阅读说明：本技术 一种接枝改性鱼肉蛋白-糖耦联复合物的制备方法 (Preparation method of graft modified fish protein-sugar coupling compound ) 是由 陈跃文 刘飞建 董秀萍 沈诗珂 胡豪犇 丁致文 于 2021-09-02 设计创作，主要内容包括：本发明公开了一种接枝改性鱼肉蛋白-糖耦联复合物的制备方法,包括,将蛋白溶解于高离子强度缓冲液,经高速剪切后获得均一的肌原纤维蛋白溶液；将肌原纤维蛋白溶液经高压均质预处理使蛋白展开后,其α-螺旋含量提高,糖基化位点增加,然后与氨基葡萄糖在水相中进行湿法糖基化反应,利用大分子拥挤理论稳定天然蛋白质结构,提高糖基化程度；将糖基化反应后的产物进行透析脱糖,并进行真空冷冻干燥,得到本发明的接枝改性蛋白-糖耦联复合物。相比于传统糖基化方式,本发明所阐述的制备方法可显著提高肌原纤维蛋白与糖基供体的接枝度以及在低离子溶剂中的溶解度,其抗氧化能力也得到提升。(The invention discloses a preparation method of a graft modified fish protein-sugar coupled compound, which comprises the steps of dissolving protein in a buffer solution with high ionic strength, and obtaining a uniform myofibrillar protein solution after high-speed shearing; after the myofibrillar protein solution is subjected to high-pressure homogenization pretreatment to expand protein, the alpha-helix content is increased, glycosylation sites are increased, then the myofibrillar protein solution and glucosamine are subjected to wet glycosylation reaction in a water phase, a natural protein structure is stabilized by utilizing a macromolecule crowding theory, and the glycosylation degree is improved; and (3) dialyzing and desugarizing the product after the glycosylation reaction, and carrying out vacuum freeze drying to obtain the graft modified protein-sugar coupled compound. Compared with the traditional glycosylation mode, the preparation method provided by the invention can obviously improve the grafting degree of the myofibrillar protein and the glycosyl donor and the solubility in a low-ion solvent, and the oxidation resistance of the preparation method is also improved.)

1. A preparation method of a graft modified fish protein-sugar coupled compound is characterized by comprising the following steps:

1) dissolving the protein in a phosphate buffer solution to enable the mass concentration of the protein to be 20-40 mg/mL;

2) carrying out high-speed shearing pretreatment on the protein solution obtained in the step 1), and then carrying out secondary high-pressure homogenization treatment;

3) adding a glycosyl donor into the protein solution obtained in the step 2), and reacting for 7-9 hours at the temperature of 35-40 ℃;

4) dialyzing the solution obtained in the step 3) to remove the excessive glycosyl donor;

5) and (4) carrying out vacuum freeze drying on the solution without the glycosyl donor obtained in the step (4) to obtain the target grafted modified protein-carbohydrate coupled compound.

2. The method for preparing a graft-modified fish meat protein-sugar coupled complex according to claim 1, wherein in step 1), the protein is sturgeon myofibrillar protein.

3. The method for preparing the graft modified fish protein-sugar coupled compound according to claim 1, wherein the phosphate buffer solution in step 1) is 0.4-0.8 mol/L.

4. The method for preparing a graft-modified fish protein-sugar coupled complex according to claim 1, wherein the high-speed shearing conditions in step 2) are as follows: the shear rate is 8000-10000 rpm, and the time is 1-2 minutes.

5. The method for preparing the graft-modified fish protein-sugar coupled compound according to claim 1, wherein the homogenizing pressure in step 2) is 400 to 600 bar.

6. The method for preparing a graft-modified fish protein-sugar coupled complex according to claim 1, wherein in step 3), the sugar donor is glucosamine.

7. The preparation method of the graft modified fish protein-sugar coupled compound according to claim 1, wherein in the step 3), the molar ratio of the protein to the sugar-based donor in the protein solution is 1: 1-1: 3.

8. The method for preparing a graft-modified fish protein-sugar coupled complex according to claim 1, wherein the dialysis solution used in the step 4) is phosphate buffered saline.

9. The method for preparing the graft-modified fish protein-sugar coupled compound according to claim 8, wherein the concentration of the phosphate buffer is 0.05-0.2 mol/L.

10. The method for preparing the graft-modified fish protein-sugar coupled complex according to claim 1, wherein the dialysis time in step 4) is 20-24 hours.

Technical Field

The invention relates to the field of modification research of salt-soluble animal protein, and in particular relates to a preparation method of a grafted modified fish protein-sugar coupling compound.

