A method for regulating the structure of glycosylated cod protein and its application in preparing high internal phase emulsion

Abstract

A method for regulating the structure of glycosylated codfish protein and its application in preparing a high internal phase emulsion. The present invention belongs to the field of protein structure regulation. The purpose of the present invention is to solve the technical problems of freeze-thaw instability of the current high internal phase emulsion, high glycemic index of the remaining free sugar of the glycosylated protein, and large sugar dosage. The present invention mixes the dialyzed glycosylated codfish protein solution with low-calorie sugar, and then freeze-dries for more than 14 hours to complete the regulation of the protein structure and obtain a protein-low-calorie sugar complex. And use it to prepare a high internal phase emulsion. Based on the dialyzed glycosylated codfish protein solution, the present invention regulates the secondary structure of the glycosylated codfish protein by freeze-drying with the assistance of low-calorie sugar, and uses a protein-low-calorie sugar complex with a new structure as a wall material to prepare a high internal phase emulsion, and the obtained high internal phase emulsion has lower free sugars, and the new protein structure gives the high internal phase emulsion higher freeze-thaw stability.

Description

Technical Field

[0001] The invention belongs to the field of protein structure regulation and application thereof, and specifically relates to a method for regulating the structure of glycosylated codfish protein and application thereof in preparing a high internal phase emulsion. Background Art

[0002] High internal phase emulsion refers to a type of emulsion with a dispersed phase volume of more than 74.05%. It has a broad application prospect in replacing solid or semi-solid oils in food. At present, high internal phase emulsions stabilized by protein particles as emulsifiers occupy an important position in food-grade high internal phase emulsions. However, high internal phase emulsions have the disadvantage of poor freeze-thaw stability, which greatly affects their application in various industries such as food, medicine, and bioengineering.

[0003] The high internal phase emulsion stabilized by codfish protein has good rheological properties and can be used as a new type of 3D printing material. However, the poor antifreeze properties of codfish protein high internal phase emulsion limit its use in frozen foods such as ice cream. How to improve the antifreeze properties of codfish protein high internal phase emulsion has become an important technical problem in promoting the application of codfish protein high internal phase emulsion. Glycosylated modified proteins can usually be used as an effective method to improve the freeze-thaw stability of emulsions. Usually, glucose can modify codfish protein quickly, cheaply and efficiently. However, a large amount of unreacted free sugars such as glucose will remain in the glycosylation reaction process, and the application in food will lead to the risk of increased blood sugar. Usually, dialysis treatment can effectively remove unreacted free sugars and eliminate the impact on blood sugar, but after the glycosylated codfish protein treated by this technology is freeze-dried, the high internal phase emulsion prepared loses its antifreeze properties. How to solve the problem of glycosylated codfish protein having antifreeze properties and maintaining a low glycemic index has become a new technical problem in the application of codfish protein high internal phase emulsion.

[0004] Studies have found that proteins can undergo secondary structural changes during freeze drying (i.e., lyophilization), but such changes usually lead to adverse effects such as protein insolubility and loss of activity. In traditional processes, various technical means are used to minimize the effects of freeze drying on protein structure, thereby maintaining the original structure and properties of the protein. Currently, there is no prior art report on regulating the secondary structure of a protein through freeze drying to give it new functional properties, especially giving it the ability to prepare a freeze-thaw stable high internal phase emulsion. Summary of the invention

[0005] In order to solve the technical problems of freeze-thaw instability of the above-mentioned high internal phase emulsion, high glycemic index of residual free sugar of glycosylated protein, and large amount of sugar, the present invention provides the following technical solutions: a method for regulating the structure of glycosylated cod protein and its application in preparing a high internal phase emulsion.

[0006] One of the purposes of the present invention is to provide a method for regulating the structure of glycosylated cod protein, comprising:

[0007] S1. Mix the dialyzed glycosylated cod protein solution with low-calorie sugar and stir for 1 to 2 hours at a pH of 7 to 10.5;

[0008] S2. Freeze-drying for more than 14 hours at a temperature of 0-40° C. and a vacuum degree of 0-15 Pa to complete the regulation of the structure of the glycosylated codfish protein and obtain a glycosylated codfish protein-low-calorie sugar complex.

