Preparation method of myosin frozen gel
By adding NaCl and EGCG to the myosin solution, the formation of myosin frozen gel is solved by inducing freezing to form myosin frozen gel, and the problem of insufficient research on the formation mechanism of myosin frozen gel is achieved, efficient gel preparation and quality improvement are achieved, ensuring the safety and quality of meat products.
Patent Information
- Application Number
- CN202510492156.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-18
- Publication Date
- 2025-08-12
AI Technical Summary
In the prior art, there are few studies on the formation mechanism and quality regulation of myosin frozen gels, especially the impact under the synergistic effect of NaCl-EGCG is still blank, affecting the processing quality and safety of meat products.
By adding NaCl and EGCG solutions to myosin solution, the myosin frozen gel is formed by freezing induction, and its gel quality under freezing conditions is regulated. The pH value is adjusted using phosphate buffer, and a stable protein network structure is formed through the freezing-thawing process.
It realizes efficient preparation of myosin frozen gel, reduces thawing losses, improves gel strength and water holding capacity, and ensures the safety and quality of meat processing.
Smart Images
Figure CN120458256A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of meat processing and relates to a method for preparing myosin cryogel. Background Art
[0002] Myosin is a salt-soluble protein with excellent gel-forming ability. Changes in its physicochemical properties and functional characteristics during the production process play a crucial role in the quality of gel-forming meat products. Currently, research on myosin gels primarily focuses on the formation mechanism of heat-induced gels and how to improve gel quality. However, little research has been conducted on the formation mechanism of myosin cryogels and their quality control under freezing conditions. Epigallocatechin-3-gallate (EGCG) is a natural, potent antioxidant and the most abundant polyphenol in green tea. Due to its excellent antioxidant and antimicrobial properties, EGCG is often used in meat processing to improve its quality. Sodium chloride (NaCl) is an essential additive in meat processing, enhancing its taste, flavor, and texture. NaCl can influence the physicochemical properties and functional characteristics of meat proteins by affecting their surface charge distribution, thereby promoting the formation of a good gel structure during heating. Currently, most research focuses on using NaCl and / or EGCG to improve the quality of heat-induced meat gels. However, there is still a lack of research on the effect of NaCl-EGCG synergistic effect on the gel properties of myosin cryogels. Summary of the Invention
[0003] The purpose of the present invention is to provide a method for preparing myosin cryogel. The synergistic effect of NaCl and EGCG in the present invention is used to explain the formation mechanism of myosin aggregation to form cryogel using ice as a template under freezing conditions and the regulation rules of gel quality, thereby providing a new preparation method for the development of innovative minced meat products.
[0004] In one aspect, the present invention provides a method for preparing a myosin cryogel, comprising: sequentially mixing a phosphate buffer solution, a NaCl solution, and an EGCG solution with myosin to obtain a myosin solution, and subjecting the myosin solution to freezing induction to obtain a myosin cryogel.
[0005] Furthermore, the present invention also provides a method for preparing phosphate buffer salt, wherein the phosphate buffer salt is prepared by mixing 20 mM sodium dihydrogen phosphate and 20 mM disodium hydrogen phosphate, and the pH value of the phosphate buffer salt is 6.0-7.0.
[0006] Furthermore, the NaCl solution used in the myosin cryogel prepared by the present invention is prepared by dissolving NaCl in phosphate buffer; the concentration of the NaCl solution is 0.1-0.6M.
[0007] Furthermore, the EGCG solution used in the myosin cryogel prepared by the present invention is prepared by dissolving EGCG in phosphate buffer; the concentration of the EGCG solution is 100-300 ppm.
[0008] Furthermore, the present invention extracts myosin from chicken breast and uses it to prepare myosin cryogel.
[0009] Furthermore, the method of preparing myosin cryogel by freezing-inducing a myosin solution in the present invention includes: reacting the myosin solution at 2-5°C to prepare a myosin gel solution; freezing the myosin gel solution at -20--15°C and thawing it at 2-5°C to obtain a myosin cryogel precursor; and storing the myosin cryogel precursor at 2-5°C to obtain the myosin cryogel.
[0010] Furthermore, the present invention adds NaCl solution and EGCG solution so that when the final concentration of NaCl in the myosin gel is 0.1 M, the final concentration of EGCG is 0 to 1000 ppm;
[0011] When the final concentration of NaCl in the myosin gel solution is 0.3 M, the final concentration of EGCG is 100 to 1000 ppm;
[0012] When the final concentration of NaCl in the myosin gel solution is 0.6 M, the final concentration of EGCG is 250-1000 ppm.
