Detection method of molecular interaction between muscle protein and terpenoid compounds in spices

Abstract

The present invention discloses a detection method for the molecular interaction between muscle protein and terpenoid compounds in spices, comprising the following steps: step 1, extracting muscle protein from fresh meat raw materials to obtain a protein solution; step 2, detecting and analyzing the performance changes of muscle protein before and after heating treatment; step 3, dissolving terpenoid compounds in a solvent to prepare a flavor substance reserve solution, sealing and mixing it with the protein solution, and determining the adsorption capacity of muscle protein for terpenoid compounds; step 4, molecularly docking the three-dimensional structural model of muscle protein with the three-dimensional structural model of terpenoid compounds, selecting the conformation with the lowest free binding energy, analyzing the binding mode and site of muscle protein and terpenoid compounds, and clarifying the interaction mechanism between the two. The present invention provides a theoretical basis for the interaction mechanism between muscle protein and flavor substances in thermal processing, and has guiding significance for improving the flavor of meat products.

Description

Technical Field

[0001] The present invention relates to the technical field of meat product processing, and more specifically, to a method for detecting the interaction between muscle protein and terpenoid compound molecules in spices. Background Art

[0002] Myofibrillar protein (MP) and sarcoplasmic protein (SP) are important components of muscle protein, accounting for 50-55% and 30-34% of muscle protein content, respectively. Protein itself has no flavor, but it can bind to flavor substances, thus affecting flavor perception. The factors affecting the binding of protein and flavor substances mainly include the properties of the protein and flavor substances themselves, as well as external factors such as temperature, ionic strength, and pH. Among them, protein structure (such as amino acid sequence and secondary, tertiary, and quaternary structures) is the basic factor affecting the binding behavior of flavor compounds. Heat treatment is the most common processing method in meat processing. It is reported that muscle protein denatures and unfolds its polypeptide chain after heating, exposing the hydrophobic flavor binding sites buried inside the protein, thereby improving the protein's ability to bind flavor. However, the current reports on the interaction between muscle protein and flavor are mainly based on the MP system, while the research on SP is limited. For example, the prior art "Research Progress on the Adsorption of Volatile Flavor Substances by Myofibrillar Proteins", Meat Research, China Meat Food Comprehensive Research Center, 2020, Vol. 34, No. 09 105-112" only reports on the adsorption capacity of myofibrillar proteins for typical flavor compounds such as alcohols, aldehydes, ketones, and esters. Therefore, it is necessary to clarify the effect of heat-induced denaturation on the binding of different types of muscle proteins to flavors.

[0003] Spices such as star anise, cardamom, pepper, prickly ash, cinnamon, fennel, etc. are usually added during meat processing to remove fishy smells, enhance color and enhance fragrance. Terpenoids have been identified as one of the main components of volatile flavor compounds in spices. Among them, compounds such as 3-carene, limonene, and linalool can give meat products resin, mint, and lemon aromas, making an important contribution to the flavor of meat products. At present, there have been many reports on the interaction between flavor substances such as aldehydes, ketones, alcohols, esters, furans, and pyrazines and muscle proteins, but the binding mechanism of terpenoids and muscle proteins is still unclear. Summary of the invention

[0004] One object of the present invention is to provide a method for detecting the interaction between muscle protein and terpenoid compound molecules in spices, which has guiding significance for improving the flavor of meat products.

[0005] In order to achieve these purposes and other advantages according to the present invention, according to one aspect of the present invention, the present invention provides a method for detecting the interaction between muscle protein and terpenoid molecules in spices, comprising the following steps:

[0006] Step 1: extracting muscle protein from fresh meat raw materials, purifying it, and heating it to obtain a protein solution;

[0007] Step 2: Detect and analyze the performance changes of muscle protein before and after heating treatment;

[0008] Step 3, dissolving the terpenoid compound in a solvent to prepare a flavor substance stock solution, mixing the flavor substance stock solution with the protein solution in a sealed manner, and determining the adsorption capacity of muscle protein for the terpenoid compound;

[0009] Step 4: Perform molecular docking between the three-dimensional structural model of muscle protein and the three-dimensional structural model of terpenoid compounds, select the conformation with the lowest free binding energy, analyze the binding mode and site of muscle protein and terpenoid compounds, and clarify the interaction mechanism between the two.

[0010] Preferably, the muscle protein is myofibrillar protein and sarcoplasmic protein.

[0011] Preferably, the extraction method of the myofibrillar protein and sarcoplasmic protein is:

[0012] Take fresh meat, remove visible fat and connective tissue, mince, add 50mmol / L phosphate buffer with pH 7.0, homogenize in an ice bath, centrifuge at 4℃, and filter the supernatant through gauze to obtain sarcoplasmic protein;

[0013] Take the precipitate and add 4 times the volume of separation buffer with a pH of 7.0, wherein the separation buffer is a mixed solution of 0.1 mol / L NaCl, 2 mmol / L MgCl2 and 1 mmol / L EGTA. After ice bath homogenization, centrifuge at 4°C. Repeat the above steps 3 times, collect the precipitate, add 4 times the volume of 0.1 mol / L NaCl solution, homogenize in an ice bath, and centrifuge at 4°C. Repeat 4 times. Before the last centrifugation, filter the precipitate through gauze. The precipitate after centrifugation is myofibrillar protein.