Background

The meat of the Russian sturgeon is delicious in taste and fleshy, the content of crude protein in the meat of the Russian sturgeon accounts for 15.20-17.25% of the muscle, the meat of the Russian sturgeon mainly contains myofibrillar protein, 18 amino acids are contained, the highest amino acid content is glutamic acid, lysine, leucine and aspartic acid, 8 essential amino acids of human bodies account for 39.87-43.09% of the total amino acid content, the content of the essential amino acids is higher than the FAO/WHO standard (35.38%), 4 amino acids with flavor are contained, and the meat of the Russian sturgeon has a better biological value. According to the method, the culture amount of sturgeons in China accounts for 85% of the global culture amount, the sturgeon meat resources are rich, but primary processed products are mainly used, so that how to fully utilize the sturgeon meat and how to realize the intensive processing of the myofibrillar proteins of the sturgeons becomes a research hotspot for improving the application value of the sturgeon meat. However, myofibrillar proteins are poorly soluble in low ionic strength media, thus limiting their use in aqueous systems and development as nutritional supplements, and thus failing to achieve widespread processing.

Glucosamine is a natural amino monosaccharide, mainly participates in the construction of human tissues and cell membranes, and is an important component of proteoglycan in human articular cartilage matrix. Glucosamine also has the physiological activities of resisting inflammation, relieving osteoarthritis pain and the like, so the glucosamine is often used as a food supplement, has wide market prospect and is also deeply valued in research.

The glycosylation technology is a protein modification method based on Maillard reaction between free amino acid and reducing sugar of protein, and is helpful for improving functional characteristics and oxidation resistance of food protein. At present, the glycosylation modification of protein is mainly performed by a dry method and a wet method, and although the dry heat method can effectively improve the functional characteristics of food protein, the preparation process is complex, the reaction conditions are strict and difficult to control, and the organoleptic characteristics of the product are influenced due to browning of the product. The wet glycosylation reaction has mild conditions and short time, can obviously improve the functional characteristics of protein, reduces the generation of harmful byproducts of dry reaction under high temperature, and has good industrial application prospect. However, the effect of single glycosylation modification on improving the functional properties of protein is very limited, so that non-thermal processing technology and glycosylation composite modification are increasingly emphasized in the field of food processing, such as auxiliary modification of ultrasonic waves, pulsed electric fields, dynamic high-pressure micro-jet and the like. The high-pressure homogenization technology is a processing method for changing the physical, chemical and structural properties of macromolecules by using high pressure, can induce the unfolding of myofibrillar protein, expose internal amino acid groups, increase glycosylation sites and improve the protein modification degree, thereby improving the functional characteristics of the myofibrillar protein, and therefore, the high-pressure homogenization combined glycosylation composite modification has good application prospect.

Disclosure of Invention

The invention aims to provide a preparation method of a graft modified fish protein-sugar coupling compound. The wet glycosylation has the outstanding advantages of low cost, simple and efficient operation, short reaction time, mild reaction conditions, easy separation of synthesized conjugates and the like, can overcome the defects of the traditional process, obviously improves the functional characteristics of the myofibrillar protein, and has wide development prospect.

A method for preparing a graft-modified fish protein-sugar coupled complex, the method comprising the steps of:

1) dissolving the protein in a phosphate buffer solution to enable the mass concentration of the protein to be 20-40 mg/mL;

2) carrying out high-speed shearing pretreatment on the protein solution obtained in the step 1), and then carrying out secondary high-pressure homogenization treatment;

3) adding a glycosyl donor into the protein solution obtained in the step 2), and reacting for 7-9 hours at the temperature of 35-40 ℃;

4) dialyzing the solution obtained in the step 3), wherein the dialysis time is 20-24 hours, and removing redundant glycosyl donors;

5) and (4) carrying out vacuum freeze drying on the solution obtained in the step (4) and removing the glycosyl donor to obtain the target graft modified protein-sugar coupled compound (namely the graft modified fish protein-sugar coupled compound).

According to the method, after the myofibrillar protein solution is subjected to high-pressure homogenization pretreatment to expand the protein, the alpha-helix content is increased, glycosylation sites are increased, then the myofibrillar protein solution and glucosamine are subjected to a wet glycosylation reaction in a water phase, a natural protein structure is stabilized by utilizing a macromolecule crowding theory, and the glycosylation degree is improved; and (3) dialyzing and desugarizing the product after the glycosylation reaction, and carrying out vacuum freeze drying to obtain the graft modified protein-sugar coupled compound. Compared with the traditional glycosylation mode, the preparation method provided by the invention can obviously improve the grafting degree of the myofibrillar protein and the glycosyl donor and the solubility in a low-ion solvent, and the oxidation resistance of the preparation method is also improved.

