Method for improving water solubility of myofibrillar protein

Abstract

The invention discloses a method for improving water solubility of myofibrillar protein, and belongs to the technical field of sucrose ester application. The method comprises the following steps: dissolving sucrose ester in a phosphate buffer solution to prepare a sucrose ester solution; dissolving myofibrillar protein in a phosphate buffer solution to prepare a myofibrillar protein solution; and adding the sucrose ester solution into the myofibrillar protein solution, and carrying out high-pressure homogenization treatment to obtain a sucrose ester-myofibrillar protein compound solution. The method can be used for effectively modifying and improving the functional properties of the myofibrillar protein and improving the dispersion uniformity of a myofibrillar protein solution system, and is relatively low in cost, safe, reliable and convenient to operate. The sucrose ester-myofibrillar protein compound solution prepared by the method can be used as a food additive.

Description

Technical Field

[0001] The present invention belongs to the technical field of sucrose ester applications, and particularly relates to a method for improving the water solubility of myofibrillar proteins. Background Art

[0002] Sucrose fatty acid esters, abbreviated as sucrose esters, are non-ionic surfactants synthesized by the esterification reaction of sucrose (β-D-fructofuranosyl α-D-glucopyranoside) with fatty acids (such as steric acid, palmitic acid, lauric acid, and oleic acid, etc.). These non-ionic surfactants are considered "green" because they are both non-toxic and biodegradable.

[0003] Myofibrillar protein is the main structural protein in muscle tissue and is mainly composed of myosin and actin. Myofibrillar protein plays an important role in the processing of emulsified meat products. However, the emulsifying ability of myofibrillar protein is usually limited by various external factors such as ionic strength and pH value. Although the use of chemical and physical modification strategies (amino acid addition, glycosylation modification, high-intensity ultrasound, and high-pressure homogenization modification) can improve the dispersion characteristics and emulsifying ability of myofibrillar protein, due to operational difficulties and safety issues, these emerging technologies have not been widely applied in the production and processing of emulsified meat products. From a more practical perspective, adding surfactants to optimize the emulsion decomposition and dispersion state of myofibrillar protein is a convenient and economical processing method.

[0004] It is of great significance to improve the emulsifying performance and dispersion state of myofibrillar protein and develop a preparation method for a sucrose ester-myofibrillar protein complex solution. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for improving the water solubility of myofibrillar protein in view of the deficiencies of the prior art.

[0006] To achieve the above object, the present invention provides a method for improving the water solubility of myofibrillar protein, which includes:

[0007] S1. Preparation of sucrose ester solution: Dissolve sucrose ester in phosphate buffer solution and vortex to mix evenly to obtain a sucrose ester solution with a concentration of 0.02 - 2.0 mM;

[0008] S2. Preparation of myofibrillar protein solution: Disperse myofibrillar protein into phosphate buffer solution to obtain a myofibrillar protein solution with a concentration of 40 mg / mL;

[0009] S3. Preparation of sucrose ester-myofibrillar protein complex solution: The sucrose ester solution prepared in step S1 and the myofibrillar protein solution prepared in step S2 are mixed at a volume ratio of 1:1, and then subjected to high-pressure homogenization treatment to obtain a sucrose ester-myofibrillar protein complex solution, which improves the dispersion characteristics of myofibrillar protein in the aqueous phase.

[0010] In an embodiment of the present invention, the components of the phosphate buffer solution in steps S1 and S2 include: 10 mM Na2HPO4, 0.3 M NaCl, and the pH of the phosphate buffer solution is 7.0.

[0011] Preferably, the concentration of the sucrose ester solution in step S1 is 0.2 mM.

[0012] In an embodiment of the present invention, after the sucrose ester solution is prepared in step S1, it needs to be hydrated overnight at room temperature (25 °C) before use.