[0009] As a preferred embodiment of the method for regulating the structure of glycosylated codfish protein of the present invention, the low-calorie sugar in S1 is one of oligosaccharides, stevioside or sugar alcohol products.

[0010] As a further preferred embodiment of the method for regulating the structure of glycosylated codfish protein of the present invention, the sugar alcohol product comprises xylitol, arabinose, erythritol and maltitol.

[0011] As a preferred embodiment of the method for regulating the structure of glycosylated cod protein of the present invention, the mass ratio of glycosylated cod protein to low-calorie sugar in the glycosylated cod protein solution in S1 is 1:(0.8-2).

[0012] As a preferred embodiment of the method for regulating the structure of glycosylated cod protein of the present invention, the dialysis method described in S1 is as follows: the glycosylated cod protein solution cooled to below 30°C is placed in a dialysis bag with a cutoff of 3500Da, and dialyzed in deionized water at 4°C for 36 to 60 hours until the free glucose content is below 0.3 mg / mL.

[0013] As a preferred embodiment of the method for regulating the structure of glycosylated codfish protein of the present invention, wherein: freeze-drying is performed at 20° C. and 5 Pa for more than 14 hours in S2.

[0014] The second object of the present invention is to provide a glycosylated cod protein-low-calorie sugar complex obtained by the above method.

[0015] The third object of the present invention is to provide an application of the glycosylated cod protein-low-calorie sugar complex obtained by the above method in the preparation of a high internal phase emulsion. The specific application process steps are as follows:

[0016] S1, mixing the glycosylated cod protein-low-calorie sugar complex with deionized water, adjusting the pH value to 7-10.5, and obtaining a composite solution;

[0017] S2. Add edible vegetable oil to the composite solution, and after homogenization, obtain a high freeze-thaw resistance high internal phase emulsion.

[0018] As a preferred embodiment of the application of the present invention, the concentration of glycosylated codfish protein in the composite solution obtained in S1 is 30-70 mg / mL.

[0019] As a preferred embodiment of the application of the present invention, the edible vegetable oil in S2 includes but is not limited to soybean oil.

[0020] As a preferred embodiment of the application of the present invention, the rotation speed of the homogenization in S2 is 5k-16k rpm, and the time is 30-180s.

[0021] As a preferred embodiment of the application of the present invention, the concentration of edible vegetable oil in the high freeze-thaw resistance high internal phase emulsion obtained in S2 is 76-88 vol%.

[0022] A fourth object of the present invention is to provide a high freeze-thaw resistance high internal phase emulsion obtained by the above method.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention utilizes the ability of freeze-drying to regulate protein structural changes, and reasonably adds a protein structure regulator to assist in controlling the structural changes of glycosylated cod protein during the freeze-drying process, so that the secondary structure of the glycosylated cod protein falls within a reasonable parameter range, thereby achieving the antifreeze property of the glycosylated cod protein after dialysis and freeze-drying. The specific advantages are as follows:

[0025] 1) The present invention is based on the dialyzed glycosylated cod protein solution, and with the assistance of low-calorie sugar, the secondary structure of the glycosylated cod protein is regulated by freeze-drying (the β-fold content increases by 1.18 times, and the β-turn content decreases by 5 times), and a newly constructed glycosylated cod protein-low-calorie sugar complex is used as the wall material to prepare a stable high internal phase emulsion. The obtained high internal phase emulsion has lower free sugar, and the new protein structure gives the high internal phase emulsion higher freeze-thaw stability.

[0026] 2) The high internal phase emulsion prepared by the present invention using the structurally modified glycosylated codfish protein can still maintain a stable emulsion structure after three freeze-thaw cycles. This provides a new direction for the preparation of high internal phase emulsions, makes up for the disadvantage of poor freeze-thaw stability of existing high internal phase emulsions, and expands the application scope of high internal phase emulsions in the field of frozen foods.

[0027] 3) The technical solution adopted by the present invention significantly reduces the usage equivalent of low-calorie sugar in the antifreeze high internal phase emulsion. Compared with the solution of directly freeze-drying glycosylated cod protein after dialysis and then adding low-calorie sugar, the usage equivalent of low-calorie sugar is reduced from 20% to 4% to prepare a freeze-thaw stable high internal phase emulsion, which greatly reduces the sugar intake and provides a new material for the preparation of low-sugar and healthy food.