[0013] Preferably, the final concentration of NaCl in the myosin gel solution is 0.1-0.6 M, and the final concentration of EGCG is 250 ppm.
[0014] Furthermore, in the method for preparing myosin cryogel provided by the present invention, the reaction time of the reaction at 2 to 5° C. is 1 to 4 hours;
[0015] The freezing time at -20 to -15°C is 24 to 72 hours;
[0016] The thawing time at 2-5°C is 10-24h;
[0017] The storage time at 2-5° C. is 10-24 hours.
[0018] In another aspect, the present invention provides a myosin cryogel.
[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:
[0020] (1) The method of the present invention verifies the possibility of myosin forming cryogel under freezing conditions and reveals the mechanism and law of the synergistic effect of NaCl and EGCG in regulating the quality of myosin cryogel, providing an innovative strategy for the development of new meat paste products.
[0021] (2) The myosin cryogel prepared by the present invention requires only the addition of natural EGCG and NaCl during preparation, and a simple ice-templating method can achieve myosin gelation under freezing conditions. This method has the advantages of simple preparation and mild conditions, and can effectively ensure the safety of meat products during processing.
[0022] (3) The experiments of the present invention verified that the synergistic effect of NaCl and EGCG can affect the interaction between myosin molecules and the formation of fine network structure of frozen gel during freezing by regulating the structural changes of myosin, thereby reducing the thawing loss of gel and improving gel strength. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 Figure 2 shows the appearance of myosin cryogels prepared with different final concentrations of NaCl and EGCG.
[0024] Figure 2 Figure 2 shows the thawing loss of myosin cryogels prepared with different final concentrations of NaCl and EGCG. Different lowercase letters indicate significant differences.
[0025] Figure 3 Figure 2 shows the gel strength of myosin cryogels prepared with different final concentrations of NaCl and EGCG. Different lowercase letters indicate significant differences.
[0026] Figure 4 The water distribution of myosin cryogels prepared with different final concentrations of NaCl and EGCG. The final concentration of NaCl in A is 0.1 M; the final concentration of NaCl in B is 0.3 M; and the final concentration of NaCl in C is 0.6 M.
[0027] Figure 5 Figure 2 shows the microstructure of myosin cryogels prepared with different final concentrations of NaCl and EGCG.
[0028] Figure 6The following are graphs showing changes in the secondary structure of myosin in cryogels prepared with different final concentrations of NaCl and EGCG. A shows the CD spectra of myosin cryogels prepared with a final NaCl concentration of 0.1 M and different final EGCG concentrations; B shows the CD spectra of myosin cryogels prepared with a final NaCl concentration of 0.3 M and different final EGCG concentrations; C shows the CD spectra of myosin cryogels prepared with a final NaCl concentration of 0.6 M and different final EGCG concentrations; D shows the secondary structure content of myosin cryogels prepared with a final NaCl concentration of 0.1 M and different final EGCG concentrations; E shows the secondary structure content of myosin cryogels prepared with a final NaCl concentration of 0.3 M and different final EGCG concentrations; and F shows the secondary structure content of myosin cryogels prepared with a final NaCl concentration of 0.6 M and different final EGCG concentrations. Different lowercase letters indicate significant differences.
[0029] Figure 7 The solubility diagram of myosin in myosin cryogels prepared with different final concentrations of NaCl and EGCG. Different lowercase letters indicate significant differences.
[0030] Figure 8 Figure 2 shows the myosin thiol content of myosin cryogels prepared with different final concentrations of NaCl and EGCG. Different lowercase letters indicate significant differences.
[0031] Figure 9 The myosin surface hydrophobicity maps of myosin cryogels prepared with different final concentrations of NaCl and EGCG. Different lowercase letters indicate significant differences. DETAILED DESCRIPTION
[0032] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.
[0033] Example 1
[0034] This embodiment provides a method for preparing myosin cryogel based on the synergistic regulation of NaCl-EGCG.
[0035] Myosin in this example was extracted from chicken breast. A phosphate buffer solution was prepared by mixing 20 mM sodium dihydrogen phosphate and 20 mM sodium dihydrogen phosphate. The pH of the solution was 6.0-7.0. NaCl and EGCG were dissolved in the phosphate buffer solution to prepare mother solutions. The concentrations of the NaCl mother solutions were 0.1 M, 0.3 M, and 0.6 M, and the concentration of the EGCG mother solution was 100-300 ppm.
