-oxidation simulation system increased from 65.96 to 153.40, representing a Schiff base formation rate 2.21 times that of the myofibrillar protein oxidation system alone. The total thiol loss rate was 1.3-1.7 times that of the myofibrillar protein oxidation system alone. Specifically, the total thiol concentration in the myofibrillar protein oxidation system decreased from 0.03153 μmol / mL to 0.02281 μmol / mL, while that in the co-oxidation simulation system decreased from 0.03477 μmol / mL to 0.02106 μmol / mL, representing a total thiol loss rate 1.48 times that of the myofibrillar protein oxidation system alone. The carbonyl growth rate was 4-5 times that of the myofibrillar protein oxidation system alone. Specifically, the carbonyl concentration in the myofibrillar protein oxidation system increased from 0.00205 μmol / mL to 0.00583 μmol / mL, while the carbonyl concentration in the co-oxidation simulation system increased from 0.06298 μmol / mL to 0.07413 μmol / mL. The carbonyl growth rate was 4.78 times that of the myofibrillar protein oxidation system alone, and after 8 days of refrigeration, the carbonyl level in the co-oxidation simulation system was approximately 12.72 times that of the myofibrillar protein oxidation system alone.

[0067] Regarding the selection of derivatizing agents and the setting of elution gradients in derivatization HPLC methods, those skilled in the art can refer to conventional chromatographic analysis standards. Setting elution gradients is a well-known technique in the field and will not be elaborated further in the patent documents. Regarding the colorimetric reaction conditions and absorbance measurement procedures of the kits for detecting total thiol and carbonyl content, those skilled in the art can follow the instructions for use of the corresponding kits. Using these kits to determine biochemical indicators is a well-known technique in the field and will not be elaborated further in the patent documents. Regarding the setting of excitation and emission wavelengths in the fluorescence spectroscopy method for detecting Schiff base content, those skilled in the art can refer to conventional biochemical fluorescence detection standards. Setting fluorescence detection wavelengths is a well-known technique in the field and will not be elaborated further in the patent documents.

[0068] Based on the chemical and biochemical measurement data obtained in step S5 above, the following details the correlation analysis of the dynamic changes of key volatile odor substances in real sturgeon meat samples and co-oxidation simulation systems, as well as the technical details of evaluating the risk of odor formation based on the correlation analysis results, lipid oxidation bridging index, and protein oxidation index.

[0069] Step S601: Correlation matching is performed on the measurement data of real sturgeon meat samples and the co-oxidation simulation system. The obtained dataset is organized, and values ​​not detected in either the real sturgeon meat samples or the co-oxidation simulation system are treated as 0. Data comparison is performed using 0, 2, 4, 6, and 8 days of refrigeration as benchmarks. Correlation analysis includes Pearson correlation analysis, Spearman correlation analysis, partial least squares regression, principal component analysis, or a combination thereof. Preferably, Pearson correlation analysis is used for data validation.

[0070] Step S602: Determine the characterization efficacy of the co-oxidation simulation system based on correlation analysis. When the correlation coefficient between the total amount of at least one key volatile odor substance, or at least two key volatile odor substances, in the co-oxidation simulation system and the corresponding indicator in the real sturgeon meat sample reaches a preset threshold, it is determined that the co-oxidation simulation system can characterize the formation process of refrigerated odor in real sturgeon meat. The preset threshold is the Pearson correlation coefficient or the Spearman correlation coefficient. Not less than 0.70.

[0071] Relationship as attached Figure 2 The dynamic changes and corresponding results of key off-odor substances in a simulated system of co-oxidation of real sturgeon meat and myofibrillar protein-glycerophospholipids are shown in the table below. The correlation data of these key off-odor substances are also presented.

[0072] Table 6. Correlation between real sturgeon meat and key off-odor substances in the MP+GP simulation system

[0073]

[0074] Specifically, (E)-2-octenal (with) Figure 2 In section A), the Pearson correlation coefficient for the content changes between real sturgeon meat samples and the co-oxidation simulation system was not less than 0.90; in the measured data, the Pearson correlation coefficient for (E)-2-octenal was... The significance level is 0.939. The value was 0.018, indicating a significant positive correlation. The Pearson correlation coefficient between the total contents of hexanal, 1-octen-3-ol, (E)-2-octenal, and 3-methylbutyraldehyde in the real sturgeon meat sample and the co-oxidation simulation system was not less than 0.70; in the measured data, the Pearson correlation coefficient between the total contents of hexanal, 1-octen-3-ol, (E)-2-octenal, and 3-methylbutyraldehyde was... The value is 0.757, indicating a positive correlation.