[0028] 4) After consuming the emulsion prepared by the glycosylated cod protein-low-calorie sugar complex of the present invention, the blood sugar rise after 1 hour was reduced by 43.33% compared with the glycosylated cod protein-glucose complex control group, which better meets the consumer's demand for green and healthy products.

[0029] 5) The preparation method of the present invention can expand the application of cod protein in emulsions and increase the economic added value of cod. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an appearance diagram of the high internal phase emulsion prepared in Example 1 of the present invention before freezing and thawing;

[0031] Figure 2 This is an appearance diagram of a high internal phase emulsion prepared in Example 1 of the present invention after one freeze-thaw cycle;

[0032] Figure 3 This is an appearance diagram of the high internal phase emulsion prepared in Example 1 of the present invention after three freeze-thaw cycles;

[0033] Figure 4 This is an appearance diagram of the high internal phase emulsion prepared in Example 2 of the present invention before freezing and thawing;

[0034] Figure 5 This is an appearance diagram of a high internal phase emulsion prepared in Example 2 of the present invention after one freeze-thaw cycle;

[0035] Figure 6 This is an appearance diagram of three freeze-thaw cycles of the high internal phase emulsion prepared in Example 2 of the present invention;

[0036] Figure 7 This is an appearance diagram of a high internal phase emulsion prepared in Comparative Example 1 of the present invention after one freeze-thaw cycle;

[0037] Figure 8 This is an appearance diagram of a high internal phase emulsion prepared in Comparative Example 2 of the present invention after one freeze-thaw cycle;

[0038] Fig. 9 This is an appearance diagram of the high internal phase emulsion prepared in Comparative Example 4 of the present invention before freezing and thawing;

[0039] Fig.10 This is an appearance diagram of one freeze-thaw cycle of the high internal phase emulsion prepared in Comparative Example 4 of the present invention. DETAILED DESCRIPTION

[0040] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0041] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0042] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0043] The cod protein used in the following examples is the crude protein extract of Atlantic cod obtained in Example 1-S1 of the invention patent with publication number CN113040369; other raw materials are obtained through commercial purchase.

[0044] Ion chromatography determination: 1 mL of glycosylated cod protein was purified using a C18 solid phase extraction cartridge (Agela Technologies, USA) and then filtered through a 0.22 μm membrane. The remaining glucose in the sample was quantified using an ion chromatography Thermo Fisher Scientific Dionex ICS-5000+ system, including a pump (Dionex ICS-5000+DP), a column oven, a detector (Dionex ICS-5000+DC), and a PA1 column (250×2 mm; Thermo Fisher Scientific, USA). Mobile phase A was ultrapure water, and mobile phase B was 250 mmol / L sodium hydroxide solution. The mobile phase was set as follows: 5% B for 0-20 min; 5% B increased to 100% B for 20-26 min; 100% B for 26-35 min; 100% B linear gradient decreased to 5% B for 35-36 min; 5% B for 36-45 min; the flow rate was 0.25 mL min / L, and the injection volume was 25 μL.

[0045] Example 1

[0046] S1

[0047] (1) Preparation of glycosylated codfish protein solution: ① Use water to re-dissolve codfish protein into a protein solution with a protein concentration of 50 mg / mL; ② Add glucose to the protein solution to obtain a mixed solution; wherein the concentration ratio of codfish protein to glucose in the protein solution is 1:4; ③ The mixed solution is reacted in an oil bath at 120°C for 20 minutes, and then cooled to room temperature to obtain a glycosylated codfish protein solution.

[0048] (2) Dialysis: The glycosylated cod protein solution cooled to below 30°C was placed in a dialysis bag with a cutoff of 3500D and dialyzed in deionized water at 4°C for 48 hours. The free glucose content was determined to be below 0.3 mg / mL by ion chromatography to obtain a dialyzed glycosylated cod protein solution.

