Association analysis method for flavor deterioration of chicken with different quality grades

Abstract

The invention provides a correlation analysis method for flavor deterioration of chicken with different quality grades, and relates to the technical field of food science and meat quality analys.The correlation analysis method comprises the steps that chicken samples with different quality grades are grouped to obtain first samples for volatile flavor substance and sensory analysis; and a second sample for non-volatile metabolite analysis; carrying out detection and qualitative and quantitative analysis on the volatile organic compounds in the first sample by adopting a gas chromatography-ion mobility spectrometry technology; carrying out non-targeted detection and qualitative and quantitative analysis on the non-volatile metabolites in the second sample by adopting a liquid chromatography-tandem mass spectrometry technology; carrying out sensory evaluation on the color, texture, appearance and smell of the chicken samples with different quality grades; by combining a multivariate statistical analysis method, volatile organic compounds, non-volatile metabolites and sensory evaluation data are comprehensively analyzed, and flavor characteristics, metabolite changes and correlation of chicken with different lignification degrees are analyzed.

Description

Technical Field

[0001] This invention relates to the fields of food science and meat quality analysis technology, specifically to a correlation analysis method for flavor degradation of chicken of different quality grades. Background Technology

[0002] Chicken breast is the most consumed poultry product globally due to its low fat, low calorie, high protein, and low cost. With the rapid development of intensive poultry farming, improving broiler growth rate and farming efficiency has become a core industry goal. However, this has also led to a high incidence of muscle abnormalities, with wooden breast (WB) being a typical example and a key issue restricting the quality upgrade of the broiler industry. WB is a growth rate-related muscle disease that primarily occurs in the pectoralis major muscle of chickens. Typical structural features of this disease include muscle fiber rupture and disordered arrangement, as well as abnormal proliferation of connective tissue, often accompanied by increased muscle hardness and surface bleeding, resulting in a deterioration in appearance. These structural abnormalities directly lead to changes in the chemical composition of muscle tissue, thereby affecting the formation and accumulation of flavor compounds, ultimately reducing the sensory quality and edible value of the meat.

[0003] Flavor is a core indicator of meat quality, determined by both volatile organic compounds and non-volatile metabolites, and its formation is closely related to lipid oxidation, amino acid metabolism, and energy metabolism in muscle tissue. With consumers' increasing demands for meat quality, the sensory characteristics, flavor quality, and nutritional value of chicken breast have received considerable attention. Although current research on lignified chicken breast covers areas such as nutritional regulation and pathological characteristics, and existing studies largely focus on the analysis of its physicochemical properties and optimization of processing performance, the dynamic evolution of flavor compounds during the lignification process, and the intrinsic relationship between these flavor compounds and endogenous metabolites in muscle, remain incompletely elucidated.

[0004] In recent years, the combination of metabolomics and flavoromics technologies has provided an effective means to deeply elucidate the molecular mechanisms of meat flavor formation. Through systematic analysis of non-volatile metabolites and volatile flavor compounds, the intrinsic link between metabolic networks and flavor characteristics can be revealed. This method has made significant progress in flavor studies of pork, beef, and some poultry. However, research on the correlation between flavor compounds and metabolites in chicken breast with different degrees of lignification still needs to be strengthened, especially regarding the understanding of how key metabolic pathways regulate flavor compound synthesis.

[0005] This invention uses 41-day-old Kobo broiler chicken breasts as the research object, dividing them into normal (NB), lightly lignified (LWB), moderately lignified (MWB), and heavily lignified (SWB) groups according to their degree of lignification. It comprehensively utilizes sensory evaluation, headspace-gaschromatography-ionmobility spectrometry (HS-GC-IMS), and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) techniques, combined with multivariate statistical analysis methods, to systematically explore the flavor characteristics, metabolite changes, and intrinsic relationships of chicken breasts with different degrees of lignification. The aim is to reveal the regulatory mechanism of flavor compound formation by differences in the metabolic network of lignified chicken breasts, elucidate the correlation pathways between key metabolites and characteristic volatile flavor compounds, and provide a theoretical basis and scientific support for improving the flavor quality of lignified chicken breasts and developing targeted quality control technologies. Summary of the Invention

[0006] This invention proposes a correlation analysis method for flavor degradation of chicken of different quality grades, comprising the following steps: Chicken samples were divided into normal group, mild lignification group, moderate lignification group and severe lignification group based on appearance and palpation. The chicken samples were grouped to obtain a first sample for volatile flavor compounds and sensory analysis, and a second sample for non-volatile metabolite analysis. Gas chromatography-ion mobility spectrometry was used to detect and perform qualitative and quantitative analysis of volatile organic compounds in the first sample; Non-volatile metabolites in the second sample were detected and analyzed qualitatively and quantitatively using liquid chromatography-tandem mass spectrometry. Sensory evaluation was performed on the color, texture, appearance, and odor of the chicken samples; By combining multivariate statistical analysis methods, the volatile organic compounds, non-volatile metabolites, and sensory evaluation data were comprehensively analyzed to analyze the flavor characteristics, metabolite changes, and correlations of chicken with different degrees of lignification.

[0007] Furthermore, the normal group had no white streaks or hard parts on the surface of the chicken; the mild lignification group had slight changes at the head and near the tail of the chicken, with a slightly hard feel at the head; the moderate lignification group had a hard head and elasticity from the middle to the tail; and the severe lignification group had a hard overall appearance, with bleeding and exudate on the surface of the lignified chicken.