[0075] Step S701: Standardize the lipid oxidation bridging index, protein oxidation index, and odor activity (OAV) values ​​of key volatile odor substances. The Z-score method is used to standardize the raw measurement data. Standardization calculations are performed on the content of 4-hydroxy-2-hexenal, the content of 4-hydroxy-2-nonenal, the hydroxyl radical level, the carbonyl content, the Schiff base content, the total thiol content, and the sum of the OAV values ​​of key volatile odor substances. The specific calculation formula for the Z-score method is as follows:

[0076]

[0077] In the formula: Indicates the standardized value; This represents the measured value of the corresponding indicator; This represents the average measurement value of the corresponding indicator at multiple time points; This represents the standard deviation of the corresponding indicator at multiple time points.

[0078] Step S702: Calculate the co-oxidation odor index. The co-oxidation odor index is used to assess odor formation risk by combining lipid oxidation bridging indices and protein oxidation indices. The co-oxidation odor index is obtained by standardizing and weighting the lipid oxidation bridging indices, protein oxidation indices, and the odor activity (OAV) values ​​of key volatile odor substances. The co-oxidation odor index is calculated using the following formula:

[0079]

[0080] In the formula: Indicates the co-oxidation odor index; Standardized values ​​representing the content of 4-hydroxy-2-hexenal; Standardized values ​​representing the content of 4-hydroxy-2-nonenal; Standardized values ​​representing hydroxyl radical levels; Standardized values ​​representing carbonyl content; This represents the standardized value of Schiff's base content; This represents the standardized value of the sum of the odor activity (OAV) values ​​of key volatile odor substances. The standardized value representing the total thiol content; to This represents the weighting coefficient for each standardized value.

[0081] Weighting coefficient to The weighting coefficients are determined based on principal component analysis loadings or regression coefficients. Before establishing a large-sample evaluation model, the weighting coefficients... to The values ​​are all set to 1.

[0082] Step S703: Output the evaluation results. The higher the co-oxidation off-odor index, the higher the risk of off-odor formation driven by myofibrillar protein-glycerophospholipid co-oxidation during sturgeon meat refrigeration.

[0083] For the specific statistical algorithm logic of Pearson correlation analysis or principal component analysis, those skilled in the art can consult basic statistical principles. Performing statistical operations is a well-known technique in this field and will not be elaborated upon in the patent documents.

[0084] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for evaluating the deterioration of fish flavor based on a protein-lipid co-oxidation simulation system, characterized in that, Includes the following steps: S1, take sturgeon meat samples and obtain real sturgeon meat samples at multiple refrigeration time points under refrigeration conditions; S2, Isolate myofibrillar protein from the sturgeon meat sample and obtain glycerophospholipid components from the lipid components of the sturgeon meat sample; S3, the myofibrillar protein and the glycerophospholipid component are added to a buffer system to construct a myofibrillar protein-glycerophospholipid co-oxidation simulation system; S4, the myofibrillar protein-glycerophospholipid co-oxidation simulation system is treated under the refrigeration conditions corresponding to the real sturgeon meat sample, and system samples are collected at the multiple refrigeration time points; S5, detect key volatile odor substances in the real sturgeon meat sample and the system sample, and detect lipid oxidation bridging index and protein oxidation index in the system sample; the lipid oxidation bridging index includes at least one of 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, and hydroxyl radical; the protein oxidation index includes at least one of total thiol content, carbonyl content, Schiff base content, α-helix content, tryptophan endogenous fluorescence intensity, Raman spectral characteristic peak, and Fourier transform infrared spectral characteristic peak; S6, perform correlation analysis on the dynamic changes of the key volatile odor substances in the real sturgeon meat sample and the system sample to obtain the correlation analysis results; The correlation analysis includes at least one of Pearson correlation analysis, Spearman correlation analysis, partial least squares regression, and principal component analysis. When performing correlation analysis on the dynamic changes of the key volatile odor substances in the real sturgeon meat sample and the system sample, if the correlation coefficient between the total amount of at least one or at least two key volatile odor substances in the system sample and the corresponding index in the real sturgeon meat sample reaches a preset threshold, it is determined that the myofibrillar protein-glycerophospholipid co-oxidation simulation system can characterize the formation process of refrigerated odor in real sturgeon meat. The preset threshold is that the correlation coefficient is not less than 0.70; S7. Based on the correlation analysis results, the lipid oxidation bridging index, and the protein oxidation index, evaluate the risk of off-flavor formation in the sturgeon meat sample during refrigeration driven by the co-oxidation of the myofibrillar protein and the glycerophospholipid components. Based on the correlation analysis results, the lipid oxidation bridging index, and the protein oxidation index, when evaluating the risk of off-odor formation driven by the co-oxidation of myofibrillar protein and glycerophospholipid components during the refrigeration of sturgeon meat samples, the co-oxidation off-odor index is used to evaluate the off-odor formation risk. The lipid oxidation bridging index, the protein oxidation index, and the odor activity value (OAV) of the key volatile odor substances were standardized. The co-oxidation odor index is calculated by weighting the standardized lipid oxidation bridging index, the standardized protein oxidation index, and the standardized odor activity value (OAV). The higher the value of the co-oxidation odor index, the higher the risk of odor formation driven by the co-oxidation of the myofibrillar protein and the glycerophospholipid components. When standardizing the lipid oxidation bridging index, the protein oxidation index, and the odor activity value (OAV) of the key volatile odor substances, the Z-score method was used to standardize the original measurement data. The standardized contents of 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, hydroxyl radical level, carbonyl content, Schiff base content, and odor activity value (OAV) were obtained separately. After being assigned corresponding weight coefficients, a weighted summation was performed. The total thiol content after standardization was then subtracted from the result of the weighted summation to obtain the final calculated value of the co-oxidized odor index. The values ​​of the corresponding weight coefficients were all set to 1.