[0049] (3) Preparation of glycosylated cod protein-low-calorie sugar complex: The dialyzed glycosylated cod protein solution was mixed with galacto-oligosaccharide at a mass ratio of 1:1, stirred at pH 10 for 1 h, and then transferred to a culture dish and placed in a freeze dryer. It was freeze-dried at 20°C and 5 Pa for 24 h to obtain a glycosylated cod protein-low-calorie sugar complex. The protein structure detection results are shown in Table 1.

[0050] S2

[0051] The glycosylated codfish protein-low-calorie sugar complex was mixed with deionized water, and the pH value was adjusted to 10 with NaOH solution to obtain a complex solution with a glycosylated codfish protein-low-calorie sugar complex concentration of 100 mg / mL (wherein the glycosylated codfish protein concentration was 50 mg / mL) as the aqueous phase.

[0052] S3

[0053] Soybean oil was added to the composite solution, and homogenized with a homogenizer at 8000 rpm for 90 seconds to obtain a high freeze-thaw resistance high internal phase emulsion, wherein the soybean oil content in the high freeze-thaw resistance high internal phase emulsion was 80 vol%.

[0054] The high freeze-thaw resistance high internal phase emulsion prepared in this example was taken and gavaged into mice at 6 g / kg. It was measured that the blood sugar of the mice increased by 51.56% 1 hour after the meal. The specific results are shown in Table 2.

[0055] 9 g of the low-sugar, high freeze-thaw resistance, high internal phase emulsion prepared in this example was placed in a 10 mL centrifuge tube, frozen at -30°C for 22 h, and thawed in a 37°C water bath for 2 h. The freezing and thawing process was considered as one freeze-thaw cycle, and a total of 3 cycles were performed. The appearance of the high internal phase emulsion before and after freezing and thawing is shown in the figure below: Figure 1-3 As shown, it can be seen that after three cycles of repeated freezing and thawing, the emulsion system has no obvious changes, no oil layer is precipitated on the surface, and there is no demulsification phenomenon, indicating that the emulsion has good freeze-thaw stability.

[0056] Example 2

[0057] S1

[0058] (1) Preparation of glycosylated codfish protein solution: ① Use water to re-dissolve codfish protein into a protein solution with a protein concentration of 50 mg / mL; ② Add glucose to the protein solution to obtain a mixed solution; wherein the concentration ratio of codfish protein to glucose in the protein solution is 1:4; ③ The mixed solution is reacted in an oil bath at 120°C for 20 minutes, and then cooled to room temperature to obtain a glycosylated codfish protein solution.

[0059] (2) Dialysis: The glycosylated cod protein solution cooled to below 30°C was placed in a dialysis bag with a cutoff of 3500D and dialyzed in deionized water at 4°C for 48 hours. The free glucose content was determined to be below 0.3 mg / mL by ion chromatography to obtain a dialyzed glycosylated cod protein solution.

[0060] (3) Preparation of glycosylated cod protein-low-calorie sugar complex: The dialyzed glycosylated cod protein solution was mixed with trehalose at a mass ratio of 1:1, stirred at pH 10 for 1 h, and then transferred to a culture dish and placed in a freeze dryer. Glycosylated cod protein-low-calorie sugar complex was obtained at 20°C and 5 Pa. The protein structure detection results are shown in Table 1.

[0061] S2

[0062] The glycosylated codfish protein-low-calorie sugar complex was mixed with deionized water, and the pH value was adjusted to 10 with NaOH solution to obtain a complex solution with a glycosylated codfish protein-low-calorie sugar complex concentration of 100 mg / mL (wherein the glycosylated codfish protein concentration was 50 mg / mL) as the aqueous phase.

[0063] S3

[0064] The soybean oil was added into the composite solution, and homogenized for 120 seconds at 6000 rpm by a homogenizer to obtain a high freeze-thaw resistance high internal phase emulsion, wherein the soybean oil content in the high freeze-thaw resistance high internal phase emulsion was 80 vol%.

[0065] The high freeze-thaw resistance high internal phase emulsion prepared in this example was taken and gavaged into mice at 6 g / kg. It was measured that the blood sugar of the mice increased by 54.69% 1 hour after the meal. The specific results are shown in Table 2.