[0008] Furthermore, the preparation of the first sample specifically involves: vacuum packaging 40g of the sample and heating it in a 95°C water bath until the center temperature reaches 75°C to lignify the chicken meat.

[0009] Furthermore, the preparation of the second sample is as follows: weigh 50±5mg of sample, add pre-cooled 80% methanol, grind and pulverize, let stand at -20°C for 30min, and then centrifuge to obtain the supernatant to lignify the chicken meat.

[0010] Furthermore, the conditions for gas chromatography-ion mobility spectrometry analysis were as follows: an MXT-WAX capillary column was used at a column temperature of 60°C; the sample was incubated at 80°C and 500 r / min for 15 min; and the IMS conditions were: tritium source as the ionization source and migration tube temperature of 45°C for lignified chicken meat.

[0011] Furthermore, the conditions for liquid chromatography-tandem mass spectrometry analysis were as follows: an ACQUITY UPLCT3 column was used at a column temperature of 40°C; mobile phase A consisted of 5 mM ammonium acetate + 5 mM acetic acid + water, and mobile phase B consisted of acetonitrile; the mass spectrometer was equipped with an electrospray ionization source, and data were acquired in both positive and negative ion modes.

[0012] Furthermore, the multivariate statistical analysis includes principal component analysis, partial least squares discriminant analysis, Spearman correlation analysis, and metabolic pathway enrichment analysis based on the KEGG database; the partial least squares discriminant analysis evaluates the robustness of the model using 5-fold cross-validation and permutation tests.

[0013] Furthermore, data integration and analysis also include: using Spearman correlation analysis to determine the correlation between differential metabolites and characteristic flavor compounds, and screening key volatile substances and differential metabolites based on variable importance projection and P<0.05, and performing redundancy analysis.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for systematically analyzing the flavor degradation of chicken breast at different degrees of lignification. By comprehensively utilizing sensory evaluation, HS-GC-IMS, and UPLC-MS / MS techniques, combined with multivariate statistical analysis, this invention can deeply explore the dynamic changes in flavor characteristics and metabolite profiles of chicken breast at different lignification stages. Its core beneficial effect lies in the fact that it systematically reveals for the first time the intrinsic correlation mechanism between flavor substances and endogenous metabolites during the lignification process, elucidating how key metabolic pathways regulate the synthesis of characteristic flavor substances. This not only fills the gap in existing research on the mechanism of flavor degradation but also provides a solid theoretical basis and scientific support for improving the flavor quality of lignified chicken breast and developing targeted quality control technologies. Attached Figure Description

[0015] Figure 1 Classification criteria and sensory evaluation of different grades of chicken breast; Figure 2 SPME-GC-IMS analysis of different grades of chicken breast; Figure 3 LC-MS analysis of different grades of chicken breast; Figure 4 Spearman correlation analysis and RDA analysis were performed to investigate the differences in volatile organic compounds and metabolites among different grades of chicken breast. Figure 5 This is a diagram of the metabolic pathways involved in the formation of KEGG. Detailed Implementation

[0016] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0017] 1. Test Samples and Grouping The test samples were obtained from Jinluo Wenrui Company in Linyi, Shandong Province. The broiler chickens were of the Kobo white-feathered breed, 41 days old, with an average body weight of about 2.89 kg, and were slaughtered and processed according to the company's conventional slaughtering and processing procedures. Two to three hours after slaughter, the chicken breasts were graded on the chicken breast processing line based on appearance and palpation, with slight adjustments made according to the grading standards of Tijaree et al. (2016) and B. Li et al. (2024).

[0018] The specific sampling standards are as follows: Normal meat: Chicken breast has no white streaks or hard parts on the surface. Woody meat is divided into 3 grades: Mild: Slight changes are observed at the head and near the tail of the chicken breast, with a slightly hard feel at the head; Moderate: The entire head of the chicken breast exhibits a hard characteristic, but the middle to tail is elastic; Severe: In addition to exhibiting a hard characteristic overall, the surface of the chicken breast is accompanied by bleeding and exudate.

[0019] The chicken breast samples were heat-treated according to the method described in Trithavisup et al. (2024). Each sample (40g) was individually vacuum-packed in a plastic bag and cooked in a 95°C water bath until the core temperature reached 75°C. The heat-treated chicken breast samples were used for HS-GC-IMS and sensory analysis. Untreated chicken breast samples were analyzed by UPLC-MS / MS. Four treatment groups were established, with five chicken breasts in each group. In subsequent measurements, each chicken breast was randomly divided into two portions, and each portion was analyzed in triplicate to ensure accuracy.

[0020] 2. Sensory evaluation The sensory characteristics of chicken breast, including color, texture, and appearance, were evaluated using a scoring method (Table 1). Each group of chicken breast samples was randomly coded using a blind evaluation method. The sensory evaluation team consisted of 10 teachers and students (5 males and 5 females) trained in sensory evaluation. Evaluators were required to avoid consuming foods that significantly affect taste before the evaluation. Evaluators rinsed their mouths with water before evaluating different groups of chicken breast samples. Evaluations were conducted independently, without communication. Three chicken breasts were evaluated for each group, and the average score for each indicator was calculated based on the members' scores.

