阅读说明：本技术 一种贻贝肌原纤维蛋白的改性方法 (Method for modifying mussel myofibrillar protein ) 是由 于翠平 孙爽 邹赫楠 闫慧佳 李思慧 张秀敏 于 2021-09-17 设计创作，主要内容包括：本发明提供了一种贻贝肌原纤维蛋白的改性方法,包括使用提取缓冲液溶解贻贝组织匀浆液,搅拌后离心得肌原纤维蛋白沉淀；用盐溶液洗涤；用磷酸盐缓冲液溶解,用酸溶液调节pH,获得1％(w/v)的贻贝肌原纤维蛋白分散液,再对其进行超声波改性处理。本发明方法提高表面疏水性、游离巯基含量、zeta电位绝对值,增大溶解度,提高乳液稳定性；在600W超声波处理下游离巯基含量增加到17.3μmol/g,zeta电位绝对值增加至79.9mV,溶解度增大到33.4％,起泡活性和起泡稳定性分别增加到77.2％和68.0％；本发明显著改变了贻贝肌原纤维蛋白的结构特性,改善了肌原纤维蛋白的功能特性,安全、环保、低成本。(The invention provides a method for modifying mussel myofibrillar protein, which comprises the steps of dissolving mussel tissue homogenate by using an extraction buffer solution, stirring and centrifuging to obtain myofibrillar protein precipitate; washing with a salt solution; dissolving with phosphate buffer solution, adjusting pH with acid solution to obtain 1% (w/v) mussel myofibrillar protein dispersion, and performing ultrasonic modification treatment. The method improves the surface hydrophobicity, the free sulfydryl content and the zeta potential absolute value, increases the solubility and improves the emulsion stability; the free thiol content increased to 17.3. mu. mol/g, the zeta potential absolute value increased to 79.9mV, the solubility increased to 33.4%, and the foaming activity and foaming stability increased to 77.2% and 68.0%, respectively, after 600W ultrasonic treatment; the invention obviously changes the structural characteristics of the mussel myofibrillar protein, improves the functional characteristics of the myofibrillar protein, and has the advantages of safety, environmental protection and low cost.)

1. A method for modifying mussel myofibrillar protein is characterized by comprising the following steps:

s1, crushing mussels, mixing with the extraction buffer solution, uniformly stirring to obtain an extracting solution, and centrifuging to obtain a precipitate;

s2, washing the precipitate obtained in the step S1 with a NaCl solution, and centrifuging to obtain the precipitate; dissolving with phosphate buffer solution, and adjusting pH with hydrochloric acid solution to obtain mussel myofibrillar protein dispersion;

and S3, performing ultrasonic treatment.

2. The method for modifying mussel myofibrillar protein according to claim 1, comprising the steps of:

s1, crushing mussels, mixing with the extraction buffer solution according to the proportion of 1:4w/v, uniformly stirring to obtain an extracting solution, and centrifuging to obtain a precipitate;

s2, washing the precipitate obtained in the step S1 by 0.1M NaCl solution according to the proportion of 1:4v/v, and centrifuging to obtain the precipitate; dissolving with 0.05mol/L phosphate buffer solution with pH of 7.0, and adjusting pH to 6.0 with 0.1mol/L hydrochloric acid solution to obtain 1% (w/v) mussel myofibrillar protein dispersion;

s3, carrying out ultrasonic treatment under the conditions of ultrasonic power of 600W and 20kHz and temperature of 4 ℃;

the ultrasonic treatment method comprises the following steps: the ultrasonic working time is 2s, the rest time is 2s, and the duration is 16 min.

3. The method for modifying mussel myofibrillar protein according to any one of claims 1 to 2, wherein the extraction buffer in step S1 comprises a combination of sodium, magnesium and phosphate solutions.

4. The method for modifying myofibrillar proteins of mussels according to claim 3, wherein the extraction buffer comprises 0.1mol/L NaCl, 2mmol/LMgCl of₂1mmol/L EDTA2Na, 6.1mmol/L Na₂HPO₄And 3.9mmol/L NaH₂PO₄The pH was adjusted to 7.0.