[0013] In an embodiment of the present invention, the myofibrillar protein in step S2 uses porcine myofibrillar protein, and the extraction method of porcine myofibrillar protein includes the following steps:

[0014] (1) Remove the fat and connective tissue attached to the pork tenderloin, and then cut it into small pieces. Then mix it with the pre-cooled phosphate buffer solution, homogenize, filter, and centrifuge. Discard the supernatant and collect the precipitate.

[0015] (2) Mix the precipitate obtained in step (1) with the salt buffer solution, homogenize, filter, and centrifuge. Discard the supernatant and collect the precipitate to obtain porcine myofibrillar protein.

[0016] In an embodiment of the present invention, the volume ratio of the small piece of meat tissue to the pre-cooled phosphate buffer solution in step (1) is 1:4, and the pre-cooled phosphate buffer solution refers to the phosphate buffer solution pre-cooled at 4 °C.

[0017] In an embodiment of the present invention, the components of the phosphate buffer solution in step (1) include 10 mM Na2HPO4, 0.1 M NaCl, 2.0 mM MgCl2, and 1.0 mM EGTA, and the pH of the phosphate buffer solution is 7.0.

[0018] In an embodiment of the present invention, the steps of mixing with the pre-cooled phosphate buffer solution, homogenizing, filtering, centrifuging, discarding the supernatant, and collecting the precipitate in step (1) should be repeated three times.

[0019] In an embodiment of the present invention, the volume ratio of the precipitate to the salt buffer solution in step (2) is 1:4, and the salt buffer solution refers to a NaCl solution with a concentration of 0.1 M.

[0020] In one embodiment of the present invention, in step (2), the precipitate obtained in step (1) is mixed with a salt buffer solution, homogenized, filtered, and then centrifuged. The step of discarding the supernatant and collecting the precipitate should be repeated twice.

[0021] In one embodiment of the present invention, in step S3, high-pressure homogenization is performed three times using a high-pressure homogenizer. The rotation speed of the high-pressure homogenizer is set to 6000 rpm, the time for each homogenization treatment is 40 s, and the temperature is 4°C.

[0022] In one embodiment of the present invention, in step S3, the concentration of sucrose ester in the sucrose ester-myofibrillar protein complex solution is 0.1 mM, and the concentration of myofibrillar protein is 20 mg / mL.

[0023] As a second aspect, the present invention provides a sucrose ester-myofibrillar protein complex solution prepared by the above method.

[0024] As a third aspect, the present invention provides an application of the sucrose ester-myofibrillar protein complex solution in the preparation of food additives.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) In the present invention, sucrose ester is combined with myofibrillar protein. Sucrose ester is non-toxic and biodegradable, and myofibrillar protein is a food-grade raw material. Both are abundant in source and the applicable population is not restricted. Therefore, the application fields and applicable population of the product can be expanded.

[0027] (2) In the present invention, a complex is formed through the interaction between sucrose ester and myofibrillar protein, reducing salt intake and improving the dispersion state of myofibrillar protein under low-salt conditions.

[0028] (3) The complex formed by the interaction between sucrose ester and myofibrillar protein in the present invention endows the complex solution with good stability, making the product develop in the direction of nutrition and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the features and advantages of the present invention will become more obvious.

[0030] Figure 1 It is the particle size distribution diagram in Test Example 1 of the present invention;

[0031] Figure 2 It is the turbidity result diagram in Test Example 2 of the present invention;

[0032] Figure 3 It is the laser confocal microscope result diagram in Test Example 3 of the present invention;

[0033] Figure 4 It is the graph of surface hydrophobicity results in Test Example 4 of the present invention;

[0034] Figure 5 It is the graph of ultraviolet spectrum results in Test Example 5 of the present invention;

[0035] Figure 6 It is the graph of fluorescence spectrum results in Test Example 6 of the present invention;

[0036] Figure 7 It is the graph of molecular docking results in Test Example 7 of the present invention; wherein, Figure 7 in (A) is the surface hydrophobicity model of sucrose ester and myosin (the main protein in myofibrillar protein), Figure 7 in (B) is the docking model of the binding interface between sucrose ester and myosin, Figure 7 in (C) is the interaction force model of sucrose ester and myosin. Detailed implementation manners