[0066] 9 g of the high freeze-thaw resistance high internal phase emulsion prepared in this example was placed in a 10 mL centrifuge tube, frozen at -30°C for 22 h, and thawed in a 37°C water bath for 2 h. The freezing and thawing process was considered as one freeze-thaw cycle, and a total of 3 cycles were performed. The appearance of the high internal phase emulsion before and after freezing and thawing is shown in the figure below: Figure 4-6 As shown, it can be seen that after three cycles of repeated freezing and thawing, the emulsion system has no obvious changes, no oil layer is precipitated on the surface, and there is no demulsification phenomenon, indicating that the emulsion has good freeze-thaw stability.

[0067] Comparative Example 1

[0068] S1

[0069] (1) Preparation of glycosylated codfish protein solution: ① Use water to re-dissolve codfish protein into a protein solution with a protein concentration of 50 mg / mL; ② Add glucose to the protein solution to obtain a mixed solution; wherein the concentration ratio of codfish protein to glucose in the protein solution is 1:4; ③ The mixed solution is reacted in an oil bath at 120°C for 20 minutes, and then cooled to room temperature to obtain a glycosylated codfish protein solution.

[0070] (2) Freeze-drying: The glycosylated codfish protein solution was placed in a culture dish and freeze-dried in a freeze dryer at -80°C and 5 Pa for 24 hours to obtain the glycosylated codfish protein.

[0071] S2

[0072] The glycosylated cod protein was mixed with deionized water, and then galactoligosaccharide was added at a mass ratio of galactoligosaccharide to glycosylated cod protein of 1:1, and stirred for 1 hour at a pH of 10 to obtain a composite solution with a glycosylated cod protein concentration of 50 mg / mL as the aqueous phase, wherein the protein structure detection results are shown in Table 1.

[0073] S3,

[0074] Soybean oil was added to the composite solution and homogenized with a homogenizer at 8000 rpm for 120 seconds to obtain a high internal phase emulsion, wherein the soybean oil content in the high internal phase emulsion was 80 vol%.

[0075] 9 g of the high internal phase emulsion prepared in this comparative example was placed in a 10 mL centrifuge tube, frozen at -30°C for 22 h, and thawed in a 37°C water bath for 2 h. The appearance of the high internal phase emulsion after freeze-thaw is shown in the figure below: Figure 7 As shown, it can be seen that after the freeze-thaw cycle, the emulsion is separated into oil and water layers and demulsification occurs, indicating that the emulsion is not freeze-thaw stable.

[0076] Comparative Example 2

[0077] S1

[0078] (1) Preparation of glycosylated codfish protein solution: ① Use water to re-dissolve codfish protein into a protein solution with a protein concentration of 50 mg / mL; ② Add glucose to the protein solution to obtain a mixed solution; wherein the concentration ratio of codfish protein to glucose in the protein solution is 1:4; ③ The mixed solution is reacted in an oil bath at 120°C for 20 minutes, and then cooled to room temperature to obtain a glycosylated codfish protein solution.

[0079] (2) Freeze-drying: The glycosylated codfish protein solution was placed in a culture dish and freeze-dried in a freeze dryer at -80°C and 5 Pa for 24 hours to obtain the glycosylated codfish protein.

[0080] S2

[0081] The glycosylated cod protein was mixed with deionized water, and then trehalose was added at a mass ratio of trehalose to glycosylated cod protein of 1:1, and stirred for 1 hour at a pH of 10 to obtain a composite solution with a glycosylated cod protein concentration of 50 mg / mL as the aqueous phase, wherein the protein structure detection results are shown in Table 1.

[0082] S3,

[0083] Soybean oil was added to the composite solution and homogenized with a homogenizer at 8000 rpm for 120 seconds to obtain a high internal phase emulsion, wherein the soybean oil content in the high internal phase emulsion was 80 vol%.

[0084] 9 g of the high internal phase emulsion prepared in this comparative example was placed in a 10 mL centrifuge tube, frozen at -30°C for 22 h, and thawed in a 37°C water bath for 2 h. The appearance of the high internal phase emulsion after freeze-thaw is shown in the figure below: Figure 8 As shown, it can be seen that after the freeze-thaw cycle, the emulsion is separated into oil and water layers and demulsification occurs, indicating that the emulsion is not freeze-thaw stable.