5. The method for modifying mussel myofibrillar protein according to any one of claims 1 to 2, wherein the phosphate solution in step S2 comprises a combination of a disodium hydrogen phosphate solution and a sodium dihydrogen phosphate solution.

6. The method for modifying myofibrillar proteins of mussels according to claim 5, wherein the phosphate buffer formulation comprises NaH at a concentration of 0.05mol/L₂PO₄And Na in a concentration of 0.05mol/L₂HPO₄The pH of the mixed solution prepared according to the ratio of 2:3v/v is 7.0.

7. The method for modifying mussel myofibrillar protein according to any one of claims 1 to 2, wherein the centrifugation conditions in steps S1, S2 comprise centrifugation at 5000g at 4 ℃ for 15 min.

8. The method for modifying mussel myofibrillar protein according to any one of claims 1 to 2, wherein the ultrasonication in step S3 is carried out 3 times.

9. The method for modifying mussel myofibrillar protein according to any one of claims 1 to 2, wherein the stirring in step S1 is carried out by stirring at 10000rpm for 30S to obtain the extract and then stirring for 2 h.

Technical Field

The invention relates to the field of food processing, in particular to a method for modifying mussel myofibrillar protein.

Background

Mussels, commonly known as sea red, are common edible bivalve mollusks living in the ocean and are produced in Liaoning, Jiangsu, Zhejiang, Fujian, Shandong, Guangdong, Guangxi and other provinces of China, wherein the yield of Shandong, Zhejiang and Fujian is the highest. Mussels are named as eggs in the sea because of abundant nutrients such as protein. Mussels contain 8 essential amino acids such as valine, leucine and the like, B vitamins and a plurality of trace elements, the content of the essential amino acids is higher than that of plant protein and a plurality of animal proteins, and the mussels can be used as a high-quality raw material for researching the characteristics of proteins. Mussels are abundant in variety, and the annual yield is over 87 ten thousand tons at present. Through appropriate modification treatment, the structure and functional characteristics of the mussel protein are improved, and the utilization value of the high-quality marine resource can be improved.

Myofibrillar proteins are the largest constituent of proteins in muscle tissue, accounting for 50% -55% of muscle proteins, and are the major subject of food protein processing studies. The myofibrillar protein can be used as an emulsifier, and has hydrophilicity and lipophilicity, so that the interfacial tension between oil and water can be reduced, and the stability of the emulsion is enhanced; the gel network can be formed, the gel network has water retention capacity, and the research on the structure and the functional characteristics of the myofibrillar protein occupies an important position in the research on the aquatic protein, so that the physicochemical functional characteristics of the myofibrillar protein are widely concerned, and the proper modification method has important significance for the application of the myofibrillar protein in the field of food. In recent years, researchers of various countries have been increasing on the research of myofibrillar proteins, and the research of appropriate modification treatment methods has become a problem of great attention in rational application in the food industry. Therefore, the invention changes the structure of myofibrillar protein by a certain method, and further improves the functional characteristics of myofibrillar protein.

The processing process may result in denaturation of the protein, resulting in changes in the functional properties of the protein. With the increasing research on protein properties, how to modify proteins becomes a hot topic. In recent years, non-thermal treatment techniques are gradually replacing conventional thermal treatment techniques and are widely used in studies of protein properties. The ultrasonic wave is used as a novel safe, environment-friendly and low-cost non-heat treatment technology, improves the physical and chemical functional characteristics of protein on the premise of basically not influencing the nutritional quality of food, and has wide application prospect. The ultrasonic treatment is applied to the modification research of the protein, and the functional characteristics of the protein are changed by a physical or chemical method based on the strong physical force such as shearing force, shock wave, turbulent flow and the like generated by the cavitation. The ultrasonic treatment is reported to change the structure and function of various proteins including milk protein, vegetable protein, chicken protein and the like, and the research on shellfish protein is relatively few, wherein mussel protein, especially myofibrillar protein is rarely reported.