[0036] Phosphate buffer was added to myosin, followed by NaCl and EGCG stock solutions. Mix thoroughly to obtain a myosin solution, which was then incubated at 2–5°C for 1–4 hours to produce a myosin gel solution. The final concentration of myosin in the myosin gel solution was 20 mg / mL, the final concentrations of NaCl were 0.1 M, 0.3 M, and 0.6 M, and the final concentrations of EGCG were 0 ppm, 100 ppm, 250 ppm, 500 ppm, and 1000 ppm. Equal volumes of the myosin gel solutions of varying concentrations were transferred to a beaker and frozen at -20–15°C for 24–72 hours. The frozen solutions were then thawed at 2–5°C for 10–24 hours to produce myosin cryogel precursors. The myosin cryogel was gently scraped along the inner wall of the beaker with a syringe needle until the gel separated from the beaker wall. The beaker was tilted downward and stored at 2-5°C for 10-24 hours to replenish the water lost during the thawing process and allow the gel to reach equilibrium to obtain a stable myosin cryogel.
[0037] Example 2
[0038] This example provides an experiment to measure the thawing loss and gel strength of myosin cryogels.
[0039] Figure 1 Figure 1 shows the appearance of myosin cryogels prepared with different final concentrations of NaCl and EGCG. Figure 1 It can be found that when the final concentrations of NaCl were 0.1M, 0.3M, and 0.6M, and the final concentration of EGCG was 250ppm, the volume of the prepared myosin cryogel was larger, indicating that the synergistic effect of NaCl and EGCG can induce the formation of a myosin gel network structure under freezing conditions and increase the network structure's ability to retain water molecules. However, when the final concentrations of NaCl in the myosin gel were 0.3M, 0ppm, 0.6M, 0ppm, and 0.6M, 100ppm, no cryogel was formed after freezing. This indicates that under high doses of NaCl (0.3M and 0.6M), the interaction between myosin and water molecules is enhanced, hindering the interaction of myosin molecules during the freezing process.
[0040] Figure 2 The figure shows the thawing loss of myosin cryogels prepared with different final concentrations of NaCl and EGCG. Figure 2As shown, when the final NaCl concentration was 0.1 M, the thaw loss of myosin cryogels initially decreased as the final EGCG concentration increased from 0 ppm to 250 ppm. When the final EGCG concentration continued to increase to 1000 ppm, the thaw loss of myosin cryogels increased. Furthermore, when the final NaCl concentrations were 0.3 M and 0.6 M, the thaw loss of myosin cryogels increased with increasing EGCG concentrations. These data suggest that a final EGCG concentration of 100–250 ppm can induce myosin unfolding, promote the formation of an ordered network gel during freezing, and reduce the thaw loss of myosin cryogels. However, a final EGCG concentration of 500–1000 ppm resulted in the formation of excessive aggregates in the cryogels, disrupting the interaction between myosin and water molecules and leading to increased thaw loss.
[0041] Figure 3 The figure shows the gel strength of myosin cryogels prepared with different final concentrations of NaCl and EGCG. Figure 3 As shown, when the final NaCl concentration was 0.1 M, the gel strength did not change significantly as the final EGCG concentration increased from 0 ppm to 1000 ppm. At low salt concentrations, myosin exists as thin filaments, and the interactions between myosin molecules are weak, preventing the formation of effective cross-linking structures. However, when the final NaCl concentrations were 0.3 M and 0.6 M, the gel strength of myosin cryogels decreased as the final EGCG concentration increased from 0 ppm to 1000 ppm. The ordered aggregation of myosin facilitates cryogel formation, while treatment with a final EGCG concentration of 500 to 1000 ppm resulted in excessive myosin aggregation and precipitation, hindering the formation of a fine gel network during freezing and leading to a decrease in the gel strength of myosin cryogels.
[0042] Example 3
[0043] This example provides the water distribution and microstructure of myosin cryogels.