[0021] Table 1 Sensory evaluation criteria for chicken breast samples 3. HS-GC-IMS Volatile organic compounds from chicken breasts with four different degrees of lignification were detected using an MXT-WAX capillary column (Restek, USA) and an IMS instrument (FlavourSpec®, Gesellschaft fr Analytische Sensorsysteme GmbH, GAS, Dortmund, Germany).

[0022] Accurately weigh 2g of sample into a 20mL headspace vial and incubate at 80℃ and 500r / min for 15min. Then extract 500µL of headspace gas and inject it into the syringe at 105℃ using a heated syringe. Chromatographic conditions: Column: MXT-WAX capillary column (15mm×0.53mm, 1.0μm); Column temperature: 60℃; Carrier gas: Nitrogen (purity >99.999%). Carrier gas flow rate: 2mL / min, maintained for 2min; gradually increased to 10mL / min from 2 to 10min; gradually increased to 100mL / min from 10 to 20min; maintained for 10min; total run time: 30min. IMS conditions: Ionization source: tritium source (… 3 H), migration tube length 53 mm, temperature 45℃, drift gas nitrogen (purity >99.999%), flow rate 75.0 mL / min. Each sample was measured three times.

[0023] The RI and drift time (DT) of volatile organic compounds were compared with the GC retention index database (NIST2020) and the IMS migration time database. Qualitative identification was performed using n-ketones (2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone, and 2-nonanone (all analytical grade, Aladdin)) as internal standards. The relative contents of volatile flavor components were determined using the area normalization method and calculated using equation (1): (1); 4. UPLC-MS / MS Four chicken breast samples with different degrees of lignification were accurately weighed (50±5 mg each) and placed into 2 mL centrifuge tubes. 500 µL of pre-cooled 80% methanol was added, and the samples were ground using a grinder with steel beads. The mixtures were then placed in a -20°C freezer for 30 min to precipitate proteins. The samples were centrifuged at 20000×g for 15 min, and 400 μL of the supernatant was transferred to another EP tube. After centrifugation at 20000×g for 15 min, the supernatant was transferred to a sample vial. 10 μL of each sample extract was taken for analysis. Each sample was analyzed in triplicate.

[0024] Non-volatile metabolites were analyzed using a Vanquish FlexUPLC system (Thermo Fisher Scientific, Bremen, Germany) equipped with an ACQUITY UPLCT3 column (100 × 2.1 mm, 1.8 μm, Waters, UK). Chromatographic conditions were: column temperature 40 °C, flow rate 0.35 mL / min, injection volume 10 μL. The mobile phase was 5 mM ammonium acetate + 5 mM acetic acid + water (A) and acetonitrile (B). The gradient elution program was: 0–0.5 min, 5% B; 0.5–6.0 min, 5%–95% B; 6.0–7.5 min, 95% B; 7.5–7.6 min, 95%–100% B; 7.6–8.0 min, 100% B; 8.0–8.1 min, 100%–5% B; 8.1–10 min, 5% B.

[0025] Metabolite identification was performed using an Orbitrap Exploris 120 high-resolution tandem mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), equipped with an electrospray ionization (ESI) source. Data were acquired in both positive and negative ion modes. The spray voltage was set to 3.80 kV for positive mode and -3.40 kV for negative mode. The capillary temperature was 350 °C, and the first-order resolution was set to 60 K. The ion scan range was 200 m / z. The top four signal ions with a signal accumulation intensity exceeding 5000 were selected from the first-order spectrum for second-order fragmentation scanning. The second-order resolution was 15.0 K. Redundant MS / MS signals were removed using a dynamic exclusion method with a dynamic exclusion setting of 6 s.

[0026] 5. Data Analysis After standardization, multivariate analysis was performed on the data using the R software package (ropls), including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). PLS-DA assessed the robustness of the model using 5-fold cross-validation and permutation tests, and calculated the projected importance (VIP) value of each variable in the model to indicate its contribution to classification. A t-test was used to determine the significance of differences between the two independent groups (P < 0.05). Finally, Spearman correlation analysis was used to determine the correlation between the two variables.

[0027] All measurements were performed in triplicate, and the results are expressed as mean ± standard deviation. One-way ANOVA was performed using SAS 9.2 software, and the Tukey multiple comparison test was used to examine the significance of differences between different treatment groups (P < 0.05).

[0028] Example 2 1. Sensory evaluation analysis Figure 1 The classification criteria and sensory evaluation of different grades of chicken breast are shown in (a) from left to right: normal chicken breast (NB), lightly lignified chicken breast (LWB), moderately lignified chicken breast (MWB), and heavily lignified chicken breast (SWB); (b) sensory evaluation.

[0029] The NB group maintained high scores across all sensory indicators, with significantly higher scores in color and texture than the lignified group (P<0.05). Regarding taste and aroma, there was no significant difference between the NB and LWB groups (P>0.05), but both groups scored significantly higher than the MWB and SWB groups (P<0.05). The SWB group had the lowest scores in all sensory indicators, particularly in appearance and aroma (scores decreased by 42-58%). This is closely related to the damage to muscle fiber structure and the accumulation of lipid oxidation products in lignified chicken breast. There were no significant differences in taste and appearance scores between adjacent lignification grades (e.g., LWB and MWB) (P>0.05), indicating that sensory evaluation has limitations in resolving lignification.

[0030] Based on the above results, this invention further employs HS-GC-IMS technology to analyze volatile matter spectra to characterize flavor features, and combines UPLC-MS / MS non-targeted metabolomics to explore metabolite changes, in order to reveal the intrinsic mechanism of texture deterioration and flavor decay in lignified chicken breast.