Disclosure of Invention

The invention provides a method for modifying myofibrillar protein of mussel, which aims to solve the problem of low protein content in the myofibrillar protein of mussel

In order to achieve the above object, the present invention provides a method for modifying mussel myofibrillar protein, comprising the steps of:

s1, crushing mussels, mixing with the extraction buffer solution, uniformly stirring to obtain an extracting solution, and centrifuging to obtain a precipitate;

s2, washing the precipitate obtained in the step S1 with a NaCl solution, and centrifuging to obtain the precipitate; dissolving with phosphate buffer solution, and adjusting pH with hydrochloric acid solution to obtain mussel myofibrillar protein dispersion;

and S3, performing ultrasonic treatment.

In a preferred mode, the method for modifying the mussel myofibrillar protein comprises the following steps:

s1, crushing mussels, mixing with the extraction buffer solution according to the proportion of 1:4w/v, uniformly stirring to obtain an extracting solution, and centrifuging to obtain a precipitate;

s2, washing the precipitate obtained in the step S1 by 0.1M NaCl solution according to the proportion of 1:4v/v, and centrifuging to obtain the precipitate; dissolving with 0.05mol/L phosphate buffer solution with pH of 7.0, and adjusting pH to 6.0 with 0.1mol/L hydrochloric acid solution to obtain 1% (w/v) mussel myofibrillar protein dispersion;

s3, carrying out ultrasonic treatment under the conditions of ultrasonic power of 600W and 20kHz and temperature of 4 ℃;

the ultrasonic treatment method comprises the following steps: the ultrasonic working time is 2s, the rest time is 2s, and the duration is 16 min.

Preferably, the extraction buffer in step S1 includes a combination of sodium salt, magnesium salt, and phosphate solution.

Preferably, the extraction buffer comprises 0.1mol/L NaCl and 2mmol/L MgCl₂1mmol/L EDTA2Na, 6.1mmol/L Na₂HPO₄And 3.9mmol/L NaH₂PO₄The pH was adjusted to 7.0.

Preferably, the phosphate solution in step S2 includes a combination of a disodium hydrogen phosphate solution and a sodium dihydrogen phosphate solution.

Preferably, the phosphate buffer solution formula comprises NaH with the concentration of 0.05mol/L₂PO₄And Na in a concentration of 0.05mol/L₂HPO₄The pH of the mixed solution prepared according to the ratio of 2:3v/v is 7.0.

Preferably, the centrifugation conditions in steps S1, S2 comprise centrifugation at 5000g, 4 ℃ for 15 min.

Preferably, the ultrasonic treatment is performed 3 times in step S3.

The method has the beneficial effects that:

the raw material used by the invention is the common raw material of the common mussel in food industry, the content of protein is rich, but the heat stability and the chemical stability of the mussel protein are lower than those of plant protein and vertebrate protein, the functional characteristics of the protein are influenced, and the utilization and the development of the mussel protein are restricted. At present, the development and utilization of mussel protein mostly stay in the experimental stage, and large-scale production and application are not yet carried out, so that a large amount of experimental research assistance is needed for the efficient utilization of the marine resources. The ultrasonic treatment method used in the modification treatment of the mussel myofibrillar protein belongs to a novel non-heat treatment method, has the advantages of safety, environmental protection and low cost, can discuss the potential value of the mussel myofibrillar protein as an emulsifier and a foaming agent through the ultrasonic treatment method, and provides a theoretical basis for improving the functional characteristics of the mussel myofibrillar protein.

The structural characteristics (such as surface hydrophobicity and free sulfydryl content) of the mussel myofibrillar protein are changed by the method, the functional characteristics (such as potential, solubility, foamability and emulsion stability) of the mussel myofibrillar protein can be improved, and the method is safe, environment-friendly and low in cost; when the ultrasonic treatment power reaches 600W, the structural characteristics and the functional characteristics of the mussel myofibrillar protein can be changed, the content of free sulfydryl is increased to 17.3 mu mol/g from 6.0 mu mol/g, the absolute value of zeta potential is increased to 79.9mV from 56.2mV, the solubility is increased to 33.4% from 11.1%, and the foaming activity and the foaming stability are respectively increased to 77.2% and 68.0% from 28.3% and 21.9%.

