CN109336962B - Method for increasing fish muscle globulin hydrophobicity - Google Patents
Method for increasing fish muscle globulin hydrophobicity Download PDFInfo
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- CN109336962B CN109336962B CN201811298275.2A CN201811298275A CN109336962B CN 109336962 B CN109336962 B CN 109336962B CN 201811298275 A CN201811298275 A CN 201811298275A CN 109336962 B CN109336962 B CN 109336962B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
The invention provides a method for increasing myosin solubility of fish meat, which is characterized by comprising the following steps: s1, slaughtering a fresh fish to obtain myosin stock solution; s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with the pH of 7.0-10.0 for dialysis, wherein the treatment solution comprises 150-400mmol/L salt solution and 5-25mmol/L amino acid solution, and dialyzing for 1-3h; s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 160-380mmol/L; s4, heating the treatment fluid to 40-60 ℃ by adopting a gradient heating mode, and continuously dialyzing for 7-12h; s5, pouring out the dialyzed myosin stock solution, and naturally cooling to room temperature. The invention provides a method for increasing the myosin solubility of fish meat, which is a feasible scheme for solubilizing fish protein (especially tilapia protein), can provide theoretical basis for deep processing of tilapia resource, and further expands the application range of aquatic protein.
Description
Technical Field
The invention relates to the technical field of aquatic product quality improvement, in particular to a method for increasing the hydrophobicity of myosin of fish meat (especially tilapia meat).
Background
Myosin is the structural protein and contractile protein with the most content in various myocytes, is the constitutive protein of the muscle myofibril thick filament, is the protein which is most abundantly expressed by striated muscle cells, accounts for about 25 percent of the total protein library, and is the main bearer of the functional characteristics of fish protein. The globulin is a bean sprout-shaped molecule which comprises two special structures, namely two spherical heads and a rod-shaped tail, is shaped like a Y, has a myosin molecular weight of about 470 k Da, and consists of two heavy chains (MHC) with a molecular weight of 220 k Da and four light chains (LHC) with a molecular weight of 17-22k Da.
Myosin is subject to disruption of intermolecular electrostatic interactions in high salt solutions in vitro (e.g. 0.6 mol/L KCl), high solubility of molecules in monomeric or soluble oligomeric form, and myosin aggregates into fibrils in insoluble state in low salt solutions in vitro (< 0.2 mol/L), thereby limiting the processing characteristics of muscle proteins. The solubility of the protein is related to the amino acid composition and structure of the protein in addition to the p H, ionic strength, temperature and protein concentration of the external environment. The hydrophobicity and the ionicity of amino acids in the protein are key factors influencing the solubility of the protein, and the interaction between the protein and the protein can be enhanced along with the increase of the hydrophobicity, so that the solubility of the protein in water is reduced. Ionic interactions promote the interaction between the protein and the solvent, which allows the protein to be better dispersed in solution, thereby improving the solubility of the protein in water. Meanwhile, the solubility of food protein is susceptible to physical action or chemical modification in the food processing process. Myosin is a major contributor to the functional properties of fish protein and is an important subject of food protein research. The research on the solubility and the functional characteristics of the myosin is beneficial to expanding the application of aquatic protein related food systems, and provides a certain theoretical basis for deeply developing aquatic protein resources.
Disclosure of Invention
In view of the above, the invention provides a method for increasing the hydrophobicity of fish myosin, and the invention is a feasible scheme for solubilizing fish protein (especially tilapia protein), which can provide theoretical basis for the deep processing of tilapia resource, and further expand the application range of aquatic protein.
The technical scheme of the invention is as follows: a method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with the pH value of 7.0-10.0 for dialysis, wherein the treatment solution comprises 150-400mmol/L salt solution and 5-25mmol/L amino acid solution, and dialyzing for 1-3h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 160-380mmol/L;
s4, heating the treatment fluid to 40-60 ℃ by adopting a gradient heating mode, and continuing dialysis for 7-12h;
and S5, pouring out dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S4 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 5-15min. In the present invention, a Supercritical Fluid Process (SCF) is used, and can be realized by any conventional technique. A supercritical fluid is a fluid having a temperature and pressure above its critical temperature (Tc) and critical pressure (Tp), and has both liquid and gaseous properties. At near-critical temperatures, supercritical fluids are highly compressible, and very small pressure changes can greatly change the density of the fluid, while the mass transfer characteristics of the fluid determine the dissolving capacity of the fluid to a great extent. In the present invention, by using supercritical fluid treatment, the particle size of the protein dissolved in SCF can be reduced, and the solubility of fish myosin can be further improved.