[0031] 2. HS-GC-IMS Analysis 2.1 Analysis of Volatile Organic Compounds in Chicken Breast with Different Degrees of Lignification To investigate the flavor differences in chicken breast with different degrees of lignification, HS-GC-IMS was used to analyze the volatile organic compounds (VOCs) in the samples. Figure 2 SPME-GC-IMS analysis of different grades of chicken breast included: (a) topographic map of volatile organic compounds; (b) comparison map of volatile organic compounds; (c) fingerprint map of volatile organic compounds; (d) pie chart showing the classification of volatile organic compounds; (e) stacked bar chart of relative values ​​of different compound categories; (f) principal component analysis score plot; (g) partial least squares discriminant analysis score plot; (h) permutation test of partial least squares discriminant analysis; (i) VIP score of partial least squares discriminant analysis; and (j) heatmap analysis of differential volatile organic compounds.

[0032] To visualize the differences in volatile organic compounds among chicken breast samples with different degrees of lignification, fingerprint patterns were constructed. Figure 2 (c) By comparing with the NIST database, a total of 62 compounds were identified in four chicken breast samples with different degrees of lignification, including aldehydes (18), alcohols (11), ketones (7), esters (6), acids (2) and furans (1). Figure 2 (d). The relative contents of volatile components in detail are shown in Table 2.

[0033] Figure 2 In zone (c)A (NB), the high VOC content in normal chicken breast is mainly composed of unsaturated aldehydes ((E)-2-heptenal, (E)-2-pentenal, 1-octanal) and saturated aldehydes (hexanal, 1-nonanal). These lipid oxidation products impart a fresh, fatty aroma and grassy notes, which are hallmarks of excellent flavor (Parketal., 2025). Characteristic alcohols (1-octen-3-ol, 1-hexanol) and esters (ethylhexanoate, sec-butylacetate) are abundant. The former originates from normal lipid oxidation (with a mushroom aroma), while the latter reflects normal co-metabolism of amino acids and fatty acids (with a fruity aroma). The high concentration of benzaldehyde (with bitter almond, cherry, and nutty notes) indicates active amino acid metabolism (Bleicher et al., 2022).

[0034] The above characteristics indicate that the orderly progression of lipid oxidation and amino acid degradation pathways is the foundation for the formation of normal chicken breast flavor. Region B (LWB) retains some characteristics of NB, both rich in 3-methylbutyraldehyde (chocolate flavor, fatty flavor) and 2-methyl-2-hepten-6-one (apple flavor, fruity flavor), indicating that amino acid degradation and lipid oxidation are not completely disrupted during mild lignification. Region C (LWB) shows the addition of substances such as n-butyraldehyde (spicy flavor, fruity flavor), acetic acid (spicy flavor), and 3-hydroxy-2-butanone (butter, creamy flavor). These changes, along with slight damage to surface muscle fibers, may lead to lipid oxidation and the generation of short-chain products, while simultaneously increasing ketones. Although the flavor deviates from normal, it still retains some desirable properties. The characteristic VOCs in region D (MWB) are mainly small-molecule aldehydes, alcohols, and ketones, including n-pentanal (banana flavor), n-heptanal (fatty flavor), ethanol (aromatic flavor), n-pentanol (spice flavor), and butanone (fruity flavor, camphor flavor). Meanwhile, the amount of long-chain unsaturated aldehydes ((E)-2-heptenal) associated with superior flavor was significantly reduced, indicating that moderately lignified chicken meat exhibits fermented and rancid flavor characteristics. Region E (SWB) is characterized by sulfur-containing compounds and abnormally accumulated long-chain unsaturated aldehydes / esters. Significantly elevated levels of 3-(methylsulfonyl)propanal (a methionine degradation product with a sulfurous taste), (E)-2-octenal (a marker of severe lipid oxidation), as well as butanol, n-propanol, and propyl acetate, confirm that heavily lignified chicken meat accumulates more sulfurous and rancid flavor precursors, resulting in a significant deterioration in flavor quality.

[0035] Figure 2 (e) reveals the compositional characteristics of volatile flavor compounds in chicken breast with different degrees of lignification. Aldehydes (45.85–46.39%), ketones (22.15–22.40%), and alcohols (19.82–21.51%) were present in relatively high amounts in all treatment groups, followed by esters (7.11–8.19%). Based on their relative content and diversity, aldehydes, ketones, and alcohols were identified as the major volatile flavor compounds in chicken breast.

[0036] 2.2 Multivariate statistical analysis of HS-GC-IMS data To further examine and differentiate the flavor differences among four chicken breast samples with different degrees of lignification, multivariate statistical analysis was performed on the HS-GC-IMS data. In the PCA, the first two principal components, PC1 (67.66%) and PC2 (10.70%), accounted for 78.36% of the total variance, indicating that most of the information in all samples could be elucidated by the first two principal components. Figure 2 (f). PLS-DA analysis further validated the differences between groups. Figure 2 The model accuracy (g) was 82.30%. After 200 permutation tests, the R-value of the model was... 2 =0.3853,Q2 =-1.2436, confirming that the model did not overfit. Figure 2 (h). Based on variable importance projection (VIP>1), 16 key VOCs were screened, including propyl acetate, ethyl 2-methylvalerate, ethyl 3-methylbutyrate, (E)-2-octenal, decanal, 1-butyric acid, (E)-2-nonenal, benzaldehyde, (Z)-4-heptenal, 3-(methylthio)propanal, 1-octanol, ethanol, butanol, (E)-2-hexen-1-ol, sec-butyl acetate, and 3-pentanol. Figure 2 (i) Of the 16 VOCs, 8 had the highest concentrations in the NB group, while 5 VOCs had the highest concentrations in the MWB and SWB groups. Thirteen of these VOCs were confirmed as significantly different by t-test (P<0.05). Figure 2 (j)