[0014] Preferably, the specific process of purification in step 1 is: dialyzing the extracted muscle protein in a phosphate buffer containing 0.6 mol / L NaCl using a dialysis bag with a cutoff of 8 to 14 kDa at 4° C. for 24 hours, the concentration of the phosphate buffer is 50 mmol / L, and the pH is 7.0. The phosphate buffer is replaced every 4 hours during the dialysis process to obtain purified muscle protein.

[0015] Preferably, the specific process of the heating treatment in step 1 is: adjusting the mass concentration of the purified muscle protein to 4 mg / mL with a phosphate buffer containing 0.6 mol / L NaCl, heating at 85°C for 1 to 60 min and then immediately cooling in an ice bath to obtain the protein solution, wherein the concentration of the phosphate buffer is 50 mmol / L and the pH is 7.0.

[0016] Preferably, the terpene compounds are 3-carene, limonene and linalool, which are dissolved in methanol at a mass ratio of 1:1:1 to prepare a fragrance substance reserve solution with a mass concentration of 100 mg / L.

[0017] Preferably, the method for determining the adsorption capacity of muscle protein for terpenoids in step 3 is:

[0018] Experimental group: Take a protein solution with a mass concentration of 4 mg / mL, add a flavor substance stock solution with a mass concentration of 100 mg / L to it, so that the final mass concentration of terpenoid compounds in the mixed solution is 1 mg / L, seal and mix, and equilibrate at 4°C for 12 hours;

[0019] Blank group: Take a phosphate buffer solution containing 0.6 mol / L NaCl, wherein the concentration of the phosphate buffer solution is 50 mmol / L and the pH is 7.0, add a 100 mg / L aroma substance stock solution thereto, so that the final mass concentration of terpenoid compounds in the mixed solution is 1 mg / L, seal and mix, and balance at 4°C for 12 hours;

[0020] Step c, using solid phase microextraction combined with gas chromatography-mass spectrometry to determine the headspace concentration of terpenoid compounds, using a 75 μm CAR / PDMS extraction fiber, adsorbing the equilibrated mixed solution at 25° C. for 30 min, desorbing at 250° C. for 3 min, and then analyzing by GC-MS;

[0021] Among them, GC-MS conditions: DB-WAX column (30m×0.18mm×0.18μm) was used, and the sample was injected in splitless mode; the temperature program: the column temperature was maintained at 35℃ for 3min and increased to 210℃ at 5℃ / min; the mass spectrometer ion source temperature was 200℃, the electron energy was 70eV, and the mass scanning range was m / z 35-500;

[0022] The adsorption capacity of muscle protein for terpenoids is expressed by the free ratio of terpenoids in the mixed solution:

[0023]

[0024] Preferably, the molecular docking process in step 4 is: after the three-dimensional structural model of muscle protein and the three-dimensional structural model of terpenoid compounds are optimized by the CHARMm force field, the CDOCKER module in the Discovery Studio 2019 software is used for molecular docking.

[0025] Preferably, the detection of changes in muscle protein properties before and after the heat treatment in step 2 includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis detection, surface hydrophobicity detection, total thiol content detection, particle size detection, and secondary structure content detection.

[0026] The present invention includes at least the following beneficial effects: In order to clarify the interaction law and mechanism between muscle protein and terpenoid compounds during heat treatment, the present invention adopts gas chromatography-mass spectrometry and other methods to explore the correlation between the adsorption capacity of myofibrillar protein and sarcoplasmic protein for three terpenoid compounds (3-carene, limonene, and linalool) and protein conformational changes under different heating times, and reveals the binding mechanism of muscle protein and terpenoid compounds through molecular docking. The research results can provide a theoretical basis for the interaction mechanism between muscle protein and flavor substances in thermal processing, which is of guiding significance for improving the flavor of meat products.

[0027] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The electrophoretic diagram of MP(A) at different heating times in Example 2;

[0029] Figure 2 The electrophoretic diagram of SP (B) at different heating times in Example 1;

[0030] Figure 3 The surface hydrophobicity change diagram of SP and MP at different heating times in Example 1 and Example 2;

[0031] Figure 4 The total thiol content of SP and MP at different heating times in Example 1 and Example 2 is a graph showing the change in total thiol content of SP and MP at different heating times;

[0032] Figure 5 The graphs are the particle size changes of SP and MP at different heating times in Example 1 and Example 2;

[0033] Figure 6 The graphs are the changes in the adsorption capacity of MP and SP for 3-carene (A), limonene (B) and linalool (C) at different heating times in Example 1 and Example 2;

[0034] Figure 7The docking conformations and docking site diagrams of myosin and three terpenoid compounds in Example 2 (A, B, and C represent the docking of myosin with 3-carene, limonene, and linalool, respectively; the numbers 1, 2, and 3 represent the optimal docking mode, docking 3D diagram, and docking 2D diagram, respectively);

[0035] Figure 8 The docking conformations and docking site diagrams of myoglobin and three terpenoid compounds in Example 1 (a, b, and c represent the docking of myoglobin with 3-carene, limonene, and linalool, respectively; the numbers 1, 2, and 3 represent the optimal docking mode, docking 3D diagram, and docking 2D diagram, respectively). DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can implement the invention with reference to the description.

[0037] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0038] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified; in the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "set" should be understood in a broad sense, for example, they can be fixedly connected, set, or detachably connected, set, or connected and set in one piece. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood in specific circumstances. The orientation or position relationship indicated by the terms "lateral", "longitudinal", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc. is based on the orientation or position relationship shown in the accompanying drawings, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation of the present invention.