Drawings

FIG. 1 is a graphical representation of the effect of power of sonication on the free sulfhydryl content of mussel myofibrillar protein in example 6;

FIG. 2 is a schematic illustration of the effect of power of sonication on the surface hydrophobicity of mussel myofibrillar proteins in example 6;

FIG. 3 is a schematic illustration of the effect of power of sonication on the myofibrillar potential of mussels in example 6;

FIG. 4 is a graphical representation of the effect of power of sonication on mussel myofibrillar protein solubility in example 6;

FIG. 5 is a graphical representation of the effect of power of sonication on mussel myofibrillar protein foamability in example 6;

FIG. 6 is a graphical representation of the effect of power of sonication on the stability of the mussel myofibrillar protein emulsion in example 6.

Detailed Description

For a better understanding of the present invention, the following description will be given with reference to examples 1 to 6.

A method for modifying mussel myofibrillar protein comprises the following specific steps:

mixing mussel with extraction buffer (1:4, w/v), pulverizing, and homogenizing (10000rpm, 30s) to obtain extractive solution, and stirring for 2 hr on magnetic stirrer. Centrifuging the extractive solution twice (5000g, 15min, 4 deg.C) to obtain myofibrillar protein precipitate. The resulting myofibrillar protein precipitate was washed with 0.1M NaCl solution at a ratio of 1:4v/v and centrifuged three times under the same conditions. Before the final centrifugation, the suspension was filtered through three layers of gauze to obtain a precipitate, the precipitate was dissolved with phosphate buffer (0.05mol/L, pH 7.0), and the pH was adjusted to 6.0 with 0.1mol/L hydrochloric acid solution. To obtain 1% (w/v) mussel myofibrillar protein dispersion, which is then subjected to ultrasonic modification treatment.

Ultrasonic treatment: the mussel myofibrillar proteins were sonicated using a Scientz-IID ultrasound probe processor. A sample of myofibrillar protein, 100mL, was taken and placed in a 150mL conical flask and immersed in a beaker of ice water with the probe extending approximately 20mm below the surface of the liquid. The sarcoplasmic protein sample is treated by ultrasonic treatment under the condition of 20kHz and 16min, and the ultrasonic power is 600W. The temperature measuring probe is immersed in the solution to measure the temperature of the sample, and the temperature of the sample is controlled to be about 4 ℃ by using an ice water bath. (ultrasound working time 2s, rest time 2s, duration 16 min).

The ultrasonic treatment power is preferably 600W.

The extraction buffer mainly comprises a combination of sodium salt, magnesium salt and phosphate solution.

The phosphate solution includes a combination of a disodium hydrogen phosphate solution, a sodium dihydrogen phosphate solution.

The extraction buffer solution formula is 0.1mol/L NaCl and 2mmol/L MgCl₂1mmol/L EDTA2Na, 6.1mmol/L Na₂HPO₄And 3.9mmol/L NaH₂PO₄，pH＝7.0。

The formulation of the phosphate buffer solution is 39mL of NaH with the concentration of 0.05mol/L₂PO₄61mL of Na having a concentration of 0.05mol/L₂HPO₄，pH＝7.0。

Example 1

Extraction of mussel myofibrillar protein: removing shell and meat of fresh mussel purchased from local market, removing byssus, pulverizing 200g thawed mussel with a blender, dissolving in 800mL extraction buffer solution, continuously stirring for 2h, and centrifuging the obtained dispersion at 5000g for 15min for 2 times. The resulting myofibrillar protein precipitate was washed with 0.1mol/L sodium chloride solution, centrifuged at 5000g for 15min and 3 times. The resulting precipitate was dissolved using a phosphate buffer (0.05mol/L, pH 7.0) and adjusted to pH 6.0 with 0.1mol/L hydrochloric acid solution. The final concentration of 1% (w/v) of the dispersion was stored at 4 ℃.