[0037] The results showed that as the lignification of chicken breast deepened, the content of aldehydes and esters decreased. This may be due to the damage to the muscle fiber structure caused by lignification, resulting in an imbalance in cell metabolism and the loss of specific flavors. Among them, 3-(methylsulfonyl)propionaldehyde was found in high amounts in SWB. This substance has a pungent sulfur smell and is a marker of the degradation of sulfur-containing amino acids during severe lignification, directly leading to flavor deterioration.

[0038] 3. UPLC-MS / MS analysis 3.1 Metabolomics Results This invention employs UPLC-MS / MS non-targeted metabolomics combined with multivariate statistical analysis to analyze the characteristics of non-volatile metabolites in chicken breasts with four different degrees of lignification: NB, LWB, MWB, and SWB. A total of 1097 metabolites were detected, classified into 16 categories: lipids and lipid-like molecules (333), organic heterocyclic compounds (223), organic acids and their derivatives (185), phenylpropanoid heterocyclic compounds (144), organic oxygen-containing compounds (62), phenylpropanoids and polyketides (40), organic nitrogen-containing compounds (37), nucleosides, nucleotides and their analogues (30), alkaloids and their derivatives (21), organosulfur compounds (9), homogeneous nonmetallic compounds (5), lignans, neolignans and related compounds (2), organic 1,3-dipolar compounds (2), organophosphorus compounds (1), and others (3). Among these, lipids and lipid-like molecules, organic heterocyclic compounds, organic acids and their derivatives, and phenylpropanoid heterocyclic compounds were the most abundant compound categories, with an average relative content greater than 20%. Figure 3For LC-MS analysis of different grades of chicken breast, (a) a pie chart showing metabolite classification; (b) principal component analysis score plot; (c) partial least squares discriminant analysis score plot; (d) permutation test of partial least squares discriminant analysis; (e) VIP score of partial least squares discriminant analysis; (f) heatmap bubble chart analysis of the top 30 differentially expressed metabolites; (g) bar charts of upregulation and downregulation of differentially expressed metabolites in different groups; (h) Venn diagram of differentially expressed metabolites in different groups; (i)~(k) volcano plots showing differentially expressed metabolites in different groups. The x-axis and y-axis represent the effect on the pathway (in logarithmic 2-fold change) and the significant changes in the pathway by the detected metabolites (in -log10), respectively; (l)~(n) inter-group pathway analysis for significantly different metabolites. In the scatter plot, the x-axis represents the effect on the pathway, while the y-axis represents the significant changes in the pathway by the detected metabolites. The results showed that there were significant differences in metabolite expression profiles among the four groups. In the SWB group, a large number of metabolites were significantly upregulated (log2FC>1, P<0.05), which may be related to the destruction of cell membrane structure caused by the high lignification of muscle fibers, resulting in the loss of flavor precursor substances.

[0039] 3.2 Identification of Differential Metabolites To further understand the changes in metabolites in chicken breast with different degrees of lignification, the NB group was used as a control. Differential metabolites in each lignification group were screened and their numbers were statistically analyzed. The screening criteria for differential metabolites were FC ≥ 1.2, P < 0.05, and VIP ≥ 1. Figure 3 From (g), we can see that LWB vs NB: 25 upregulated metabolites and 12 downregulated metabolites. MWB vs NB: 56 upregulated metabolites and 22 downregulated metabolites. SWB vs NB: 72 upregulated metabolites and 25 downregulated metabolites. Furthermore, through Venn diagrams, Figure 3 The number of shared and unique differential metabolites among the groups was compared (h), revealing six shared differential metabolites: 1,1,1-trifluoroheptadecane-2-one, 11,12-epoxyeicosatotrienoic acid, 16-hydroxydihydrocyclohexene, irrigin trimethyl ether, 15(S)-hydroxyeicosatotrienoic acid, and methanesulfonic acid. The SWBvsNB group had the most unique differential metabolites (50), followed by the MWBvsNB group (28), and the LWBvsNB group had the fewest (24). Among them, MWBvsNB had the most overlap with the other two groups, with 5 and 39 shared differential metabolites with the LWBvsNB group and the SWBvsNB group, respectively. This result indicates that the intermediate lignification stage is a transitional state of the metabolic network, exhibiting some of the differential characteristics of the mild lignification stage while beginning to transition to the severe lignification stage, resulting in more complex metabolite changes.