[0037] The present invention provides a method for improving the water solubility of myofibrillar protein, comprising the following steps:

[0038] S1. Preparation of sucrose ester solution: Dissolve sucrose ester in phosphate buffer solution, and vortex and mix evenly to obtain a sucrose ester solution;

[0039] S2. Preparation of porcine myofibrillar protein solution: Disperse porcine myofibrillar protein into phosphate buffer solution to obtain a myofibrillar protein solution;

[0040] S3. Preparation of sucrose ester-myofibrillar protein complex solution: After mixing the sucrose ester solution prepared in step (1) with the myofibrillar protein solution prepared in step (2), perform high-pressure homogenization treatment to obtain a complex solution;

[0041] In the present invention, the components of the phosphate buffer solution in steps S1 and S2 include: 10 mM Na2HPO4, 0.3 M NaCl, and the pH of the phosphate buffer solution is 7.0.

[0042] In the present invention, after the sucrose ester solution is prepared in step S1, it needs to be hydrated overnight at room temperature (25 °C) before use.

[0043] In the present invention, the extraction method of the porcine myofibrillar protein in step S2 includes the following steps:

[0044] (1) Remove the fat and connective tissues attached to the pork tenderloin, and then cut it into small pieces. Chop the meat pieces in a tissue homogenizer with four times the volume of phosphate buffer solution, homogenize the homogenate at 4000 rpm for 15 minutes at 4 °C, and discard the supernatant. Then resuspend the precipitate in four times the volume of the same phosphate buffer solution and centrifuge again under the same conditions. Repeat this process three times.

[0045] (2) Mix the precipitate obtained in step (1) with the salt buffer solution, homogenize, filter and then centrifuge, discard the supernatant, collect the precipitate, and repeat this process twice.

[0046] (3) Resuspend the precipitate obtained in step (2) in four times the volume of phosphate buffer solution and filter through four layers of coarse cotton cloth. Centrifuge the filtrate under the above conditions, discard the supernatant, and collect the precipitate to obtain myofibrillar protein.

[0047] In one embodiment of the present invention, the components of the phosphate buffer solution in step (1) include 10 mM Na2HPO4, 0.1 M NaCl, 2.0 mM MgCl2 and 1.0 mM EGTA, and the pH of the phosphate buffer solution is 7.0.

[0048] In the present invention, the salt buffer solution in step (2) refers to a NaCl solution with a concentration of 0.1 M.

[0049] In the present invention, the components of the phosphate buffer solution in step (3) include 10 mM Na2HPO4, and the pH of the phosphate buffer solution is 6.0.

[0050] In the present invention, in step S2, the concentration of porcine myofibrillar protein in the myofibrillar protein solution is 40 mg / mL.

[0051] In the present invention, in step S3, high-pressure homogenization is carried out three times using a high-pressure homogenizer. Set the rotation speed of the high-speed homogenizer to 6000 rpm, the time for each homogenization treatment to be 40 s, and the temperature to be 4 °C.

[0052] In the present invention, in step S3, the sucrose ester-myofibrillar protein complex solution is mixed with the sucrose ester solution and the myofibrillar protein solution at a volume ratio of 1:1.

[0053] In one embodiment of the present invention, in step S3, the concentration of myofibrillar protein in the sucrose ester-myofibrillar protein complex solution is 20 mg / mL.

[0054] Example 1

[0055] The concentration of the sucrose ester solution in step S1 is 0.02 mM.

[0056] In step S3, the concentration of sucrose ester in the sucrose ester-myofibrillar protein complex solution is 0.01 mM.

[0057] Example 2

[0058] The concentration of the sucrose ester solution in step S1 is 0.2 mM.

[0059] In step S3, the concentration of sucrose ester in the sucrose ester-myofibrillar protein complex solution is 0.1 mM.

[0060] Example 3

[0061] The concentration of the sucrose ester solution in step S1 is 2.0 mM.