Preferably, the supercritical fluid treatment process conditions in the present invention are: carbon dioxide, nitrogen and the like are used as supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is 15MPa, the action is carried out for 5-15min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the amino acid solution comprises the following components in parts by weight: 5-18 parts of lysine, 6-14 parts of histidine, 3-10 parts of glycine, 8-19 parts of arginine and 3-7 parts of glycerol. According to the amino acid solution, the inventor of the invention prepares a formula with obvious solubilizing effect on fish myosin by compounding various amino acids through a large number of creative tests.
Further, the mediating solution comprises the following components in parts by weight: 23-35 parts of tea polyphenol, 9-17 parts of gallic acid and 4-10 parts of sodium citrate. In particular, the tea polyphenol, the gallic acid and the sodium citrate are all food grade. According to the invention, the adopted mediating solution mainly plays a role in assisting dissolution in the whole system, wherein the inventor discovers through a great deal of creative work that the compounding of tea polyphenol and gallic acid can form weak interaction with internal structural components of protein, including hydrogen bonds (acting force of X-H … Y type), double hydrogen bonds, dipole-dipole force, complexation and the like, and meanwhile, sodium citrate can improve the co-dissolution performance of the system to a certain extent. The inventor obtains the mediating solution with high selective solubilization effect through a large number of creative tests, and can obviously improve the solubility of fish myosin in the dialysis process.
Further, the gradient temperature rise heats the treatment fluid to 40 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, heating to 35 deg.C for 0-30min, and heating to 40 deg.C for 2-5 min.
Further, the gradient temperature rise heats the treatment fluid to 50 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, and 50 deg.C for 3-7 min.
Further, the gradient temperature rise heats the treatment fluid to 55 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 55 deg.C for 3-7 min.
Further, the gradient temperature rise heats the treatment fluid to 60 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 60 deg.C for 3-10 min.
In the invention, a gradient temperature rise mode is adopted, the solubility of myosin is improved to a certain extent along with the rise of temperature, on the other hand, the irreversible change of the internal structure of protein caused by the sudden change of temperature can be avoided, and meanwhile, the sodium citrate in the medium and the glycerol in the amino acid solution in the medium can improve the thermal stability of myosin and a system and ensure the solubilization effect.
Further, the salt solution is any one of KCl or NaCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A in weight of fish, homogenizing at high speed at 20000r/min for 30s, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And detecting, and then performing centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, pH6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E contained 18mmol/L PBS, 1mol/L NaCl, pH6.8.
The inventor of the present application has found that the solubility is mainly influenced by the structure of the protein molecule and the molecular aggregation behavior, and the structure and aggregation of the protein molecule are also influenced by the characteristics of the external solution, and generally, strong non-covalent interactions, hydrophobic interactions, hydrogen bonds and the like between the protein and the protein are the most important roles of the solvent property of the protein. Therefore, the invention provides a method which does not destroy the protein structure, is non-toxic and harmless and is used for promoting the increase of the myosin solubility, further introduces natural and non-toxic substances into the solution, and is an effective way for improving the solubility and the dispersibility by changing the non-covalent interaction between proteins.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with the pH of 7.0 for dialysis, wherein the treatment solution comprises 150 mmol/L salt solution and 5mmol/L amino acid solution, and dialyzing for 1h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 160 mmol/L;
s4, heating the treatment fluid to 40 ℃ by adopting a gradient heating mode, and continuing dialysis for 7h;
and S5, pouring out dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S4 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 5min.
The process conditions of the supercritical fluid treatment in the invention are as follows: nitrogen is adopted as the supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is controlled to be 15MPa, the reaction is carried out for 5min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the amino acid solution comprises the following components in parts by weight: lysine 5, histidine 6, glycine 3, arginine 8 and glycerol 3.
Further, the mediating solution comprises the following components in parts by weight: tea polyphenol 23, gallic acid 9 and sodium citrate 4.
Further, the gradient temperature rise heats the treatment fluid to 40 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, heating to 35 deg.C for 0-30min, and heating to 40 deg.C for 2-5 min.
Further, the salt solution is KCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A by weight of fish, homogenizing at high speed for 30s at 20000r/min, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And (4) detecting, and then carrying out centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, pH6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E contained 18mmol/L PBS, 1mol/L NaCl, pH6.8.