[0040] To more intuitively illustrate the changing trends of metabolite abundance under different degrees of lignification, the differentially expressed metabolites among the comparison groups are displayed in the form of a volcano plot. Figure 3 (i)~(k), and the screened differential metabolites are further used for metabolic pathway analysis. Figure 3 The results show that in the LWBvsNB group... Figure 3 (i) The main upregulated differential metabolites were urocanic acid, amisifrine, and hexadecanoylcarnitine, while the main downregulated differential metabolites were 1,1,1-trifluoroheptadecane, 9-octadecanoamide, and sphinganamine. Hexadecanoylcarnitine is involved in fatty acid metabolism; its abnormally high level may lead to disordered lipolysis, indirectly affecting the synthesis of short-chain flavor aldehydes and thus diminishing the meat's flavor. Sphinganine is involved in cell membrane construction; its reduction may disrupt sphingolipid metabolism, increasing cell membrane permeability and causing the loss of umami nucleotides, thereby reducing the umami flavor of the meat.

[0041] In the MWB vs NB group Figure 3 The levels of 11,12-epoxyethyl hydantoin, acetamide, 2-((2-hydroxyethyl)thio)-N-(3-(3-(1-piperidinylmethyl)phenoxy)propyl)- and 5(S)-hydroxyeicosatetraenoic acid were significantly upregulated, while 2-methyl-5-(methylthio)furan and oxalic acid were significantly downregulated. Among them, 11,12-epoxyeicosatetraenoic acid is an oxidation product of arachidonic acid. Its increased content may competitively bind to esterases, reduce the synthesis of ethyl hexanoate, and reduce the fruity aroma characteristics brought by esters in meat. Furans are important aroma substances. The formation mechanism of 2-methyl-5-(methylthio)furan is related to the degradation reaction of thiamine. Its reduction will directly inhibit the formation of disulfide, a key meat aroma substance, and weaken the characteristic aroma of meat products.

[0042] In the SWB vs NB group Figure 3 The (k) group showed a significant increase in differentially expressed metabolites. 11,12-epoxyeicosatetraenoic acid, 11-(3-pentylepoxyethylene-2-yl)undec-9-enoic acid, and 13,14-dihydro-15-ketoprostaglandin F2α were significantly upregulated, while 1,1,1-trifluoroheptadec-2-one was significantly downregulated. Among these, the abnormally elevated level of 13,14-dihydro-15-ketoprostaglandin F2α, as a terminal product of non-enzymatic lipid peroxidation, exacerbates the fatty acid oxidation cascade, leading to the formation of large amounts of hexanal, nonanal, and other oxidized aldehydes / ketones, thus intensifying the rancidity of the fat. 1,1,1-trifluoroheptadec-2-one is a ketone, and ketones are important flavor compounds for "creamy" and "fruity" aromas; its reduction directly results in a monotonous aroma profile, making the heavily woody chicken breast taste bland.

[0043] 3.3 Pathway enrichment analysis Pathway enrichment analysis was performed based on the KEGG database to visually demonstrate the relationships between differential metabolites in chicken breast with different degrees of lignification, such as... Figure 3 As shown in (l)~(n). In this invention, most of the enriched metabolic pathways are related to amino acid metabolism, energy metabolism, and lipid metabolism.

[0044] During the mild lignification stage, pathways such as histidine metabolism, tricarboxylic acid cycle, and sphingolipid metabolism are enriched to a higher degree. Figure 3 (l). Histidine, as a precursor to umami amino acids, undergoes metabolic abnormalities that promote its breakdown into histamine, reducing the umami substances in chicken. The tricarboxylic acid cycle participates in energy metabolism; abnormalities in this pathway can disrupt energy supply to muscle cells, indirectly affecting the synthesis of flavor precursors such as organic acids and amino acids. Sphingolipid metabolism disorders lead to damage to cell membrane integrity, accelerating the loss of umami substances in meat. During moderate lignification of chicken breast, the core pathways enriched include histidine metabolism, linoleic acid metabolism, arachidonic acid metabolism, and nitrogen metabolism. Figure 3 (m). The oxidation of linoleic acid and arachidonic acid, two types of fatty acids, produces aldehydes, ketones, and other flavor and aroma precursors. Abnormal metabolic pathways in these compounds can lead to the accumulation of oxidation products such as hexanal and nonanal, resulting in off-flavors like rancidity. Abnormal nitrogen metabolism inhibits the synthesis of umami amino acids such as glutamic acid and aspartic acid, causing a loss of meat flavor. In cases of severe lignification in chicken breast, glycerophospholipid metabolism, linoleic acid metabolism, alanine, aspartic acid, and glutamic acid metabolism are in an enriched state. Figure 3 (n). Among them, the significant accumulation of glycerophospholipids leads to the disruption of cell membrane integrity, and the released fatty acids are oxidized to produce off-flavors. The metabolic disorders of alanine, aspartic acid, and glutamate work synergistically with membrane damage to cause a significant reduction in umami substances, ultimately resulting in bland flavor in chicken breast.

[0045] 3.3 Correlation analysis between key volatile substances and differential metabolites In lignified chicken breast, metabolic disorders in muscle tissue produce flavor-influencing metabolites, including organic acids (OAs), volatile compounds (such as aldehydes, alcohols, and other VOCs), and amino acid derivatives. These metabolites collectively shape the flavor characteristics of the meat. Therefore, Spearman correlation analysis was first performed to determine the correlation between differential metabolite composition and characteristic flavor compounds. Figure 4Spearman correlation analysis (a) and RDA analysis (b) were performed to analyze the differentially expressed volatile organic compounds (VOCs) and metabolites of different grades of chicken breast. The results showed significant positive and negative correlations between key VOCs and differentially expressed metabolites (|r|>0.8, P<0.05). These included 11 lipids and lipid-like molecules, 6 organic heterocyclic compounds, 4 organic acids and their derivatives, 1 benzene compound, 1 nucleoside, nucleotide and analogue, 1 organic oxygen-containing compound, 1 phenylpropane compound, and 1 polyketide compound.