[0039] At present, the key terpenoid compounds in the disclosed spices include but are not limited to estragole, eugenol, anethole, 3-carene, citral, geraniol, α-terpineol, linalool, β-phellandrene, myrcene, β-caryophyllene, eucalyptol, β-pinene, limonene, etc., and their aroma descriptions and sources are shown in Table 1. Among them, compounds such as 3-carene, limonene, and linalool can give meat products resin, mint, and lemon aromas, and make an important contribution to the flavor of meat products.

[0040] Table 1 Key terpenoid compounds in spices

[0041]

[0042] Example 1

[0043] A method for detecting the molecular interaction between muscle protein and terpenoid compounds in spices, wherein the muscle protein is sarcoplasmic protein (SP), and the terpenoid compounds are 3-carene, limonene and linalool, and the mass ratio of the three is 1:1:1, as follows:

[0044] Step 1: Take 40 g of fresh longissimus dorsi muscle of pig, remove visible fat and connective tissue, mince, add 80 mL of phosphate buffer (50 mM, pH 7.0), homogenize in an ice bath (10000 rpm, 1 min), and then centrifuge for 20 min (4°C, 6000×g), and filter the supernatant through 4 layers of gauze to obtain SP;

[0045] The extracted SP was dialyzed in phosphate buffer (50 mM, pH 7.0) containing 0.6 M NaCl at 4°C using a dialysis bag with a cutoff of 8-14 kDa for 24 h, and the phosphate buffer was replaced every 4 h. The mass concentration of the purified protein was determined by the BCA method and refrigerated at 4°C and used within 12 h.

[0046] The mass concentration of SP was adjusted to 4 mg / mL with phosphate buffer (50 mM, pH 7.0) containing 0.6 M NaCl, and the mixture was heated at 85°C for 1, 5, 10, 30, and 60 min, respectively, and then immediately cooled in an ice bath to obtain SP solutions;

[0047] Step 2: The concentration of SP solution was adjusted to 2 mg / mL, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed for detection and analysis, with a separation gel concentration of 12.5%, a concentrated gel concentration of 5%, a sample volume of 10 μL, a concentrated gel voltage of 80 V, and a separation gel voltage of 120 V. After electrophoresis, the sample was stained with Coomassie Brilliant Blue R-250 for 2 h, decolorized with distilled water, and imaged with a gel imager;

[0048] The surface hydrophobicity of SP was determined by ANS fluorescent probe method. The concentration of SP solution was diluted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively. 4 mL of SP solution was mixed with 50 μL of 8-aniline-1-naphthalenesulfonic acid ANS (8 mM), and the mixture was kept in dark for 10 min. The fluorescence intensity was determined. The fluorescence determination conditions were as follows: excitation wavelength 374 nm, emission wavelength 480 nm, slit 5 nm, and phosphate buffer (50 mM, pH 7.0) containing 0.6 M NaCl was used as blank. The surface hydrophobicity was expressed by the slope of the curve corresponding to the fluorescence intensity and protein concentration.

[0049] Determination of total thiol content of protein: Take 1 mL of SP solution and add 9 mL of 50 mM phosphate buffer (containing 0.6 M KCl, 10 mM EDTA, 8 M urea, pH 7.0) and mix well. Take 5 mL of the mixed solution and add 0.5 mL of DTNB (0.2 mM). React at 25 °C for 25 min and measure the absorbance at 412 nm. The molar extinction coefficient is 13600 mol -1 cm -1 Calculate the total thiol content;

[0050] After the SP solution was diluted to 1 mg / mL, the average particle size was measured using a laser particle size analyzer, with a measurement temperature of 25 °C and an equilibrium time of 1 min;

[0051] After the SP solution was freeze-dried, the protein secondary structure content was determined by multi-bounce attenuated total reflection (MB-ATR) combined with Fourier transform infrared (FT-IR) spectroscopy in the scanning range of 400-4000 cm -1 , resolution 4cm -1 , scanned 100 times, and the infrared spectra were subjected to Fourier deconvolution processing by PeakFit 4.12 software to calculate the content of protein secondary structure (α-helix, β-sheet, β-turn, random coil);

[0052] Step 3, dissolving 3-carene, limonene and linalool in methanol at a mass ratio of 1:1:1 to prepare a fragrance substance stock solution with a mass concentration of 100 mg / L;

[0053] Experimental group: Take a 4 mg / mL SP solution, add a 100 mg / L aroma substance stock solution to it, so that the final concentration of terpenoid compounds in the mixed solution is 1 mg / L, seal and mix, and equilibrate at 4°C for 12 hours;

[0054] Blank group: Take a phosphate buffer solution containing 0.6 mol / L NaCl, wherein the concentration of the phosphate buffer solution is 50 mmol / L and the pH is 7.0, add a 100 mg / L aroma substance stock solution thereto, so that the final mass concentration of terpenoid compounds in the mixed solution is 1 mg / L, seal and mix, and balance at 4°C for 12 hours;

[0055] Step c, using solid phase microextraction combined with gas chromatography-mass spectrometry to determine the headspace concentration of terpenoid compounds, using a 75 μm CAR / PDMS extraction fiber, adsorbing the equilibrated mixed solution at 25° C. for 30 min, desorbing at 250° C. for 3 min, and then analyzing by GC-MS;

[0056] Among them, GC-MS conditions: DB-WAX column (30m×0.18mm×0.18μm) was used, and the sample was injected in splitless mode; the temperature program: the column temperature was maintained at 35℃ for 3min and increased to 210℃ at 5℃ / min; the mass spectrometer ion source temperature was 200℃, the electron energy was 70eV, and the mass scanning range was m / z 35-500;