[0044] 1. Water Distribution in Myosin Cryogels
[0045] like Figure 4 As shown, about 0 to 10 ms (T 2b )、0~100ms(T 21 )、100~1000ms(T 22 ) and 1000~10000ms(T 23 ) corresponds to the four T2 distribution peaks of tightly bound water, bound water, fixed water and free water, respectively. 22It is the main water in all frozen gel samples. When the final concentration of NaCl is 0.1M, 0.3M and 0.6M, the final concentration of EGCG is 100-250ppm, which shortens the T 2b and T 21 , indicating that treatment with a final concentration of 100-250 ppm EGCG enhanced the water-holding capacity of myosin cryogels. However, treatment with a final concentration of 500-1000 ppm EGCG resulted in excessive aggregation of myosin, which increased the relaxation time of the cryogels and reduced the affinity between myosin and water molecules. Consequently, a disordered gel network formed during the freezing process was unable to effectively retain water molecules, leading to an increase in free water content.
[0046] 2. Microstructure of Myosin Cryogel
[0047] The microstructure of myosin cryogels was observed using a scanning electron microscope. The frozen gels were cut into small pieces (5 mm × 5 mm × 5 mm) and soaked in 2.5% glutaraldehyde for 72 hours. The gels were washed four times with 0.1 M PBS (pH 7.2). The gels were then dehydrated using a gradient of 30%, 50%, 70%, 80%, 90%, and 100% ethanol. The dehydrated gels were supercritically dried and gold-sprayed for observation.
[0048] like Figure 5 As shown, at a final NaCl concentration of 0.1 M, the density of the myosin cryogel microstructure increased as the final EGCG concentration increased from 0 ppm to 250 ppm. Further increases in the EGCG concentration to 1000 ppm reduced the density of the myosin cryogel microstructure, indicating that treatment with a final EGCG concentration of 100-250 ppm favors the formation of a compact cross-linked network. At final NaCl concentrations of 0.3 M and 0.6 M, and after treatment with a final EGCG concentration of 500-1000 ppm, the originally dense gel structure transformed into a rough and loose state. This result is consistent with the results of thawing loss, water distribution, and gel strength. During the freezing process, moderate unfolding of myosin leads to the exposure of reactive groups within the myosin molecule.
[0049] Example 4
[0050] This example provides the results of myosin determination in myosin cryogels.
[0051] 1. Determination of the secondary structure of myosin
[0052] Circular dichroism (CD) spectroscopy is an effective method for determining protein secondary structure. Figure 6 A in the figure is the CD spectra of myosin cryogels with different final concentrations of EGCG and a final concentration of 0.1 M NaCl. Figure 6B in the figure is the CD spectra of myosin cryogels with a final NaCl concentration of 0.3 M and different final EGCG concentrations. Figure 6 C in the figure is the CD spectra of myosin cryogels with a final NaCl concentration of 0.6 M and different final EGCG concentrations. Figure 6 A in Figure 6 B in and Figure 6 As can be seen from Figure C, the CD spectrum of myosin shows two negative peaks near 202 nm and 218 nm, indicating the existence of α-helical structure in myosin. Figure 6 D in the figure is the secondary structure content in myosin cryogels with different final concentrations of EGCG and a final concentration of 0.1 M NaCl. Figure 6 E in the figure is the secondary structure content in myosin cryogels with different final concentrations of EGCG and a final concentration of 0.3 M NaCl. Figure 6 Figure F shows the secondary structure content in myosin cryogels treated with different final EGCG concentrations at a final NaCl concentration of 0.6 M. With increasing EGCG concentration, the α-helix content of the three myosin samples decreased from 46.31±0.16% (0.1 M NaCl), 57.14±0.45% (0.3 M NaCl), and 77.16±0.36% (0.6 M NaCl) to 11.54±0.01% (0.1 M NaCl), 12.92±0.03% (0.3 M NaCl), and 13.16±0.08% (0.6 M NaCl), respectively. In contrast, β-sheets, β-turns, and random coils showed an increasing trend. These data demonstrate that EGCG can induce the unfolding of myosin α-helical structure in a dose-dependent manner. The abundant hydroxyl groups in EGCG form new hydrogen bonds with myosin molecules, interfering with the stability of the hydrogen bonds that stabilize the α-helical structure, causing the α-helical structure to unfold and further transform into β-sheets, β-turns and random coils.
[0053] 2. Determination of Myosin Solubility
[0054] like Figure 7 As shown in the figure, when the final concentration of EGCG was 0 ppm, the myosin solubility increased significantly when the final concentration of NaCl increased from 0.1 M to 0.6 M, indicating that high-dose NaCl (0.6 M) has a strong electrostatic shielding effect on myosin, effectively inhibiting protein aggregation by reducing electrostatic interactions between myosins, thereby increasing myosin solubility. As the final concentration of EGCG increased, the solubility of myosin samples with final NaCl concentrations of 0.1 M, 0.3 M, and 0.6 M all decreased significantly. This suggests that the addition of EGCG induces the unfolding of myosin, promotes interactions between protein molecules, causes myosin aggregation, and thus reduces solubility.