[0085] Comparative Example 3

[0086] S1

[0087] (1) Preparation of glycosylated codfish protein solution: ① Use water to re-dissolve codfish protein into a protein solution with a protein concentration of 50 mg / mL; ② Add glucose to the protein solution to obtain a mixed solution; wherein the concentration ratio of codfish protein to glucose in the protein solution is 1:4; ③ The mixed solution is reacted in an oil bath at 120°C for 20 minutes, and then cooled to room temperature to obtain a glycosylated codfish protein solution.

[0088] (2) Freeze-drying: The glycosylated codfish protein solution was placed in a culture dish and freeze-dried in a freeze dryer at -80°C and 5 Pa for 24 hours to obtain the glycosylated codfish protein.

[0089] S2

[0090] The glycosylated cod protein was mixed with deionized water, and then glucose was added at a mass ratio of glucose to glycosylated cod protein of 1:1, and the mixture was stirred for 1 hour at a pH of 10 to obtain a composite solution with a glycosylated cod protein concentration of 50 mg / mL as the aqueous phase.

[0091] S3,

[0092] Soybean oil was added to the composite solution and homogenized with a homogenizer at 8000 rpm for 120 seconds to obtain a high internal phase emulsion, wherein the soybean oil content in the high internal phase emulsion was 80 vol%.

[0093] The high internal phase emulsion prepared in this embodiment was taken and gavaged into mice at 6 g / kg. It was measured that the blood sugar of the mice increased by 93.75% 1 hour after the meal, and the blood sugar increase rate was 1.8 times higher than that of the embodiment. The specific results are shown in Table 2. The comparison shows that the product prepared by this patent can better meet the consumer's demand for green and healthy products.

[0094] Comparative Example 4

[0095] S1

[0096] (1) Preparation of glycosylated codfish protein solution: ① Use water to re-dissolve codfish protein into a protein solution with a protein concentration of 50 mg / mL; ② Add glucose to the protein solution to obtain a mixed solution; wherein the concentration ratio of codfish protein to glucose in the protein solution is 1:4; ③ The mixed solution is reacted in an oil bath at 120°C for 20 minutes, and then cooled to room temperature to obtain a glycosylated codfish protein solution.

[0097] (2) Freeze-drying: The glycosylated codfish protein solution was placed in a culture dish and freeze-dried in a freeze dryer at -80°C and 5 Pa for 24 hours to obtain the glycosylated codfish protein.

[0098] S2

[0099] The glycosylated cod protein was mixed with deionized water, and then galactoligosaccharide was added at a mass ratio of galactoligosaccharide to glycosylated cod protein of 4:1, and stirred for 1 hour at a pH of 10 to obtain a composite solution with a glycosylated cod protein concentration of 50 mg / mL as the aqueous phase.

[0100] S3,

[0101] Soybean oil was added to the composite solution and homogenized with a homogenizer at 8000 rpm for 120 seconds to obtain a high internal phase emulsion, wherein the soybean oil content in the high internal phase emulsion was 80 vol%.

[0102] 9 g of the high internal phase emulsion prepared in this comparative example was placed in a 10 mL centrifuge tube, frozen at -30°C for 22 h, and thawed in a 37°C water bath for 2 h. The freezing and thawing process was considered as one freeze-thaw cycle. The appearance of the high internal phase emulsion before and after freezing and thawing is shown in the figure below: Figure 9-10 As shown. After one freeze-thaw cycle, the oil and water layers at the bottom of the emulsion system were separated and demulsification occurred, indicating that when the freeze-drying technology proposed in this patent is not used to regulate the protein structure, adding 20% ​​galacto-oligosaccharide to the water phase still cannot make the high internal phase emulsion have freeze-thaw stability. This shows that the product prepared by this patent not only improves the freeze-thaw stability of the high internal phase emulsion, but also greatly reduces the sugar intake, providing a new material for the preparation of low-sugar and healthy foods.