[0057] The adsorption capacity of muscle protein for terpenoids is expressed by the free ratio of terpenoids in the mixed solution:

[0058]

[0059] Step 4: Select myoglobin, the main protein in SP, as a model to explore its interaction mechanism with aroma compounds. The three-dimensional structural model of pork myoglobin (PDB ID: 1MWC) was obtained from the protein database of the National Center for Biotechnology Information of the United States. The three-dimensional structures of 3-carene (ID: 26049), limonene (ID: 440917), and linalool (ID: 6549) were obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). After the CHARMm force field optimized the structures of myoglobin and three terpenoid compounds, the CDOCKER module in Discovery Studio 2019 software was used for molecular docking, and the conformation with the lowest free binding energy was selected.

[0060] Each experiment was repeated three times, and SPSS 25 software was used for statistical analysis. Duncan multiple comparisons or independent sample T test were used to analyze the differences between groups, and P < 0.05 indicated a significant difference. Origin 8.5 software was used for drawing.

[0061] Example 2

[0062] A method for detecting the molecular interaction between muscle protein and terpenoid compounds in spices, wherein the muscle protein is myofibrillar protein (MP), and the terpenoid compounds are 3-carene, limonene and linalool, as follows:

[0063] Step 1: Take 40g of fresh pig longissimus dorsi muscle, remove visible fat and connective tissue, mince, add 80mL phosphate buffer (50mM, pH 7.0), ice bath homogenize (10000rpm, 1min), and centrifuge for 20min (4℃, 6000×g), add 4 times the volume of separation buffer (0.1M NaCl, 2mM MgCl2, 1mM EGTA, pH 7.0) to the above centrifuged precipitate, ice bath homogenize (10000rpm, 1min), and centrifuge for 15min (4℃, 6000×g). Repeat the above steps 3 times, collect the precipitate, add 4 times the volume of 0.1M NaCl solution to the precipitate, ice bath homogenize (10000rpm, 1min), and centrifuge for 15min (4℃, 6000×g), and repeat 4 times. Before the last centrifugation, the precipitate is filtered through 4 layers of gauze, and the precipitate after centrifugation is MP;

[0064] The extracted MP was dialyzed in phosphate buffer (50 mM, pH 7.0) containing 0.6 M NaCl at 4°C for 24 h using a dialysis bag with a cutoff of 8-14 kDa. The phosphate buffer was replaced every 4 h. The mass concentration of the purified protein was determined by the BCA method and refrigerated at 4°C and used within 12 h.

[0065] The mass concentration of MP was adjusted to 4 mg / mL using phosphate buffer (50 mM, pH 7.0) containing 0.6 M NaCl, and then heated at 85°C for 1, 5, 10, 30, and 60 min, respectively, and then immediately cooled in an ice bath to obtain MP solutions;

[0066] Step 2: The concentration of the MP solution was adjusted to 2 mg / mL, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed for detection and analysis, with a separation gel concentration of 12.5%, a concentrated gel concentration of 5%, a sample volume of 10 μL, a concentrated gel voltage of 80 V, and a separation gel voltage of 120 V. After the electrophoresis was completed, the solution was stained with Coomassie Brilliant Blue R-250 for 2 h, decolorized with distilled water, and imaged with a gel imager;

[0067] The surface hydrophobicity of MP was determined by ANS fluorescent probe method. The concentration of MP solution was diluted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively. 4 mL of MP solution was mixed with 50 μL of 8-aniline-1-naphthalenesulfonic acid ANS (8 mM), and the mixture was kept in dark for 10 min. The fluorescence intensity was determined. The fluorescence measurement conditions were as follows: excitation wavelength 374 nm, emission wavelength 480 nm, slit 5 nm, and phosphate buffer (50 mM, pH 7.0) containing 0.6 M NaCl was used as blank. The surface hydrophobicity was expressed by the slope of the curve corresponding to the fluorescence intensity and protein concentration.

[0068] Determination of total thiol content of protein: 1 mL of MP solution was added with 9 mL of 50 mM phosphate buffer (containing 0.6 M KCl, 10 mM EDTA, 8 M urea, pH 7.0) and mixed evenly. 5 mL of the mixed solution was added with 0.5 mL of DTNB (0.2 mM), and the mixture was reacted at 25 °C for 25 min. The absorbance was measured at 412 nm, and the molar extinction coefficient was 13600 mol. -1 cm -1 Calculate the total thiol content;

[0069] After the MP solution was diluted to 1 mg / mL, the average particle size was measured using a laser particle size analyzer, with a measurement temperature of 25 °C and an equilibrium time of 1 min;

[0070] After the MP solution was freeze-dried, the protein secondary structure content was determined by multi-bounce attenuated total reflection (MB-ATR) combined with Fourier transform infrared (FT-IR) spectroscopy in the scanning range of 400 to 4000 cm -1 , resolution 4cm -1 , scanned 100 times, and the infrared spectra were subjected to Fourier deconvolution processing by PeakFit 4.12 software to calculate the content of protein secondary structure (α-helix, β-sheet, β-turn, random coil);

[0071] Step 3, dissolving 3-carene, limonene and linalool in methanol at a mass ratio of 1:1:1 to prepare a fragrance substance stock solution with a mass concentration of 100 mg / L;