Example 2
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with the pH of 10.0 for dialysis, wherein the treatment solution comprises 400mmol/L salt solution and 25mmol/L amino acid solution, and dialyzing for 3 hours;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 380mmol/L;
s4, heating the treatment fluid to 60 ℃ by adopting a gradient heating mode, and continuing dialysis for 12 hours;
s5, pouring out the dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S4 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 15min.
The process conditions of the supercritical fluid treatment in the invention are as follows: nitrogen is adopted as the supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is controlled to be 15MPa, the reaction is carried out for 15min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the amino acid solution comprises the following components in parts by weight: lysine 18, histidine 14, glycine 10, arginine 19 and glycerol 7.
Further, the mediating solution comprises the following components in parts by weight: 35 parts of tea polyphenol, 17 parts of gallic acid and 10 parts of sodium citrate.
Further, the gradient temperature rise heats the treatment fluid to 60 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 60 deg.C for 3-10 min.
Further, the salt solution is NaCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A by weight of fish, homogenizing at high speed for 30s at 20000r/min, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, and standing2h, adding 1/10 times of solution D into the mixed system, and stirring 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And (4) detecting, and then carrying out centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, pH6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E contained 18mmol/L PBS, 1mol/L NaCl, pH6.8.
Example 3
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with the pH of 8.0 for dialysis, wherein the treatment solution comprises 200 mmol/L salt solution and 10mmol/L amino acid solution, and dialyzing for 1.5h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 200 mmol/L;
s4, heating the treatment fluid to 50 ℃ in a gradient heating mode, and continuing dialysis for 9 hours;
s5, pouring out the dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S4 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 8min.
The process conditions of the supercritical fluid treatment in the invention are as follows: carbon dioxide is used as supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is controlled to be 15MPa, the action is carried out for 8min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the amino acid solution comprises the following components in parts by weight: lysine 8, histidine 8, glycine 5, arginine 10 and glycerol 4.
Further, the mediating liquid comprises the following components in parts by weight: 25 parts of tea polyphenol, 11 parts of gallic acid and 6 parts of sodium citrate.
Further, the gradient temperature rise heats the treatment fluid to 50 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, and 50 deg.C for 3-7 min.
Further, the salt solution is KCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A in weight of fish, homogenizing at high speed at 20000r/min for 30s, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And detecting, and then performing centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, and the pH value of the solution is 6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E contained 18mmol/L PBS, 1mol/L NaCl, pH6.8.
Example 4
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with pH of 9.0 for dialysis, wherein the treatment solution comprises 350 mmol/L salt solution and 20mmol/L amino acid solution, and dialyzing for 2.5h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 340mmol/L;
s4, heating the treatment fluid to 55 ℃ by adopting a gradient heating mode, and continuing dialysis for 10 hours;
s5, pouring out the dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S4 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 12min.
The process conditions of the supercritical fluid treatment in the invention are as follows: carbon dioxide is used as a supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is 15MPa, the reaction is carried out for 12min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the amino acid solution comprises the following components in parts by weight: lysine 15, histidine 12, glycine 8, arginine 17 and glycerol 6.
Further, the mediating solution comprises the following components in parts by weight: tea polyphenol 32, gallic acid 14 and sodium citrate 8.
Further, the gradient temperature rise heats the treatment fluid to 55 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 55 deg.C for 3-7 min.
Further, the salt solution is NaCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A by weight of fish, homogenizing at high speed for 30s at 20000r/min, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, and stirringEvenly stirring and then centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And (4) detecting, and then carrying out centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, pH6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E contained 18mmol/L PBS, 1mol/L NaCl, pH6.8.
Example 5
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with the pH of 8.2 for dialysis, wherein the treatment solution comprises 220 mmol/L salt solution and 13mmol/L amino acid solution, and dialyzing for 1.8h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 260mmol/L;
s4, heating the treatment fluid to 60 ℃ by adopting a gradient heating mode, and continuing dialyzing for 10 hours;
s5, pouring out the dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S4 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 10min.
The process conditions of the supercritical fluid treatment in the invention are as follows: nitrogen is adopted as the supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is controlled to be 15MPa, the reaction is carried out for 10min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the amino acid solution comprises the following components in parts by weight: lysine 12, histidine 10, glycine 5, arginine 13 and glycerol 5.
Further, the mediating solution comprises the following components in parts by weight: tea polyphenol 29, gallic acid 13 and sodium citrate 7.
Further, the gradient temperature rise heats the treatment fluid to 60 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 60 deg.C for 3-10 min.