[0062] In step S3, the concentration of sucrose ester in the sucrose ester-myofibrillar protein complex solution is 1.0 mM.

[0063] Comparative Example 1

[0064] The difference between the embodiment and the method is that it only includes steps 2 to 3 but not step 1.

[0065] The composites prepared in the above examples and comparative examples were subjected to tests 1 to 7.

[0066] Test Example 1:

[0067] The volume average particle size (D [4.3] The refractive index of the material and medium was set to 1.472 and 1.333, respectively. The sample was diluted in the instrument's built-in sample cup until the obscuration was between 2% and 20%. Particle size was determined using the automatic measurement mode.

[0068] Particle size can intuitively indicate the size of particles in the sample and, to a certain extent, characterize whether protein aggregation has occurred. Figure 1 As shown in Table 1, a clear bimodal phenomenon was observed in Comparative Example 1 (the main peak and the secondary peak appeared at about 32 μm and 200 μm, respectively), indicating that the myofibrillar protein in Comparative Example 1 was unevenly distributed and a small amount of large aggregates existed. With the addition of sucrose ester, the particle size of the samples of all examples was smaller than that of the comparative example, among which the particle size distribution of Example 2 was the most uniform. [4,3] It can be seen that as the concentration of sucrose ester increases, the particle size values of the samples of the examples show a trend of first decreasing and then increasing, wherein the particle size of Example 2 reaches the minimum value.

[0069] Table 1: Sample volume average particle size measurement results

[0070] Group <![CDATA[D [4.3] (μm)]]> Comparative Example 1 <![CDATA[32.97±0.47 a > Example 1 <![CDATA[24.75±0.03 b > Example 2 <![CDATA[23.97±0.24 c > Example 3 <![CDATA[25.44±0.03 b >

[0071] Test Example 2:

[0072] The turbidity of the samples was measured using a UV-visible spectrophotometer. Each sample was diluted to 1.0 mg / mL (based on protein concentration) with phosphate buffer, and then the absorbance was recorded at 600 nm, and the result was converted to a turbidity value.

[0073] Turbidity is usually used to analyze the dispersion characteristics of proteins in the aqueous phase. As Figure 2 shown, compared with Comparative Example 1, the turbidity of the samples in all examples decreased significantly, and the turbidity of Example 2 was the lowest, indicating that the interaction between sucrose esters and myofibrillar proteins inhibited the self-aggregation of myofibrillar proteins in the aqueous phase, and the inhibitory effect of Example 2 was the most obvious.

[0074] Test Example 3:

[0075] The microscopic morphology of the samples was observed using a laser confocal scanning microscope. 1.0 mL of the sample was mixed evenly with 25 μL of Nile blue, incubated in the dark at 25 °C for 30 minutes after uniform staining, and then the stained sample was placed on a concave microscope slide with a cover glass and observed using a 20× objective lens under 633 nm laser excitation.

[0076] The laser confocal scanning images of all samples are as Figure 3 shown. The microscopic images can more intuitively observe the aggregation phenomenon of myofibrillar proteins. In Comparative Example 1, the presence of some large particles can be seen, while the disappearance of large particles can be observed in all three examples with the addition of sucrose esters, and the distribution of myofibrillar proteins is more uniform, especially in Example 2 where the distribution of myofibrillar proteins is the most homogeneous.

[0077] Test Example 4:

[0078] The surface hydrophobicity of the samples was measured. 2.0 mL of the sample (5.0 mg / mL) was mixed with 0.4 mL of bromophenol blue solution (1.0 mg / mL) and incubated at room temperature for 10 minutes. After centrifugation at 4000×g for 15 minutes, the absorbance of the supernatant was measured at 595 nm and recorded as A sample. Phosphate buffer was used as a blank control and recorded as A blank. The surface hydrophobicity was expressed as the bromophenol blue (BPB) binding amount and calculated by the following formula: BPB binding amount (μg) = 200 × (A blank - A sample) / A blank.