The extraction buffer solution formula is 0.1mol/L NaCl and 2mmol/L MgCl₂1mmol/L EDTA2Na, 6.1mmol/L Na₂HPO₄And 3.9mmol/L NaH₂PO₄，pH＝7.0。

The formulation of the phosphate buffer solution is 39mL of NaH with the concentration of 0.05mol/L₂PO₄61mL of Na having a concentration of 0.05mol/L₂HPO₄，pH＝7.0。

Ultrasonic treatment: the mussel myofibrillar proteins were sonicated using a Scientz-IID ultrasound probe processor. A sample of myofibrillar protein, 100mL, was taken and placed in a 150mL conical flask and immersed in a beaker of ice water with the probe extending approximately 20mm below the surface of the liquid. And (3) carrying out ultrasonic treatment on the sarcoplasmic protein sample at 20kHz for 16min, wherein the ultrasonic power is 0W. The temperature of the sample is measured by immersing the temperature measuring probe in the solution, and the temperature of the sample is controlled to be about 4 ℃ by using an ice water bath (ultrasonic working time is 2s, and rest time is 2 s). The dispersion after ultrasonic treatment was used as a control group for subsequent experiments.

Example 2

Extraction of mussel myofibrillar protein: removing shell and meat of fresh mussel purchased from local market, removing byssus, pulverizing 200g thawed mussel with a blender, dissolving in 800mL extraction buffer solution, continuously stirring for 2h, and centrifuging the obtained dispersion at 5000g for 15min for 2 times. The resulting myofibrillar protein precipitate was washed with 0.1mol/L sodium chloride solution, centrifuged at 5000g for 15min and 3 times. The resulting precipitate was dissolved using a phosphate buffer (0.05mol/L, pH 7.0) and adjusted to pH 6.0 with 0.1mol/L hydrochloric acid solution. The final concentration of 1% (w/v) of the dispersion was stored at 4 ℃.

The extraction buffer solution formula comprises 0.1mol/L NaCl and 2mmol/L MgCl₂1mmol/L EDTA2Na, 6.1mmol/L Na₂HPO₄And 3.9mmol/L NaH₂PO₄Adjusting pH7.0。

The formulation of the phosphate buffer solution is 39mL of NaH with the concentration of 0.05mol/L₂PO₄61mL of Na having a concentration of 0.05mol/L₂HPO₄，pH＝7.0。

Ultrasonic treatment: the mussel myofibrillar proteins were sonicated using a Scientz-IID ultrasound probe processor. A sample of myofibrillar protein, 100mL, was taken and placed in a 150mL conical flask and immersed in a beaker of ice water with the probe extending approximately 20mm below the surface of the liquid. The sarcoplasmic protein sample is treated by ultrasonic treatment under the condition of 20kHz and 16min, and the ultrasonic power is 150W. The temperature measuring probe is immersed in the solution to measure the temperature of the sample, and the temperature of the sample is controlled to be about 4 ℃ by using an ice water bath. (ultrasonic working time 2s, rest time 2s) the dispersion after ultrasonic treatment was used as a control for the subsequent experiments.

Example 3

Extraction of mussel myofibrillar protein: removing shell and meat of fresh mussel purchased from local market, removing byssus, pulverizing 200g thawed mussel with a blender, dissolving in 800mL extraction buffer solution, continuously stirring for 2h, and centrifuging the obtained dispersion at 5000g for 15min for 2 times. The resulting myofibrillar protein precipitate was washed with 0.1mol/L sodium chloride solution, centrifuged at 5000g for 15min and 3 times. The resulting precipitate was dissolved using a phosphate buffer (0.05mol/L, pH 7.0) and adjusted to pH 6.0 with 0.1mol/L hydrochloric acid solution. The final concentration of 1% (w/v) of the dispersion was stored at 4 ℃.

The extraction buffer solution formula comprises 0.1mol/L NaCl and 2mmol/L MgCl₂1mmol/L EDTA2Na, 6.1mmol/L Na₂HPO₄And 3.9mmol/L NaH₂PO₄The pH was adjusted to 7.0.