Further, in the step 1), the phosphate buffer solution is 0.4-0.8 mol/L, and most preferably 0.6 mol/L. The protein is sturgeon myofibrillar protein.

Further, in the step 2), the conditions of the high-speed shearing are as follows: the shearing rate is 8000-10000 rpm, the time is 1-2 minutes, and the homogenizing pressure is 400-600 bar.

Further, in the step 3), the glycosyl donor is glucosamine, and the molar ratio of the protein to the glycosyl donor is 1: 1-1: 3.

Further, in the step 4), the dialysate used for dialysis is a phosphate buffer solution, and the concentration of the phosphate buffer solution is 0.05-0.2 mol/L, and most preferably 0.1 mol/L.

Compared with the prior art, the invention has the following advantages:

(1) the method overcomes the problem of insufficient modification of myofibrillar protein by the existing method, exposes more glycosylation sites through high-pressure homogenization, and greatly improves the grafting degree of glucosamine and myofibrillar protein;

(2) the invention obviously increases the solubility of the modified product in a low ionic strength medium, and simultaneously improves the oxidation resistance of the modified product;

(3) the whole modification process is simple to operate, stable in product performance and less in byproduct formation, so that the method has good industrial and large-scale application prospects.

Drawings

FIG. 1 shows the degree of grafting of myofibrillar proteins to glucosamine under different homogenization pressures;

FIG. 2 is a graph of comparative data on the oxidation resistance of linoleic acid for example 1 and comparative examples 1-3;

FIG. 3 is data comparing the radical scavenging capacity of the ABTS of example 1 and comparative examples 1-3.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying tables and drawings.

Example 1

1) Dissolving sturgeon myofibrillar protein in 0.6mol/L phosphate buffer solution to ensure that the mass concentration of the sturgeon myofibrillar protein is 30 mg/mL;

2) after the protein solution obtained in the step 1) is subjected to high-speed shearing pretreatment, the shearing rate is 8000rpm, the time is 1 minute, and then secondary high-pressure homogenization treatment is carried out, wherein the homogenization pressure is 500 bar;

3) adding glucosamine into the protein solution obtained in the step 2), wherein the molar ratio of the protein to the glycosyl donor is 1:2, and reacting for 8 hours at 37 ℃;

4) dialyzing the solution obtained in the step 3), wherein the dialyzate is 0.1mol/L phosphate buffer solution, the dialysis time is 24 hours, and removing redundant glucosamine;

5) and (4) carrying out vacuum freeze drying on the solution obtained in the step (4) and removing the glucosamine to obtain the target graft modified protein-sugar coupled compound.

Comparative example 1

1) Dissolving sturgeon myofibrillar protein in 0.6mol/L phosphate buffer solution to ensure that the mass concentration of the sturgeon myofibrillar protein is 30 mg/mL;

2) reacting the protein solution obtained in the step 1) for 8 hours at 37 ℃;

3) dialyzing the solution obtained in the step 2), wherein the dialyzate is 0.1mol/L phosphate buffer solution, and the dialysis time is 24 hours;

4) vacuum freeze drying the solution obtained in step 3) to obtain the product of comparative example 1.

Comparative example 2

1) Dissolving sturgeon myofibrillar protein in 0.6mol/L phosphate buffer solution to ensure that the mass concentration of the sturgeon myofibrillar protein is 30 mg/mL;

2) after the protein solution obtained in the step 1) is subjected to high-speed shearing pretreatment, the shearing rate is 8000rpm, the time is 1 minute, and then secondary high-pressure homogenization treatment is carried out, wherein the homogenization pressure is 500 bar;

3) reacting the protein solution obtained in the step 2) for 8 hours at 37 ℃;

4) dialyzing the solution obtained in the step 3), wherein the dialyzate is 0.1mol/L phosphate buffer solution, and the dialysis time is 24 hours;

5) and (4) carrying out vacuum freeze drying on the solution obtained in the step (4) to obtain the product of the comparative example 2.

Comparative example 3

1) Dissolving sturgeon myofibrillar protein in 0.6mol/L phosphate buffer solution to ensure that the mass concentration of the sturgeon myofibrillar protein is 30 mg/mL;

2) adding glucosamine into the protein solution obtained in the step 1), and reacting for 8 hours at 37 ℃;

3) dialyzing the solution obtained in the step 2), wherein the dialyzate is 0.1mol/L phosphate buffer solution, the dialysis time is 24 hours, and removing redundant glucosamine;

4) the glucosamine removed solution obtained in 3) was subjected to vacuum freeze-drying to obtain the product of comparative example 3.