[0079] Changes in surface hydrophobicity expose or bury the internal hydrophobic agent groups of surface proteins, thus effectively reflecting changes in protein conformation. As Figure 4As shown in the figures, the surface hydrophobicity of the embodiments is higher than that of comparative example 1, which indicates that the structure of myofibrillar protein has changed after the addition of sucrose ester. In Example 1, when a lower concentration of sucrose ester is added, the sucrose ester molecules can freely bind to the hydrophobic patches exposed on the surface of the myofibrillar protein, resulting in a slight increase in the surface hydrophobicity. In Example 2, as the concentration of sucrose ester increases, the sucrose ester molecules form micelles, which can wrap some myofibrillar proteins in the micelles, thereby slightly reducing the surface hydrophobicity value. In Example 3, the concentration of sucrose ester continues to increase until saturation is reached in the aqueous phase. The further increase in the concentration of sucrose ester causes the monomers and micelles to work closely together, promotes the unfolding of the protein structure, exposes more hydrophobic groups, and then causes the surface hydrophobicity to show an upward trend.

[0080] Test Example 5:

[0081] Interactions within the samples were scanned using a UV-visible spectrophotometer, with each sample adjusted to 0.5 mg / mL (based on protein concentration) with phosphate buffer, and scanned from 230 nm to 320 nm at a scan rate of 10 nm / s.

[0082] Ultraviolet absorption spectra mainly reflect the absorption characteristics of specific amino acid residues in proteins to ultraviolet light, and can also indirectly reveal the effects of small molecule-protein interactions on protein structure and function. Figure 5 As shown, the absorbance of myofibrillar proteins in all Examples was higher than that in Comparative Example 1. Furthermore, the absorbance of myofibrillar proteins in the Examples increased with increasing sucrose ester concentration, accompanied by a slight red shift. This increase in absorbance indicates that sucrose esters interact with the aromatic amino acid residues of myofibrillar proteins, thereby altering the UV absorption spectrum of the protein's chromophores. The red shift reflects changes in the microenvironment of these residues, with the most pronounced interaction between sucrose esters and the aromatic amino acid residues of myofibrillar proteins in Example 2.

[0083] Test Example 6:

[0084] Changes in the tertiary structure of myofibrillar proteins in the samples were measured using a fluorescence spectrophotometer. Each sample was diluted to 0.5 mg / mL (based on protein concentration) with phosphate buffer. Spectra were recorded with an excitation wavelength of 280 nm and emission wavelengths between 300 and 500 nm. The slit width was set to 5 nm and the scan speed was set to 1200 nm / min.

[0085] By studying the intrinsic fluorescence spectrum of the samples, the changes in the tertiary structure of myofibrillar proteins were further revealed. Figure 6As shown, the fluorescence intensity of the samples in all examples was lower than that of Comparative Example 1. The decrease in fluorescence intensity indicates that the interaction between sucrose esters and myofibrillar proteins may occur near the hydrophobic regions around tryptophan and tyrosine residues, and the interaction between sucrose esters and myofibrillar proteins was the strongest in Example 2.

[0086] Test Example 7:

[0087] Molecular docking was performed between sucrose esters and myosin (the main protein in myofibrillar proteins). First, the ligands present in the obtained myosin model were removed, and then water molecules were removed from the receptor protein and ligand structures. The missing hydrogen atoms were added to the myosin model using the AutoDock tool. Since the active site of the protein was unknown, the size of the search box was set to include the entire protein. A blind docking procedure was carried out with a grid size of 106×126×126 along the X, Y, and Z axes, and 50 docking poses were generated. The pose with the lowest binding energy was selected for further analysis. Finally, a visual analysis of the docking results was performed.