Further, the salt solution is KCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A by weight of fish, homogenizing at high speed for 30s at 20000r/min, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And (4) detecting, and then carrying out centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, and the pH value of the solution is 6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E comprises 18mmol/L PBS and 1mol/L NaClThe pH of the solution was 6.8.
Example 6
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with pH of 7.9 for dialysis, wherein the treatment solution comprises 270 mmol/L salt solution and 17 mmol/L amino acid solution, and dialyzing for 2.2h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 280 mmol/L;
s4, heating the treatment fluid to 55 ℃ by adopting a gradient heating mode, and continuing dialysis for 11h;
s5, pouring out the dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S4 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 12min.
The process conditions of the supercritical fluid treatment in the invention are as follows: nitrogen is adopted as the supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is controlled to be 15MPa, the reaction is carried out for 12min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the amino acid solution comprises the following components in parts by weight: lysine 14, histidine 9, glycine 6, arginine 14 and glycerol 4.5.
Further, the mediating solution comprises the following components in parts by weight: 30 parts of tea polyphenol, 15 parts of gallic acid and 7 parts of sodium citrate.
Further, the gradient temperature rise heats the treatment fluid to 55 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 55 deg.C for 3-7 min.
Further, the salt solution is NaCl.
Further, the extraction method of the myosin comprises the following steps: adding 5 times of fresh minced fishHomogenizing solution A at high speed of 20000r/min for 30s, standing for 15min, centrifuging to obtain precipitate, and repeating the above steps for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And detecting, and then performing centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, and the pH value of the solution is 6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E contained 18mmol/L PBS, 1mol/L NaCl, pH6.8.
Comparative example 1
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with pH of 7.9 for dialysis, wherein the treatment solution comprises 270 mmol/L salt solution and 17 mmol/L amino acid solution, and dialyzing for 2.2h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 280 mmol/L;
s4, heating the treatment fluid to 55 ℃ by adopting a gradient heating mode, and continuing dialysis for 11h;
s5, pouring out the dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the amino acid solution comprises the following components in parts by weight: lysine 14, histidine 9, glycine 6, arginine 14 and glycerol 4.5.
Further, the mediating solution comprises the following components in parts by weight: 30 parts of tea polyphenol, 15 parts of gallic acid and 7 parts of sodium citrate.
Further, the gradient temperature rise heats the treatment fluid to 55 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 55 deg.C for 3-7 min.
Further, the salt solution is NaCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A in weight of fish, homogenizing at high speed at 20000r/min for 30s, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And (4) detecting, and then carrying out centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, pH6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E contained 18mmol/L PBS, 1mol/L NaCl, pH6.8.
Comparative example 2
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with the pH of 8.2 for dialysis, wherein the treatment solution comprises 220 mmol/L salt solution and 13mmol/L amino acid solution, and dialyzing for 1.8h;
s3, heating the treatment fluid to 60 ℃ by adopting a gradient heating mode, and continuing dialysis for 10 hours;
and S4, pouring out dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S3 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 10min.
The process conditions of the supercritical fluid treatment in the invention are as follows: nitrogen is adopted as the supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is controlled to be 15MPa, the reaction is carried out for 10min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the amino acid solution comprises the following components in parts by weight: lysine 12, histidine 10, glycine 5, arginine 13 and glycerol 5.
Further, the gradient temperature rise heats the treatment fluid to 60 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 60 deg.C for 3-10 min.
Further, the salt solution is KCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A in weight of fish, homogenizing at high speed at 20000r/min for 30s, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to reach saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to reach saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And (4) detecting, and then carrying out centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, pH6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E comprises 18mmol/L PBS, 1mol/L NaCl, and the solution pH is 6.8.
Comparative example 3
A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with pH of 7.9 for dialysis, wherein the treatment solution comprises 270 mmol/L salt solution, and dialyzing for 2.2h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 280 mmol/L;
s4, heating the treatment fluid to 55 ℃ by adopting a gradient heating mode, and continuing dialysis for 11h;
s5, pouring out the dialyzed myosin stock solution, and naturally cooling to room temperature.
Further, the step S4 further includes, after the dialysis is finished, placing the processed protein solution into a supercritical fluid, and performing supercritical fluid processing for 12min.
The process conditions of the supercritical fluid treatment in the invention are as follows: nitrogen is adopted as the supercritical fluid, the temperature of the fluid is controlled to be 35 ℃, the reaction pressure is controlled to be 15MPa, the reaction is carried out for 12min, and an exhaust valve is opened to rapidly discharge the supercritical fluid after the reaction is finished.