[0055] 3. Determination of Myosin Thiol Groups
[0056] Figure 8 is the result of determination of sulfhydryl groups in myosin. Figure 8 As shown in the figure, as the final concentration of NaCl (0 ppm EGCG) increased from 0.1M to 0.6M, the thiol content of myosin increased from 11.22±0.18nM / mg to 14.96±0.03nM / mg. This is because the high salt environment destroys the electrostatic interaction between myosin molecules, inhibits the aggregation of myosin molecules, and thus hinders the cross-linking between myosin molecules through disulfide bonds. However, under the same NaCl concentration conditions, the thiol content of myosin decreased significantly with the increase of EGCG addition. This is because EGCG can induce the formation of disulfide bonds between myosin, promote myosin aggregation, and further shield unreacted thiol groups.
[0057] 4. Determination of Myosin Surface Hydrophobicity
[0058] Figure 9 is the result of the measurement of myosin surface hydrophobicity. Figure 9 As shown. When the final concentration of EGCG was 0 ppm, the final concentration of NaCl increased from 0.1 M to 0.6 M, and the surface hydrophobicity of myosin showed an upward trend. At the same NaCl concentration, the surface hydrophobicity of myosin decreased significantly with the increase of EGCG content. This is because the introduction of abundant hydroxyl groups in EGCG creates a strong hydrophilic environment for myosin, which in turn leads to a decrease in the surface hydrophobicity of myosin. In addition, the continuous exposure of hydrophobic groups promotes the formation of hydrophobic interactions between myosin-myosin or myosin-EGCG at different NaCl concentrations, so that the binding sites of the fluorescent probe are occupied, resulting in a further decrease in surface hydrophobicity.
[0059] The embodiments described above are some of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.
Claims
1. A method for preparing myosin cryogel, characterized in that: The preparation method comprises: sequentially mixing a phosphate buffer solution, a NaCl solution and an EGCG solution with myosin to obtain a myosin solution, and subjecting the myosin solution to freezing induction to obtain a myosin cryogel.
2. The preparation method according to claim 1, characterized in that The phosphate buffer is prepared by mixing 20 mM sodium dihydrogen phosphate and 20 mM disodium hydrogen phosphate, and the pH value of the phosphate buffer is 6.0-7.
0.
3. The preparation method according to claim 1, characterized in that The NaCl solution is prepared by dissolving NaCl in phosphate buffer; The concentration of the NaCl solution is 0.1-0.6M.
4. The preparation method according to claim 1, characterized in that The EGCG solution is prepared by dissolving EGCG in phosphate buffer; The concentration of the EGCG solution is 100-300 ppm.
5. The preparation method according to claim 1, characterized in that The myosin is extracted from chicken breast.
6. The preparation method according to claim 1, characterized in that The method for preparing the myosin cryogel by freezing-inducing the myosin solution comprises the following steps: reacting the myosin solution at 2-5° C. to prepare a myosin gel solution; freezing the myosin gel solution at -20--15° C. and thawing it at 2-5° C. to obtain a myosin cryogel precursor; and storing the myosin cryogel precursor at 2-5° C. to obtain the myosin cryogel.
7. The preparation method according to claim 6, characterized in that When the final concentration of NaCl in the myosin gel solution is 0.1 M, the final concentration of EGCG is 0 to 1000 ppm; When the final concentration of NaCl in the myosin gel solution is 0.3 M, the final concentration of EGCG is 100 to 1000 ppm; When the final concentration of NaCl in the myosin gel solution is 0.6 M, the final concentration of EGCG is 250-1000 ppm.
8. The preparation method according to claim 7, characterized in that The final concentration of NaCl in the myosin gel solution is 0.1-0.6 M, and the final concentration of EGCG is 250 ppm.
9. The preparation method according to claim 6, characterized in that The reaction time of the reaction at 2 to 5°C is 1 to 4 hours; The freezing time at -20 to -15°C is 24 to 72 hours; The thawing time at 2-5°C is 10-24h; The storage time at 2-5° C. is 10-24 hours.
10. Myosin cryogel prepared by the method according to any one of claims 1 to 9.