[0088] Molecular docking simulation is a visualization tool used to reveal the binding affinity, sites, and energies between proteins and small molecules. The molecular docking results showed that sucrose esters successfully docked with myosin, and the binding free energy was -5.9 KJ / mol. As shown in (A) of Figure 7 , the sucrose ester molecule was located inside the hydrophobic pocket of myosin, and hydrophobic interactions were formed between carbon atoms (C 17-21 , C 24-26 , and C 29，30 ) and residues (Met-93, Glu-89, Ile-88, Tyr-85, Lys-84, and Arg-24). As shown in (B) of Figure 7 , three hydrogen bonds were found between sucrose esters and Gln-149 and Glu-778 residues, with bond lengths of 2.3, 2.8, and As shown in (C) of Figure 7 , van der Waals forces were found between sucrose esters and residues Arg-18, Ala-151, Tyr-118, Pro-152, Asp-90, Gly-774, Gly-771, Met-93, Glu-89, Tyr-85, Lys-84, Gln-28, Lys-19, and Arg-24. The results showed that the main interactions between sucrose esters and myofibrillar proteins were hydrophobic interactions and van der Waals force interactions, and the dispersion characteristics of myofibrillar proteins in the aqueous phase were improved through these interactions.

[0089] It can be seen from the above experiments that: by using the method of the embodiment of the present invention to prepare the sucrose ester-myofibrillar protein complex solution, after high-pressure homogenization treatment, the tertiary structure of myofibrillar protein is changed, and the dispersion performance of myofibrillar protein in aqueous solution is improved. The surfactant sucrose ester used is non-toxic and biodegradable, realizing pure green preparation. The results show that adding sucrose ester helps to improve the dispersion characteristics of myofibrillar protein solution, which has practical significance for promoting the industrial production of meat protein foods.

Claims

1. A method for improving the water solubility of myofibrillar proteins, characterized in that, The method comprises the following steps: Dissolve the sucrose ester in phosphate buffer solution and mix well by vortexing to obtain a sucrose ester solution with a concentration of 0.02 - 2.0 mM; Disperse myofibrillar protein into phosphate buffer solution to obtain a myofibrillar protein solution with a concentration of 40 mg / mL; After mixing the sucrose ester solution and the myofibrillar protein solution at a volume ratio of 1:1, perform high-pressure homogenization treatment to obtain a sucrose ester-myofibrillar protein complex solution.

2. The method according to claim 1, characterized in that, The components of the phosphate buffer solution include: 10 mM Na2HPO4, 0.3 M NaCl, and the pH of the phosphate buffer solution is 7.

0.

3. The method according to claim 1, characterized in that, The concentration of the sucrose ester solution is 0.2 mM.

4. The method according to claim 1, characterized in that The method further includes: after obtaining the sucrose ester solution, hydrate it overnight at room temperature before use.

5. The method according to claim 1, characterized in that, The myofibrillar protein includes porcine myofibrillar protein, and the extraction method of the porcine myofibrillar protein includes the following steps: Remove the fat and connective tissue attached to the pork loin, cut it into small pieces, mix it with the phosphate buffer solution pre-cooled at 4°C at a volume ratio of 1:4, homogenize, filter, and then centrifuge. Discard the supernatant and collect the precipitate; Mix the obtained precipitate with the salt buffer solution at a volume ratio of 1:4, homogenize, filter, and then centrifuge. Discard the supernatant and collect the precipitate to obtain porcine myofibrillar protein.

6. The method according to claim 5, characterized in that, The components of the phosphate buffer solution include 10 mM Na2HPO4, 0.1 M NaCl, 2.0 mM MgCl2, and 1.0 mM EGTA, and the pH of the phosphate buffer solution is 7.0; the salt buffer solution is a NaCl solution with a concentration of 0.1 M.

7. The method according to claim 1, characterized in that, The specific high-pressure homogenization treatment is as follows: use a high-pressure homogenizer for three homogenization treatments, set the rotation speed of the high-pressure homogenizer at 6000 rpm, the time for each homogenization treatment is 40 s, and the temperature is 4°C.

8. A sucrose ester-myofibrillar protein complex solution prepared by the method according to any one of claims 1 - 7.

9. Use of the sucrose ester-myofibrillar protein complex solution according to claim 8 in the preparation of food additives.

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