[0072] Experimental group: Take MP solution with a mass concentration of 4 mg / mL, add aroma substance stock solution with a mass concentration of 100 mg / L to it, so that the final mass concentration of terpenoid compounds in the mixed solution is 1 mg / L, seal and mix, and balance at 4°C for 12 hours;

[0073] Blank group: Take a phosphate buffer solution containing 0.6 mol / L NaCl, wherein the concentration of the phosphate buffer solution is 50 mmol / L and the pH is 7.0, add a 100 mg / L aroma substance stock solution thereto, so that the final mass concentration of terpenoid compounds in the mixed solution is 1 mg / L, seal and mix, and balance at 4°C for 12 hours;

[0074] Step c, using solid phase microextraction combined with gas chromatography-mass spectrometry to determine the headspace concentration of terpenoid compounds, using a 75 μm CAR / PDMS extraction fiber, adsorbing the equilibrated mixed solution at 25° C. for 30 min, desorbing at 250° C. for 3 min, and then analyzing by GC-MS;

[0075] Among them, GC-MS conditions: DB-WAX column (30m×0.18mm×0.18μm) was used, and the sample was injected in splitless mode; the temperature program: the column temperature was maintained at 35℃ for 3min and increased to 210℃ at 5℃ / min; the mass spectrometer ion source temperature was 200℃, the electron energy was 70eV, and the mass scanning range was m / z 35-500;

[0076] The adsorption capacity of muscle protein for terpenoids is expressed by the free ratio of terpenoids in the mixed solution:

[0077]

[0078] Step 4: Select myosin, the main protein in MP, as a model to explore its interaction mechanism with aroma compounds. The amino acid sequence of pork myosin (sequence number: XP_020921876.1) was obtained from the protein database of the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / protein) of the United States. The SWISS-MODEL tool (https: / / swissmodel.expasy.org / ) was used to search and homology modeling was performed using the highly homologous protein 6FSA (homology 81.48%) as a template. The constructed pork myosin three-dimensional structure model was constructed by The PROCHECK program (http: / / servicesn.mbi.ucla.edu / PROCHECK / ) was used for verification. The number of amino acid residues located in the favorable and acceptable regions was greater than 99%, indicating that the model can be used for intermolecular interactions. The three-dimensional structures of 3-carene (ID: 26049), limonene (ID: 440917), and linalool (ID: 6549) were obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). After the structures of myosin and three terpenoids were optimized by the CHARMm force field, the CDOCKER module in the Discovery Studio 2019 software was used for molecular docking, and the conformation with the lowest free binding energy was selected;

[0079] Each experiment was repeated three times, and SPSS 25 software was used for statistical analysis. Duncan multiple comparisons or independent sample T test were used to analyze the differences between groups, and P < 0.05 indicated a significant difference. Origin 8.5 software was used for drawing.

[0080] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis detection results and analysis

[0081] The electrophoretic patterns of MP and SP at different heating times in Example 2 and Example 1 are as follows: Figure 1 (A) and Figure 2 As shown in (B). MP is mainly composed of myosin heavy chain (200kDa), actin (43kDa), tropomyosin (32kDa) and myosin light chain (22, 17kDa). SP mainly includes most enzymes of the glycolysis pathway, creatine kinase and myoglobin. As the heating time increases, the myosin heavy chain band of MP gradually becomes blurred and lighter, indicating that heating induces protein aggregation or degradation. As the heating time increases, most of the sarcoplasmic enzyme bands in SP gradually become lighter, which may be due to the conversion of some enzymes from soluble to insoluble after the SP is heated. In addition, the color of the top of the MP and SP gels gradually deepens, indicating that heating induces protein aggregation and cross-linking to form large molecular aggregates. After heating, muscle proteins can aggregate through disulfide bonds or other chemical bonds to form large molecular clusters, resulting in an increase in molecular weight.

[0082] Results and analysis of changes in protein surface hydrophobicity

[0083] The surface hydrophobicity of SP and MP changes at different heating times in Example 1 and Example 2 are as follows: Figure 3 As shown in the figure (Note: different letters af indicate significant differences between the same protein at different heating times (P<0.05); * indicates significant differences between MP and SP at the same heating time (P<0.05).). Surface hydrophobicity can reflect the number of hydrophobic groups on the protein surface and evaluate the unfolding and aggregation state of the protein. ANS can bind to the hydrophobic region of the protein and emit fluorescence, so it is widely used in the determination of protein surface hydrophobicity. Figure 3 As shown in the figure, the surface hydrophobicity of MP and SP first increases within 5 minutes of heating, and then gradually decreases. The possible reason is that within 0 to 5 minutes of heating, heating induces the protein structure to unfold, exposing the buried hydrophobic groups inside, so the content of hydrophobic groups gradually increases. As the heating time increases, protein aggregation plays a dominant role. A large number of exposed hydrophobic groups cause protein cross-linking to form soluble or insoluble aggregates. The hydrophobic residues of the protein may be embedded in the aggregates, resulting in a decrease in surface hydrophobicity. In addition, by comparing the surface hydrophobicity of MP and SP, it was found that the surface hydrophobicity of SP was higher than that of MP within 1 to 5 minutes of heating, but the surface hydrophobicity of MP remained high after continued heating. This may be because SP has a lower denaturation temperature and is easier to unfold after heating, exposing more hydrophobic amino acids. As heating continues, the aggregation rate of SP is faster, causing more hydrophobic groups to be buried. In addition, the difference in amino acid composition between MP and SP will also affect the surface hydrophobicity of proteins.