2. The method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system according to claim 1, characterized in that, When the myofibrillar protein and the glycerophospholipid component are added to the buffer system, the final concentration of the myofibrillar protein is 5-20 mg / mL, and the final concentration of the glycerophospholipid component is 1-10 mg / mL.

3. The method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system according to claim 1, characterized in that, The buffer system is a phosphate buffer or a Tris buffer, and the pH value of the buffer system is set to 6.5-7.8; the refrigeration conditions include refrigeration temperature and continuous refrigeration time period, the refrigeration temperature ranges from 0-6 ℃, and the continuous refrigeration time period ranges from 0-10 d.

4. The method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system according to claim 1, characterized in that, The key volatile odor substances include at least one of hexanal, 1-octen-3-ol, (E)-2-octenal, 3-methylbutanal, octanal, (Z)-4-heptenal, 1-hexanol, 2,3-pentanedione, and 1-penten-3-ol; when detecting the key volatile odor substances in the real sturgeon meat sample and the system sample, a specific detection instrument is used to obtain the measured concentration of the key volatile odor substances; the measured concentration of the key volatile odor substances is divided by the odor threshold of the key volatile odor substances in water to obtain the odor activity value (OAV); compounds with an OAV value greater than 1 are identified as key odor substances.

5. The method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system according to claim 1, characterized in that, When preparing the buffer system, a separate oxidation system for glycerophospholipids and a separate oxidation system for myofibrillar proteins are simultaneously established; the myofibrillar protein-glycerophospholipid co-oxidation simulation system meets at least two of the following test conditions: The formation rate of the 4-hydroxy-2-hexenal is 10-15 times that of the glycerophospholipid oxidation system alone; The formation rate of 4-hydroxy-2-nonenal is 4-6 times that of the glycerophospholipid oxidation system alone; The formation rate of the hydroxyl radicals is 3-4 times that of the glycerophospholipid oxidation system alone; The rate of Schiff base formation is 2-2.5 times that of the myofibrillar protein oxidation system alone; The rate of loss of total thiol content is 1.3-1.7 times that of the myofibrillar protein oxidation system alone; The rate of increase in carbonyl content is 4-5 times that of the myofibrillar protein oxidation system alone.

6. The method for evaluating fish flavor deterioration based on a protein-lipid co-oxidation simulation system according to claim 4, characterized in that, The specific detection instruments include HS-SPME-GC-MS, SAFE-GC-MS, or GC-IMS; The key volatile odor substances in the real sturgeon meat sample were detected by SAFE-GC-MS, and the key volatile odor substances in the system sample were detected by HS-SPME-GC-MS.

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