[0046] Alcohols (ethanol, butanol), esters (sec-butyl acetate, ethyl 2-methylpropionate), and aldehydes (decanal, 3-(methylthio)propanal) are mainly positively correlated with phenylpropane compounds and polyketides (lansameid 4, trimethyl irisin), organic oxygen-containing compounds (1-(2,4,6-trimethoxyphenyl)-1,3-butanedione, ethperisone), organic acids and their derivatives (glycolic acid, DN-(carboxyacetyl)alanine), and organic nitrogen-containing compounds (methappyridine), while they are negatively correlated with benzene compounds, lipids and lipid-like molecules, nucleosides, nucleotides and analogues, and organic heterocyclic compounds. These correlation patterns suggest that lignified chicken breast may have a metabolic bias towards amino acid decomposition and carbohydrate oxidation due to muscle fiber sclerosis. These pathways not only accumulate intermediate metabolites such as organic acids and organic oxygen-containing compounds, but also promote their further conversion into non-volatile substances such as alcohols, aldehydes, and esters, thus affecting flavor formation.

[0047] To clarify the regulatory mechanism of lignification degree on metabolites and flavor compounds, metabolites were ranked according to |r|>0.8, VIP>1, P<0.05, and the top 10 representative differentially expressed metabolites and flavor compounds were selected for redundancy analysis (RDA). Figure 4 (b) RDA1 (75.95%) and RDA2 (17.92%) collectively explained 93.87% of the variance, effectively reflecting the association between metabolites, flavor compounds, and different lignification levels in chicken breast samples. The samples showed significant clustering in the ordination space, indicating that the degree of lignification significantly affects metabolic and flavor characteristics. Ethyl isobutyrate (r 2 =0.776, P=0.003), (E)-2-nonenal (r 2 =0.635, P=0.015), 3-(methylthio)propionaldehyde (r 2=0.583, P=0.019) was strongly correlated with core differential metabolites. Specifically, 12,13-epoxy-octadecanoic acid was a key associated metabolite in the NB group, which reduced aldehyde production by inhibiting the lipoxygenase pathway and was significantly negatively correlated with decanal and (Z)-4-heptenal. In contrast, (±)11(12)-epoxy-5Z,8Z,14Z,17Z-eicosatetraenoic acid, 12,13-epoxy-octadecanoic acid, and 20-hydroxy-6Z,15Z-eicosatedienoic acid were positively correlated with the MWB and SWB groups. These are oxidation products of polyunsaturated fatty acids such as arachidonic acid and linoleic acid, indicating that severe lipid oxidation is a key metabolic feature of heavily lignified chicken breast. In addition, ethyl 2-methylpropionate (r 2 =0.776, P=0.003) showed a significant positive correlation with the low-lignification group (NB, LWB) and a significant negative correlation with the high-lignification group (MWB, SWB). Notably, Ethyl 2-methylpropionate, (E)-2-nonenal, and 3-(methylthio)propional are key characteristic indicators reflecting the differences in metabolites and flavor of lignified chicken breast.

[0048] 4. Core metabolites drive the formation of characteristic flavors and their related metabolic regulatory networks To more clearly elucidate the relationship between differential metabolites and characteristic flavor compounds in chicken breast with different degrees of lignification, functional annotation was performed using the KEGG database, and the core metabolic pathways driving flavor formation were systematically reconstructed. Figure 5 This is a metabolic pathway diagram based on the formation process of KEGG. Red represents differential metabolites, and black represents metabolites in the metabolic pathway.

[0049] In the metabolic network of lignified chicken breast, lipid oxidation cascade is the starting point for flavor formation. Linoleic acid and arachidonic acid, as the main polyunsaturated fatty acid substrates, are oxidized via pathways such as lipoxygenase (LOX) and cyclooxygenase / cytochrome P450 (CYP). Linoleic acid metabolism produces the key intermediate 12(13)-epoxyoctadecanoic acid. Arachidonic acid metabolism produces 5-hydroxyeicosatetraenoic acid and 11,12-epoxyeicosatetrienoic acid. These oxidative metabolites themselves, or the volatile aldehydes and alcohols ((E)-2-nonenal, (E)-2-octenal, 1-octanol) produced by their cleavage, are important precursors of the characteristic flavor compounds of lignified meat.

[0050] Glycolysis and the tricarboxylic acid cycle (TCA cycle) in carbon metabolism serve as the core hubs of the metabolic network, regulating the supply and flow of flavor precursors. Glucose is glycolytically converted to pyruvate, which is further converted to acetyl-CoA, a core precursor in the TCA cycle and the synthesis of many volatile substances. Simultaneously, glucose can also be metabolized via the pentose phosphate pathway to produce intermediates such as sedoheptulose-7-phosphate. The NADPH produced through this pathway is crucial for coping with oxidative stress and supporting biosynthesis. Acetyl-CoA enters the TCA cycle, converting into key node metabolites such as citric acid and fumaric acid. These metabolites provide the carbon skeleton (acetyl-CoA) for fatty acid synthesis and indirectly regulate the biosynthetic efficiency and balance of flavor compounds such as aldehydes (decanal), alcohols (1-octanol), and esters through complementation reactions and the intersection of amino acid metabolism (e.g., fumaric acid, α-ketoglutarate).