[0084] Results and analysis of changes in total thiol content of proteins

[0085] The total thiol content of SP and MP under different heating time in Example 1 and Example 2 is as follows: Figure 4As shown in Figure 2 (Note: different letters a-f indicate significant differences between the same protein at different heating times (P<0.05); * indicates significant differences between MP and SP at the same heating time (P<0.05).). The total thiol content is an important parameter reflecting the formation and cleavage of disulfide bonds between or within protein molecules. Figure 4 As shown in the figure, the total thiol content of MP and SP increased within 10 minutes of heating and then gradually decreased. The unfolding of protein structure caused by microenvironmental changes will expose cysteine ​​residues and eventually form disulfide bonds. The increase in total thiol content within 10 minutes of heating can be attributed to the cleavage of protein disulfide bonds induced by heating or the exposure of cysteine ​​residues inside the protein. As heating continues, the exposed thiol groups gradually form disulfide bonds to maintain the stability of the protein structure, so the total thiol content decreases significantly. In addition, after heating, the total thiol content of SP is always higher than that of MP, which may be because SP is mainly composed of proteases, most of which have a high content of cysteine ​​residues, so SP can expose more thiol groups after thermal unfolding.

[0086] Particle size change results and analysis

[0087] The particle size changes of SP and MP at different heating times in Example 1 and Example 2 are as follows: Figure 5 As shown (Note: different letters a-f indicate significant differences between the same protein at different heating times (P<0.05); * indicates significant differences between MP and SP at the same heating time (P<0.05).). Figure 5 As shown in the figure, as the heating time increases, the particle size of MP and SP increases continuously, indicating that heating induces protein aggregation to form macromolecular clusters, which is consistent with the SDS-PAGE results. During the heating process, protein molecules can aggregate and form clusters through chemical bonds such as hydrophobic interactions, disulfide bonds and hydrogen bonds, resulting in an increase in particle size. In the early stage of heating, the increase in protein particle size can be attributed to the unfolding of protein peptide chains, the exposure of internal hydrophobic groups, and the enhancement of hydrophobic interactions between protein molecules. As heating continues, the exposed thiol groups gradually form disulfide bonds, macromolecular aggregates are formed between protein molecules, and the particle size continues to increase. In addition, the particle size of SP after heating is always significantly higher than that of MP (P<0.05), indicating that the degree of aggregation of SP is higher, which may be related to the lower denaturation temperature of SP and its higher thiol content, which makes it easier to form disulfide bonds between protein peptide chains.

[0088] Results and analysis of changes in secondary structure content

[0089] Table 2 Changes in secondary structure content of MP and SP at different heating times

[0090]

[0091] The changes in the secondary structure content of SP and MP at different heating times in Example 1 and Example 2 are shown in Table 2. Infrared spectrum amide I band (1600-1700cm -1 ) can reflect the secondary structure information of proteins and is often used to evaluate the conformational changes of proteins. As shown in Table 1, within 0 to 60 minutes of heating, the α-helix content of MP and SP decreased from 46.36% and 50.40% to 37.89% and 31.53%, respectively, the β-fold content increased from 21.47% and 20.63% to 27.06% and 30.86%, respectively, and the random coil content increased from 14.58% and 12.10% to 16.70% and 20.75%, respectively. The results show that with the extension of heating time, the α-helix structure of MP and SP gradually transforms into β-fold and random coil structure. Heating will promote the increase of "thermal motion" of protein peptide chains, destroy the intramolecular forces that maintain the protein structure (such as hydrophobic interaction, hydrogen bonds, electrostatic interactions and disulfide bonds), and reorganize the protein structure. When not heated, the secondary structure of the protein is mainly α-helix. The stability of the α-helix structure is mainly maintained by hydrogen bonds within the polypeptide chain, and the β-fold structure is maintained by hydrogen bonds between peptide chains. After heating, the hydrogen bonds of the protein peptide chains are rearranged, resulting in the transformation of the α-helix to the β-fold. At the same time, as the degree of heating increases, the ordered structure gradually disappears, and the protein becomes a random coil structure that is close to fully unfolded, resulting in an increase in the random coil content. In addition, compared with MP, the secondary structure content of SP changes more significantly, indicating that the conformation of SP is more susceptible to thermal changes, which is consistent with the surface hydrophobicity and particle size results.

[0092] Changes in protein adsorption capacity for aroma compounds

[0093] The changes in the adsorption capacity of MP and SP for 3-carene (A), limonene (B) and linalool (C) at different heating times in Example 1 and Example 2 are shown in FIG. Figure 6 As shown. The headspace concentrations of the three aroma compounds in the MP and SP samples decreased continuously within 0-5 min of heating, indicating that heating promoted the adsorption of aroma compounds by proteins; but within 10-60 min of heating, the headspace concentrations of the three aroma compounds continued to increase, indicating that the protein's ability to adsorb aroma decreased. In addition, the free proportion of linalool in MP was greater than 100% when heated for 60 min, and the free proportion of limonene and linalool in SP was greater than 100% when heated for 30 or 60 min, indicating that the addition of protein at this time promoted the release of these compounds.