The formulation of the phosphate buffer solution is 39mL of NaH with the concentration of 0.05mol/L₂PO₄61mL of Na having a concentration of 0.05mol/L₂HPO₄，pH＝7.0。

Ultrasonic treatment: the mussel myofibrillar proteins were sonicated using a Scientz-IID ultrasound probe processor. A sample of myofibrillar protein, 100mL, was taken and placed in a 150mL conical flask and immersed in a beaker of ice water with the probe extending approximately 20mm below the surface of the liquid. The sarcoplasmic protein sample is treated by ultrasonic treatment under the condition of 20kHz and 16min, and the ultrasonic power is 300W. The temperature measuring probe is immersed in the solution to measure the temperature of the sample, and the temperature of the sample is controlled to be about 4 ℃ by using an ice water bath. (ultrasound working time 2s, rest time 2 s). The dispersion after ultrasonic treatment was used as a control group for subsequent experiments.

Example 4

Extraction of mussel myofibrillar protein: removing shell and meat of fresh mussel purchased from local market, removing byssus, pulverizing 200g thawed mussel with a blender, dissolving in 800mL extraction buffer solution, continuously stirring for 2h, and centrifuging the obtained dispersion at 5000g for 15min for 2 times. The resulting myofibrillar protein precipitate was washed with 0.1mol/L sodium chloride solution, centrifuged at 5000g for 15min and 3 times. The resulting precipitate was dissolved using a phosphate buffer (0.05mol/L, pH 7.0) and adjusted to pH 6.0 with 0.1mol/L hydrochloric acid solution. The final concentration of 1% (w/v) of the dispersion was stored at 4 ℃.

The extraction buffer formula is 0.1mol/L NaCl and 2mmol/L MgCl₂、1mmol/L EDTA2Na，6.1mmol/LNa₂HPO₄And 3.9mmol/LNaH2PO₄，pH＝7.0。

The formulation of the phosphate buffer solution is 39mL of NaH with the concentration of 0.05mol/L₂PO₄61mL of Na having a concentration of 0.05mol/L₂HPO₄，pH＝7.0。

Ultrasonic treatment: the mussel myofibrillar proteins were sonicated using a Scientz-IID ultrasound probe processor. A sample of myofibrillar protein, 100mL, was taken and placed in a 150mL conical flask and immersed in a beaker of ice water with the probe extending approximately 20mm below the surface of the liquid. The sarcoplasmic protein sample is treated by ultrasonic treatment under the condition of 20kHz and 16min, and the ultrasonic power is 450W. The temperature measuring probe is immersed in the solution to measure the temperature of the sample, and the temperature of the sample is controlled to be about 4 ℃ by using an ice water bath. (ultrasound working time 2s, rest time 2 s). The dispersion after ultrasonic treatment was used as a control group for subsequent experiments.

Example 5

Extraction of mussel myofibrillar protein: removing shell and meat of fresh mussel purchased from local market, removing byssus, pulverizing 200g thawed mussel with a blender, dissolving in 800mL extraction buffer solution, continuously stirring for 2h, and centrifuging the obtained dispersion at 5000g for 15min for 2 times. The resulting myofibrillar protein precipitate was washed with 0.1mol/L sodium chloride solution, centrifuged at 5000g for 15min and 3 times. The resulting precipitate was dissolved using a phosphate buffer (0.05mol/L, pH 7.0) and adjusted to pH 6.0 with 0.1mol/L hydrochloric acid solution. The final concentration of 1% (w/v) of the dispersion was stored at 4 ℃.

The extraction buffer solution formula comprises 0.1mol/L NaCl and 2mmol/L MgCl₂EDTA at a concentration of 1mmol/L₂Na with a concentration of 6.1mmol/L₂HPO₄And NaH at a concentration of 3.9mmol/L₂PO₄，pH＝7.0。

The formulation of the phosphate buffer solution is 39mL of NaH with the concentration of 0.05mol/L₂PO₄61mL of Na having a concentration of 0.05mol/L₂HPO₄，pH＝7.0。

Ultrasonic treatment: the mussel myofibrillar proteins were sonicated using a Scientz-IID ultrasound probe processor. A sample of myofibrillar protein, 100mL, was taken and placed in a 150mL conical flask and immersed in a beaker of ice water with the probe extending approximately 20mm below the surface of the liquid. The sarcoplasmic protein sample is treated by ultrasonic treatment under the condition of 20kHz and 16min, and the ultrasonic power is 600W. The temperature measuring probe is immersed in the solution to measure the temperature of the sample, and the temperature of the sample is controlled to be about 4 ℃ by using an ice water bath. (ultrasound working time 2s, rest time 2 s). The dispersion after ultrasonic treatment was used as a control group for subsequent experiments.