Comparative example 1 is different from the method of example 1 in that there is no high pressure homogenization and glycosylation treatment.

Comparative example 2 is different from the method of example 1 in that there is no glycosylation treatment.

Comparative example 3 is different from the method of example 1 in that there is no high-pressure homogenization treatment.

Table 1 shows the effect of different modification modes on the secondary structure content of myofibrillar proteins. Comparative data for the secondary structures of example 1 and comparative examples 1-3 are shown in Table 1.

TABLE 1

The data comparison in table 1 shows that the invention uses high-pressure homogenization and glycosylation to compositely modify myofibrillar proteins, which can significantly improve the content ratio of alpha-helix of the modified proteins, reduce the content ratio of beta-sheet, show that the main chain of the protein is opened, and more amino acid groups are exposed, thereby increasing glycosylation sites and improving the glycosylation degree of the protein.

Table 2 shows the effect of different modifications on the solubility of myofibrillar proteins in low ionic strength media. The data comparing the solubility in low ionic strength media of example 1 with comparative examples 1-3 are shown in table 2.

TABLE 2

The data comparison in the table 2 shows that the high-pressure homogenization and glycosylation composite modification technology used in the invention can significantly improve the solubility of myofibrillar protein in a low ionic strength medium, and the effect is more obvious than that of the single high-pressure homogenization or glycosylation technology, which indicates that the composite modification has a better result for improving the solubility of salt-soluble animal protein in a low ionic strength solvent.

Table 3 shows the effect of different modifications on the degree of grafting of myofibrillar proteins to glucosamine. Comparative data on the degree of grafting of example 1 versus comparative examples 1-3 are shown in Table 3.

TABLE 3

The data comparison in table 3 shows that the high-pressure homogenization and glycosylation composite modification technology used in the invention can significantly improve the grafting degree of myofibrillar protein and glucosamine, and the effect is more obvious than that of the glycosylation technology used singly, which indicates that the composite modification has better influence on improving the modification degree of myofibrillar protein.

The degree of grafting of myofibrillar proteins to glucosamine at different homogenization pressures is shown in FIG. 1. As can be seen from fig. 1, the grafting degree of myofibrillar proteins and glucosamine tends to increase first and then decrease with the increase of the homogenizing pressure, and is at a maximum value at 500bar, which indicates that the higher the homogenizing pressure is, the higher the grafting degree is, and the possible reason is that, at the homogenizing pressure of 300-500 bar, the increase of the homogenizing pressure helps the proteins to spread, more glycosylation sites are exposed, and thus the grafting degree increases; when the homogenization pressure exceeds 500bar, the high-pressure homogenization ruptures the protein, the glycosylation sites are destroyed, and the degree of grafting is reduced. Repeated experiments prove that the grafting degree of myofibrillar protein and glucosamine can be improved to the maximum degree when the homogenizing pressure is 500bar, and the modification effect is obviously enhanced.

The data comparing the oxidation resistance of linoleic acid of example 1 with that of comparative examples 1-3 are shown in FIG. 2. It can be found from the comparative data in fig. 2 that the high-pressure homogenization and glycosylation complex modification technology used in the invention can significantly improve the linoleic acid oxidation resistance of the product in example 1. The oxidation resistance of linoleic acid is lower in comparative examples 2 and 3 than in comparative example 1, probably because the protein is partially oxidized during the process, thus reducing the antioxidant activity.

Data comparing the radical scavenging ability of example 1 to comparative examples 1-3ABTS are shown in FIG. 3. As can be seen from the comparative data in fig. 3, the ABTS radical scavenging ability of comparative example 2 is lower than that of comparative example 1, and thus high pressure homogenization decreases the radical scavenging ability of myofibrillar proteins; however, comparative example 3 has a stronger scavenging ability for ABTS radicals than comparative example 1, and thus glycosylation can increase the radical scavenging ability of myofibrillar proteins; example 1 is the strongest in ABTS free radical scavenging ability, so the high pressure homogenization and glycosylation complex modification technology used in the invention can obviously improve the ABTS free radical scavenging ability of myofibrillar protein, which can not be realized by a single modification method, thus the method has substantial gain effect.

The above embodiments are merely exemplary, and the present invention is not limited to the above embodiments. All changes, modifications, substitutions, combinations, and simplifications which may be made without departing from the spirit and principles of the invention are intended to be embraced therein.