[0094] Previous studies have shown that heating can affect flavor binding sites by changing the conformation of proteins. When the temperature is high enough, the protein denatures and unfolds to expose the binding sites, but too high a temperature or too long a heating time will inhibit adsorption. Since most flavor compounds are hydrophobic in their natural state, hydrophobic interactions are often considered to be the main driving force for the binding of flavor substances to proteins. Therefore, within 0 to 5 minutes of heating, the protein unfolds and the internal hydrophobic groups are exposed, which promotes the binding of flavor compounds to proteins through hydrophobic interactions. In addition, linalool contains hydroxyl groups and may also bind to proteins through hydrogen bonds. Heating can destroy the α-helical structure of proteins and expose more hydrogen bonding sites on the protein surface. However, as heating continues, the interactions between protein peptide chains increase, proteins aggregate to form large molecular clusters, and active binding sites are re-embedded into the protein, resulting in a decrease in the protein's ability to adsorb flavors.

[0095] Table 3 Properties of three terpenoid compounds

[0096]

[0097] Note: Data source: https: / / www.chemsrc.com / . logP represents the oil-water partition coefficient or hydrophobicity coefficient. The larger the logP value, the more lipophilic the substance is.

[0098] The properties of 3-carene, limonene and linalool are shown in Table 3. In addition to the conformational changes of the protein induced by heating, the aroma compounds and the properties of the protein itself also affect the protein's ability to adsorb aroma. The free proportions of 3-carene, limonene and linalool in the MP samples at different heating times were 59.97-86.22%, 54.34-84.92% and 76.24-108.68%, respectively, and the free proportions of 3-carene, limonene and linalool in the SP samples were 61.12-108.66%, 57.87-112.22% and 72.64-121.70%, respectively. The adsorption capacity of MP and SP for 3-carene and limonene was stronger than that for linalool, which may be due to the similar hydrophobicity of 3-carene and limonene, while linalool has a lower hydrophobicity. The interaction between protein and aroma is hydrophobic in nature, and the adsorption capacity of protein for aroma substances is positively correlated with the hydrophobicity of aroma substances. In addition, since linalool contains hydrophilic hydroxyl groups, water may compete with linalool for the binding sites of protein, which also reduces the adsorption capacity of protein for linalool.

[0099] In addition, the aroma adsorption capacity of MP and SP was similar within 0-5 min of heating, but the aroma adsorption capacity of SP was significantly lower than that of MP within 10-60 min of heating. This may be because the aggregation rate of SP was greater than that of MP during the heating period of 10-60 min, resulting in more hydrophobic binding sites in SP being buried, which is consistent with the surface hydrophobicity results. In addition, due to the higher aggregation degree of SP ( Figure 5), greater steric hindrance may prevent aroma compounds from entering the hydrophobic region inside the protein, which will further weaken the aroma adsorption capacity of SP. In addition, when the heating time is long, it is observed that the addition of MP or SP has a release-promoting effect on some aroma substances, which may be due to the addition of protein reducing the surface tension of the solution, resulting in more aroma substances being distributed in the gas phase. Therefore, the release / retention of aroma in the protein matrix is ​​related to multiple factors such as protein structural characteristics (such as surface hydrophobicity, particle size, surface tension, steric hindrance) and the properties of aroma substances (such as molecular size, polarity, gas / liquid partition coefficient).

[0100] Analysis of molecular docking results

[0101] To further explore the interaction between muscle proteins and terpenoids, molecular docking technology was used to analyze the binding modes and sites of the main protein components (myosin and myoglobin) in MP and SP with 3-carene, limonene, and linalool, such as Figure 7 and Figure 8 As shown, the optimal docking modes of the protein and three terpenoids were selected, and the residues with greater influence around the binding site were listed in the docking 3D and 2D maps. Among them, four amino acid residues of myosin (Leu270, Lys273, Lys599, Val651) have hydrophobic interactions with 3-carene and limonene, and three amino acid residues (Met93, Arg716, Leu774) have hydrophobic interactions with linalool; myoglobin has eight (Val68, Ala71, Leu72, Leu89, His93, Ile99, Leu104, Ile107), six (Phe43, Val68, Leu89, His93, Ile99, Ile107), and seven (Leu29, Phe43, Val68, Leu72, Leu89, Ile107, Phe138) amino acid residues that have hydrophobic interactions with 3-carene, limonene, and linalool, respectively. No other chemical interactions were detected between myosin and myoglobin and 3-carene and limonene except for hydrophobic interactions, indicating that hydrophobic interactions are the main contribution. The Ser715 residue of myosin can also form a bond with linalool with a length of The conventional hydrogen bond between the myoglobin His93 residue and linalool forms a bond of It is generally believed that the closer the bond length is, the The stronger the hydrogen bond, the closer The weaker the hydrogen bond force, the shorter the hydrogen bond length between myosin and myoglobin and linalool, indicating the existence of a stronger hydrogen bond interaction.

[0102] This paper explores the relationship between the adsorption capacity of MP and SP for three terpenoids and protein conformational changes during heat treatment, and clarifies the interaction mechanism between muscle protein and terpenoids. The study found that within 5 minutes of heating, the heat treatment unfolded the secondary structures of MP and SP, and the hydrophobic and hydrogen bond binding sites buried inside the protein were exposed, which promoted the adsorption of aroma substances by the protein; but as the heating time increased, the protein gradually aggregated into large molecular clusters, the active binding sites were buried, and the protein's adsorption capacity for aroma substances gradually decreased. SP has a higher degree of aggregation after heating, resulting in more binding sites being buried and a higher steric hindrance effect, making its ability to adsorb aroma weaker than MP within 10 to 60 minutes of heating. In addition, due to the smaller hydrophobic coefficient (logP) of linalool, the adsorption capacity of MP and SP on it is weaker than that of 3-carene and limonene. Finally, the molecular docking results confirmed that the three terpenoids bind to muscle protein mainly through hydrophobic interactions, and linalool can also bind to muscle protein through hydrogen bonds. The research results can provide a theoretical basis for the interaction mechanism between muscle protein and flavor substances during thermal processing, and have guiding significance for improving the flavor of meat products.