Example 6

Performing various representations on the groups of dispersion liquid subjected to ultrasonic treatment obtained in the embodiments 1-5, specifically including the following steps:

s1, determination of free thiol content and surface hydrophobicity

S1.1, determination of free mercapto content

The ultrasonically treated mussel myofibrillar protein dispersions of examples 1-5 were each in Tris-glycine buffer(0.086mol/L Tris, 0.09mol/L glycine, 0.004mol/L EDTA) (pH 8.0) to 3.0 mg/mL. The dispersion was allowed to stand at room temperature for 1h, and 50.0. mu.L of Ellman's reagent (4mg/mL of DTNB (5, 5' -dithiobis-2-nitrobenzoic acid)) was added. Centrifugation was carried out at 10000g for 15min at 4 ℃. The absorbance of the supernatant was measured at 412nm and 1.36X 10 was used⁴M^-1cm^-1The free thiol content was calculated from the extinction coefficient of (g).

The free thiol content is calculated as follows:

wherein 73.53-10⁶/(1.36×10⁴)，1.36×10⁴Is the molar extinction coefficient; a. the₄₁₂Absorbance value at 412 nm; c is the protein concentration of the sample in mg/mL.

As shown in fig. 1, the free thiol content increased significantly after sonication. As the ultrasonic power was increased, the free thiol content increased from 6.0. mu. mol/g. + -. 0.6. mu. mol/g to 17.3. mu. mol/g. + -. 0.7. mu. mol/g, where the free thiol content reached a maximum under the 600W condition of example 5. Cavitation generated by ultrasound destroys disulfide bonds, reducing part of disulfide bonds to sulfhydryl groups, resulting in an increase in free sulfhydryl content.

S1.2 measurement of surface hydrophobicity

The groups of sonicated mussel myofibrillar protein dispersions of examples 1-5 were measured using 8-anilino-1-naphthalenesulfonate (ANS). Groups of sonicated mussel myofibrillar protein dispersions (10mg/mL) were diluted to 1mg/mL in 0.01mol/L PBS (pH 7.2), respectively. 40.0. mu.LANS was mixed with 4.0mL of the sample and left to stand for 3 min. The fluorescence intensity was measured by an F-2700 fluorescence spectrometer (Hitachi, Tokyo, Japan) with an excitation wavelength of 390nm, an emission wavelength of 400-650nm and a slit calibration of 5 nm. The initial slope of a standard curve obtained with the fluorescence intensity of the dilution as the ordinate and the protein concentration as the abscissa indicates the surface hydrophobicity.

Surface hydrophobicity is an important index reflecting the tertiary structure of protein, fig. 2 shows the surface hydrophobicity of mussel myofibrillar protein before and after ultrasonic treatment, and the results show that ultrasonic treatment leads to the significant increase of surface hydrophobicity. The surface hydrophobicity is increased continuously with the increase of the ultrasonic power, and is increased from 133.4 +/-0.7 to 161.5 +/-1.2, and the maximum value is reached under the condition of 600W of the embodiment 5. This phenomenon may be caused by the fact that the sonication spreads the protein molecules, exposing certain hydrophobic groups located inside the molecules, resulting in increased hydrophobicity.

S3 measurement of protein potential

Determination of protein potential: the protein potential of the sonicated mussel myofibrillar protein dispersion (10mg/mL) was measured using an ultrasonic particle size analyzer DT-1202 (Dispersion technologies, USA). The zeta potential (-38mV) was corrected with a standard calibration solution before measurement.

The absolute value of the zeta potential is related to the degree of aggregation of the protein, with higher values indicating more stable protein and lower values indicating that the protein is susceptible to flocculation. As shown in FIG. 3, it was found that the absolute value of zeta potential of Mytilus edulis myofibrillar proteins after ultrasonic treatment was significantly increased from 56.2 mV. + -. 3.5mV to 79.9 mV. + -. 1.2mV, and the absolute value of zeta potential reached the maximum under the conditions of example 5 (600W). As the ultrasonic power increases, the absolute value of the zeta potential increases. Ultrasound enhances the electrostatic repulsion of protein molecules, disrupts protein aggregates, increases surface negative charges, and results in an increase in the absolute value of the zeta potential.