[0051] Alanine, aspartic acid, and glutamate metabolism regulate flavor metabolic fluxes through amino acid derivatives. Metabolites such as N-acetyl-L-aspartic acid and L-aspartic acid participate in the dynamic adjustment of associated metabolic nodes during lignification. Simultaneously, amino acids such as aspartic acid, glutamate, and alanine can generate key flavor compounds such as aldehydes, alcohols, acids, and sulfur-containing compounds through Strecker degradation, deamination, and decarboxylation reactions. L-aspartic acid is converted to L-arginine-succinic acid, which is then associated with the formation of fumaric acid in the TCA cycle. These amino acid metabolites can act as amino donors in transamination reactions, affecting the entry of the carbon skeleton into energy metabolism or flavor precursor synthesis pathways; their derived peptides also contribute to flavor. These changes are related to oxidative stress induced by lignification.

[0052] In addition, although the metabolism of terpenoids and analogues is not directly presented in the current pathway diagram, the synthesis of isoprene precursors from acetyl-CoA produced by lipid oxidation via the mevalonate (MVA) pathway provides a potential basis for further metabolism into terpenoid flavor compounds, and their metabolic flux is affected by the degree of lignification.

[0053] The flavor characteristics of chicken breasts with varying degrees of lignification are the result of the synergistic effects of multiple pathways within their intrinsic metabolic network. Oxidative stress-induced overactivation of linoleic acid and arachidonic acid metabolism provides characteristic undesirable flavor precursors such as oxidized lipids and cleaved aldehydes / alcohols. Glycolysis and the tricarboxylic acid cycle influence the synthesis of basic flavor compounds by regulating the supply and metabolic flow of key precursors such as acetyl-CoA. Alanine, aspartic acid, and glutamate metabolism directly generate flavor compounds while dynamically adjusting associated metabolic nodes, further shaping the complex profile of flavor. The synergy and imbalance of these three core pathways ultimately drive the differentiated accumulation of characteristic flavor compounds such as aldehydes, alcohols, and sulfur-containing compounds in chicken breasts with different degrees of lignification, providing an integrated view at the metabolic pathway level for a deeper understanding of their flavor mechanisms.

[0054] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A correlation analysis method for flavor degradation of chicken of different quality grades, characterized in that, Includes the following steps: Chicken samples were divided into normal group, mild lignification group, moderate lignification group and severe lignification group based on appearance and palpation. The chicken samples were grouped to obtain a first sample for volatile flavor compounds and sensory analysis, and a second sample for non-volatile metabolite analysis. Gas chromatography-ion mobility spectrometry was used to detect and perform qualitative and quantitative analysis of volatile organic compounds in the first sample. Non-volatile metabolites in the second sample were detected and analyzed qualitatively and quantitatively using liquid chromatography-tandem mass spectrometry. Sensory evaluation was performed on the color, texture, appearance, and odor of the chicken samples; By combining multivariate statistical analysis methods, the volatile organic compounds, non-volatile metabolites, and sensory evaluation data were comprehensively analyzed to analyze the flavor characteristics, metabolite changes, and correlations of chicken with different degrees of lignification.

2. The method according to claim 1, characterized in that, The normal group has no white streaks or hard parts on the surface of the chicken; the mild lignification group has slight changes at the head and near the tail of the chicken, with a slightly hard feel at the head; the moderate lignification group has a hard head and elasticity from the middle to the tail; the severe lignification group has a hard overall appearance, with bleeding and exudate on the surface of the lignified chicken.

3. The method according to claim 1, characterized in that, The preparation of the first sample specifically involves vacuum packaging 40g of the sample and heating it in a 95°C water bath until the center temperature reaches 75°C to lignify the chicken meat.

4. The method according to claim 1, characterized in that, The preparation of the second sample is as follows: weigh 50±5mg of sample, add pre-cooled 80% methanol, grind and crush, let stand at -20°C for 30min, and then centrifuge to obtain the supernatant to lignify the chicken meat.

5. The method according to claim 1, characterized in that, The conditions for gas chromatography-ion mobility spectrometry analysis were as follows: an MXT-WAX capillary column was used at a column temperature of 60°C; the sample was incubated at 80°C and 500 rpm for 15 min; and the IMS conditions were: tritium source as the ionization source and migration tube temperature of 45°C for lignified chicken meat.

6. The method according to claim 1, characterized in that, The conditions for liquid chromatography-tandem mass spectrometry analysis were as follows: an ACQUITY UPLCT3 column was used at a column temperature of 40°C; mobile phase A consisted of 5 mM ammonium acetate + 5 mM acetic acid + water, and mobile phase B consisted of acetonitrile; the mass spectrometer was equipped with an electrospray ionization source, and data were acquired in both positive and negative ion modes.

7. The method according to claim 1, characterized in that, The multivariate statistical analysis included principal component analysis, partial least squares discriminant analysis, Spearman correlation analysis, and metabolic pathway enrichment analysis based on the KEGG database; the partial least squares discriminant analysis was used to evaluate the robustness of the model using 5-fold cross-validation and permutation test for lignified chicken meat.

8. The method according to claim 7, characterized in that, Data integration and analysis also included: using Spearman correlation analysis to determine the correlation between differential metabolites and characteristic flavor compounds, and screening key volatile substances and differential metabolites based on variable importance projection and P<0.05, and performing redundancy analysis.

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