Further, the mediating liquid comprises the following components in parts by weight: 30 parts of tea polyphenol, 15 parts of gallic acid and 7 parts of sodium citrate.
Further, the gradient temperature rise heats the treatment fluid to 55 ℃, and the specific steps are as follows: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 55 deg.C for 3-7 min.
Further, the salt solution is NaCl.
Further, the extraction method of the myosin comprises the following steps: taking fresh minced fish, adding 5 times of solution A by weight of fish, homogenizing at high speed for 30s at 20000r/min, standing for 15min, centrifuging to obtain precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until there is no SO 4 2- And (4) detecting, and then carrying out centrifugal separation to obtain supernatant, namely myosin solution.
Specifically, the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, and the pH value of the solution is 6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E contained 18mmol/L PBS, 1mol/L NaCl, pH6.8.
Test example
Determination of surface hydrophobicity
And (3) detecting the surface hydrophobicity of the soluble protein by adopting an ANS and PRODAN fluorescence probe method. The centrifuged soluble myosin solution is diluted in gradient (0.5, 0.25, 0.125, 0.0625 mg/m L), 4 ml of each solution with different concentrations is added with 20. Mu.L of ANS solution (8.0 mmol/L ANS,0.01 mol/L Tris-HCl, p H7.0.0) or 10. Mu.L of PRODAN solution (1.0 mmol/L PRODAN, methanol), mixed well, and reacted in the dark for 10min and 15min, respectively, and the Fluorescence Intensity (FI) of the sample is measured using a fluorescence spectrophotometer. The excitation wavelength and the emission wavelength of the ANS method are 374 nm and 485 nm respectively; the excitation wavelength and the emission wavelength of the detection of the PRODAN fluorescent probe are 365 nm and 465 nm respectively, and the slit width is 5nm. And subtracting the endogenous fluorescence intensity value of the corresponding sample from the fluorescence intensity value of the sample added with the probe to obtain the relative fluorescence intensity value (RFI) of the protein. RFI is plotted against protein concentration, and the slope of the initial segment is used as an indicator of the surface hydrophobicity of the protein (ANS-S0 or PRODAN-S0). Each sample was replicated three times. The fish myosin prepared in examples and comparative examples was subjected to the measurement of surface hydrophobicity, and the results are shown in table 1.
TABLE 1
| Experimental group | ANS | PRODAN |
| Example 1 | 4710 | 6360 |
| Example 2 | 4890 | 6600 |
| Example 3 | 5620 | 7870 |
| Example 4 | 5830 | 8160 |
| Example 5 | 7890 | 11440 |
| Example 6 | 8150 | 11820 |
| Comparative example 1 | 2040 | 2960 |
| Comparative example 2 | 2350 | 3410 |
| Comparative example 3 | 2270 | 3290 |
Determination of the Secondary Structure (alpha-helix) content
The treated myosin solution was diluted to 100. Mu.g/mL with the corresponding buffer and subjected to a circular dichroism scan. The wavelength scanning range is 190 to 260nm, the optical path of a sample pool is 1mm, the sample loading amount is 0.3mL, the measurement temperature is 4 ℃, the measurement resolution is 0.5mm, the scanning speed is 100nm/min, the sensitivity is 20mdeg, the response time is 0.25s, a buffer solution is used as a blank, the experimental value is the mean value of 3 scans, and the calculation formula of the alpha-helix content is as follows:
[θ] 222 =[θ]obs × 1000 × Mw/(L × C) formula (1)
α -helix (%) = [ θ [ ]] 222 X 100/(-40000) formula (2)
The fish myosin prepared in examples and comparative examples was subjected to the measurement of surface hydrophobicity, and the results are shown in table 2.
TABLE 2
| Experimental group | Alpha-helix (%) |
| Example 1 | 85.7 |
| Example 2 | 86.3 |
| Example 3 | 89.5 |
| Example 4 | 90.1 |
| Example 5 | 94.5 |
| Example 6 | 95.6 |
| Comparative example 1 | 66.3 |
| Comparative example 2 | 71.5 |
| Comparative example 3 | 70.8 |
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art. It should be noted that the technical features not described in detail in the present invention can be implemented by any prior art.