S3 determination of protein solubility

Determination of protein solubility: the sonicated mussel myofibrillar protein dispersion (10mg/mL) was centrifuged at 10000g for 20 min. The protein content was determined using the biuret method. Protein solubility can be expressed as the ratio of the protein content in the supernatant after centrifugation to the protein content in the sample before centrifugation.

The solubility is calculated as follows:

as shown in fig. 4, it can be seen that sonication can significantly increase protein solubility. The solubility increased from 11.1% + -0.3% to 33.4% + -0.5% with increasing ultrasound power, reaching a maximum under the sonication conditions of example 5 (600W), which is associated with a particle size reduction caused by sonication, which increases the particle size, resulting in an enhanced protein-water interaction. In addition, cavitation disrupts hydrophobic interactions between protein molecules and may also lead to increased solubility.

S4 measurement of protein foamability

Determination of protein foamability: the foamability of mussel myofibrillar proteins is expressed in terms of foaming capacity (FA) and Foaming Stability (FS). 20mL of the dispersion (10mg/mL, pH 7.0) was homogenized at high speed (T25, IKA, Germany) at 5,000rpm for 1 minute, the volume was measured using a graduated cylinder, the percentage of volume increase was defined as FA, and the percentage of residual foam volume after 10 minutes of storage was expressed as FS.

FA. The calculation formula of FS is as follows:

wherein V₀And V₁₀The volume of foam at 0min and the volume of residual foam at 10min are indicated, respectively.

The results of measuring the foaming properties of the ultrasonically-treated mussel myofibrillar protein dispersions of examples 1-6 are shown in fig. 5. Figure 5 shows that sonication significantly improved foaming activity and foaming stability, increasing from 28.3% ± 0.8% and 21.9% ± 5.1% to 77.2% ± 2.0% and 68.0% ± 1.5% respectively with increasing sonication power, and reaching a maximum under sonication conditions of example 5 (600W). The foaming property is influenced by the surface hydrophobicity, and the hydrophobic groups are exposed after ultrasonic treatment, so that the air-water interface interaction can be enhanced, and the protein foaming capacity is improved. In addition, the particle size reduction caused by the ultrasound can also increase the protein adsorption rate, which is beneficial to the formation of foam and the stability thereof.

S5 determination of emulsion stability

Preparation of the emulsion: the dispersion (10mg/mL) was mixed with soybean oil (9: 1, v/v) and homogenized at high speed (12000rpm, 1min) using a homogenizer (model T25, IKA Dispersion technologies, Germany). The mixture was subjected to high-pressure homogenization (40MPa, homogenization 3 times) using a homogenizer (PandaPlus 2000, GEA, Italy). To prevent microbial growth, 0.02% sodium azide was added.

Determination of emulsion stability: the emulsion was filled into a 10mL glass sample bottle and stored at room temperature for 28 hours, recording the height of the bottom clear liquid layer (Hs) and the height of the total emulsion (Ht) every 4 h. The calculation formula is as follows:

the results of measuring the emulsion stability of the ultrasonic-treated mussel myofibrillar protein dispersions of examples 1-5 are shown in fig. 6. The milk separation index can be used for representing the stability of the emulsion, and the lower the milk separation index is, the smaller the phase separation degree of the emulsion is, and the better the stability of the emulsion is shown; as can be seen from the figure, the emulsion started to be unstable after 1h of standing, when the creaming index of the non-sonicated emulsion was the highest (3.1%) and increased with the increase of the storage time, indicating that the emulsion was the least stable and decreased in stability after a period of standing. Compared with a control group, the emulsion separation index of the emulsion after ultrasonic treatment is reduced, and the emulsion stability is increased. The reason for the increased stability of the emulsion may be that the ultrasonic treatment promotes adsorption of proteins on the oil-water interface, enhances repulsion between droplets, reduces particle size, and causes a change in protein conformation.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.