[0103] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0104] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. A method for detecting the interaction between muscle protein and terpenoid molecules in spices, characterized in that: The following steps are involved: Step 1: extracting muscle protein from fresh meat raw materials, purifying it, and heating it to obtain a protein solution; Step 2: Detect and analyze the changes in the properties of muscle protein before and after heating treatment; Step 3, dissolving the terpenoid compound in a solvent to prepare a flavor substance stock solution, mixing the flavor substance stock solution with the protein solution in a sealed manner, and determining the adsorption capacity of muscle protein for the terpenoid compound; Step 4: Molecular docking of the three-dimensional structural model of muscle protein with the three-dimensional structural model of terpenoid compounds is performed, the conformation with the lowest free binding energy is selected, the binding mode and site of muscle protein and terpenoid compounds are analyzed, and the interaction mechanism between the two is clarified; Wherein, the muscle protein is myofibrillar protein and sarcoplasmic protein, the terpenoid compound is 3-carene, limonene, and linalool, the three are dissolved in methanol at a mass ratio of 1:1:1, and a flavor substance reserve solution with a mass concentration of 100 mg / L is prepared, and the performance change detection of the muscle protein before and after the heating treatment in the step 2 includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis detection, surface hydrophobicity detection, total thiol content detection, particle size detection, and secondary structure content detection; The extraction methods of the myofibrillar protein and sarcoplasmic protein are respectively: Take fresh meat, remove visible fat and connective tissue, mince, add 50 mmol / L phosphate buffer with a pH of 7.0, homogenize in an ice bath, centrifuge at 4°C, and filter the supernatant through gauze to obtain sarcoplasmic protein; Take the precipitate and add 4 times the volume of separation buffer with a pH of 7.0, wherein the separation buffer is a mixed solution of 0.1 mol / L NaCl, 2 mmol / L MgCl2 and 1 mmol / L EGTA, ice bath homogenize and centrifuge at 4°C, repeat the above steps 3 times, collect the precipitate, add 4 times the volume of 0.1 mol / L NaCl solution, ice bath homogenize and centrifuge at 4°C, repeat 4 times, filter the precipitate through gauze before the last centrifugation, and the precipitate after centrifugation is the myofibrillar protein; The method for determining the adsorption capacity of muscle protein to terpenoids in step 3 is as follows: Solid phase microextraction coupled with gas chromatography-mass spectrometry was used to determine the headspace concentration of terpenoids; The GC-MS conditions were as follows: DB-WAX column, 30 m×0.18 mm×0.18 μm, injection in splitless mode; temperature program: column temperature was maintained at 35°C for 3 min and increased to 210°C at 5°C / min; mass spectrometry ion source temperature was 200°C, electron energy was 70 eV, and mass scanning range was m / z 35~500; The adsorption capacity of muscle protein for terpenoids is expressed by the free ratio of terpenoids in the mixed solution: ； The molecular docking process in step 4 is as follows: after the three-dimensional structural model of muscle protein and the three-dimensional structural model of terpenoid compounds are optimized by the CHARMm force field, the CDOCKER module in the Discovery Studio 2019 software is used for molecular docking.

2. The method for detecting the interaction between muscle protein and terpenoid molecules in spices as claimed in claim 1, characterized in that: The specific process of purification in step 1 is: the extracted muscle protein is dialyzed in a phosphate buffer containing 0.6 mol / L NaCl using a dialysis bag with a cutoff of 8-14 kDa at 4°C for 24 h, the concentration of the phosphate buffer is 50 mmol / L, and the pH is 7.

0. The phosphate buffer is replaced every 4 h during the dialysis process to obtain purified muscle protein.

3. The method for detecting the interaction between muscle protein and terpenoid molecules in spices as claimed in claim 1, characterized in that: The specific process of the heating treatment in step 1 is: adjusting the mass concentration of the purified muscle protein to 4 mg / mL with a phosphate buffer containing 0.6 mol / L NaCl, heating at 85°C for 1 to 60 min and then immediately cooling in an ice bath to obtain the protein solution, wherein the concentration of the phosphate buffer is 50 mmol / L and the pH is 7.

0.

4. The method for detecting the interaction between muscle protein and terpenoid molecules in spices as claimed in claim 1, characterized in that: The method for determining the adsorption capacity of muscle protein to terpenoids in step 3 is as follows: Experimental group: Take a protein solution with a mass concentration of 4 mg / mL, add a flavor substance stock solution with a mass concentration of 100 mg / L to it, so that the final mass concentration of terpenoid compounds in the mixed solution is 1 mg / L, seal and mix, and equilibrate at 4°C for 12 h; Blank group: Take a phosphate buffer solution containing 0.6 mol / L NaCl, wherein the concentration of the phosphate buffer solution is 50mmol / L and the pH is 7.0, add a 100 mg / L aroma substance stock solution to it, so that the final mass concentration of terpenoid compounds in the mixed solution is 1 mg / L, seal and mix, and balance at 4°C for 12 h; Solid phase microextraction combined with gas chromatography-mass spectrometry was used to determine the headspace concentration of terpenoids. A 75 μm CAR / PDMS extraction fiber was used. The equilibrated mixed solution was adsorbed at 25°C for 30 min and desorbed at 250°C for 3 min before analysis by GC-MS.

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