Claims (8)
1. A method for increasing the hydrophobicity of fish myosin, comprising the steps of:
s1, slaughtering a fresh fish, taking a white meat part of the fish, extracting myosin, and obtaining myosin stock solution;
s2, putting myosin stock solution into a dialysis bag, putting the dialysis bag into treatment solution with the pH value of 7.0-10.0 for dialysis, wherein the treatment solution comprises 150-400mmol/L salt solution and 5-25mmol/L amino acid solution, and dialyzing for 1-3h;
s3, adding a guiding liquid into the treatment liquid, and enabling the final concentration of the guiding liquid to be 160-380mmol/L;
s4, heating the treatment fluid to 40-60 ℃ by adopting a gradient heating mode, and continuing dialysis for 7-12h;
s5, pouring out dialyzed myosin stock solution, and naturally cooling to room temperature;
the medium liquid comprises the following components in parts by weight: 23-35 parts of tea polyphenol, 9-17 parts of gallic acid and 4-10 parts of sodium citrate;
the amino acid solution comprises the following components in parts by weight: 5-18 parts of lysine, 6-14 parts of histidine, 3-10 parts of glycine, 8-19 parts of arginine and 3-7 parts of glycerol.
2. The method for increasing the hydrophobicity of fish myosin according to claim 1, wherein the gradient temperature elevation heats the treatment fluid to 40 ℃ by the following steps: heating to 25 deg.C for 0-20min, heating to 35 deg.C for 0-30min, and heating to 40 deg.C for 2-5 min.
3. The method for increasing the hydrophobicity of fish myosin according to claim 1, wherein the gradient temperature elevation heats the treatment fluid to 50 ℃ by the following steps: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, and 50 deg.C for 3-7 min.
4. The method for increasing the hydrophobicity of fish myosin according to claim 1, wherein the gradient temperature elevation heats the treatment fluid to 55 ℃, comprising the steps of: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 55 deg.C for 3-7 min.
5. The method for increasing the hydrophobicity of the myosin protein of fish meat according to claim 1, wherein the gradient temperature elevation heats the treatment fluid to 60 ℃ by the steps of: heating to 25 deg.C for 0-20min, 37 deg.C for 0-30min, 43 deg.C for 3-10min, 50 deg.C for 3-7min, and 60 deg.C for 3-10 min.
6. The method for increasing the hydrophobicity of myosin in fish meat according to claim 1, wherein the salt solution is any one of KCl or NaCl.
7. The method for increasing the hydrophobicity of fish myosin according to claim 1, wherein the myosin is extracted by the following method: taking fresh minced fish, adding 5 times of solution A by weight of fish, homogenizing at 20000r/min for 30s, standing for 15Centrifuging after min, collecting precipitate, and repeating the above process for 3 times; adding 3 times of the solution B into the obtained precipitate, uniformly stirring and centrifuging; filtering with gauze, diluting the filtrate with ice distilled water, standing, centrifuging to obtain precipitate, adding 1/5 times of solution C into the precipitate, mixing, standing for 2h, adding 1/10 times of solution D into the mixed system, and stirring for 1h; adding saturated ammonium sulfate to saturation of 40%, centrifuging to obtain supernatant, and adding saturated ammonium sulfate to saturation of 45%, centrifuging to obtain precipitate; precipitating with dialysate E, and dialyzing until SO4 is removed 2- Detecting, and then performing centrifugal separation to obtain supernatant which is myosin solution;
the solution A comprises 20mmol/L PBS, 2mmol/L PMSF and 0.02% sodium azide, and the pH value of the solution is 6.9; the solution B comprises 0.35mol/L KCl, 5mmol/L ATP and 7mmol/L MgCl 2 0.3mmol/L DTT, pH6.2; the solution C comprises 0.12mol/L Tris-maleic acid, 1.6mol/L KCl and 0.6mmol/L DTT, and the pH value of the solution is 7.4; the solution D comprises 0.13mol/L ATP and 48mmol/L MgCl 2 5.5mmol/L EGTA, pH7.5; the dialysate E comprises 18mmol/L PBS and 1mol/L NaCl, and the solution is pH6.8.
8. The method for increasing the hydrophobicity of myosin in fish meat according to claim 1, wherein the step S4 further comprises subjecting the treated protein solution to a supercritical fluid treatment for 5-15min after the dialysis.
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| 氨基酸诱导低离子强度下罗非鱼肌球蛋白增溶腥味及机制;付苇娅;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20180215(第2期);第10-12页2.3实验方法部分和第22页2.5本章小结部分 * |
| 蛋白稳定剂对罗非鱼肌球蛋白热变性聚集的抑制及机理;时娇娇;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20180415(第4期);第9-11页实验方法部分、第34-35页4.3实验方法、第26页2.5本章小结和第43页4.5本章小结 * |
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