[0103] Table 1 Protein secondary structure

[0104] α-helix β-sheet β-turn Randomcoil G-CP before freeze-drying <![CDATA[0.26±0.03 b ]]> <![CDATA[0.34±0.01 a ]]> <![CDATA[0.09±0.01 a ]]> <![CDATA[0.29±0.01 b ]]> Freeze-dried G-CP <![CDATA[0.39±0.02 a ]]> <![CDATA[0.17±0.04 b ]]> <![CDATA[0.12±0.02 a ]]> <![CDATA[0.32±0.00 a ]]> Example 1: G-CP (mixed with galacto-oligosaccharide in a ratio of 1:1 and then freeze-dried) <![CDATA[0.32±0.03 b ]]> <![CDATA[0.37±0.02 a ]]> <![CDATA[0.02±0.00 c ]]> <![CDATA[0.27±0.04 b ]]> Example 2: G-CP (mixed with trehalose in a ratio of 1:1 and then freeze-dried) <![CDATA[0.32±0.04 b ]]> <![CDATA[0.39±0.03 a ]]> <![CDATA[0.03±0.01 c ]]> <![CDATA[0.26±0.02 b ]]> Comparative Example 1: G-CP (galacto-oligosaccharide was added at a ratio of 1:1 after freeze-drying) <![CDATA[0.34±0.03 b ]]> <![CDATA[0.24±0.04 b ]]> <![CDATA[0.06±0.02 b ]]> <![CDATA[0.36±0.04 a ]]> Comparative Example 2: G-CP (adding trehalose at a ratio of 1:1 after freeze-drying) <![CDATA[0.32±0.01 b ]]> <![CDATA[0.21±0.03 b ]]> <![CDATA[0.10±0.01 a ]]> <![CDATA[0.37±0.05 a ]]>

[0105] *G-CP is glycosylated cod protein

[0106] It can be observed from Table 1 that the β-turn content of glycosylated cod protein freeze-dried with low-calorie sugar is significantly lower than that of glycosylated cod protein freeze-dried directly without sugar, while the β-folding content is significantly higher than that of glycosylated cod protein freeze-dried directly. This shows that low-calorie sugar effectively regulates the secondary structure of glycosylated cod protein during the freeze-drying process. The change in the secondary structure of the protein changes the functionality of the glycosylated cod protein, and the glycosylated cod protein with a changed secondary structure has the ability to prepare a high internal phase emulsion with freeze-thaw stability.

[0107] Table 2 Comparison of blood glucose changes in mice given high internal phase milk (mmol / L)

[0108] High internal phase emulsion wall material Fasting blood sugar Blood glucose level 0.5h after meal Blood glucose level 1 hour after meal Blood sugar level 1.5 hours after meal Comparative Example 3 6.4±0.3 8.7±0.4 12.4±0.8 8.1±0.4 Example 1 6.6±0.2 7.1±0.6 9.8±0.5 7.5±0.4 Example 2 6.4±0.4 7.7±0.5 9.9±0.7 7.2±0.6

[0109] It can be observed from Table 2 that the postprandial blood sugar rise and fluctuation range of mice gavaged with the low-sugar high internal phase emulsion prepared by the present invention are much lower than those of mice in the glucose control group, indicating that the emulsion prepared by the present invention using the glycosylated codfish protein-low-calorie sugar complex can reduce the rate of blood sugar rise after eating, reduce blood sugar fluctuations, and better meet consumers' demand for green and healthy products.

[0110] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for regulating the structure of glycosylated cod protein, characterized in that: include, S1. Mix the dialyzed glycosylated cod protein solution with low-calorie sugar, and stir for 1 to 2 hours at a pH value of 7 to 10.5, wherein the mass ratio of glycosylated cod protein to low-calorie sugar in the glycosylated cod protein solution is 1:(0.8 to 2), and the low-calorie sugar is galacto-oligosaccharide or trehalose. The dialysis method is as follows: cool the glycosylated cod protein solution below 30° C. into a dialysis bag with a cutoff of 3500 Da, and dialyze in deionized water at 4° C. for 36 to 60 hours until the free glucose content is below 0.3 mg / mL; S2. Freeze-drying for more than 14 hours at a temperature of 20° C. and a vacuum degree of 5 Pa to complete the regulation of the glycosylated cod protein structure and obtain a glycosylated cod protein-low-calorie sugar complex.

2. The glycosylated cod protein-low-calorie sugar complex obtained by the method according to claim 1.

3. Use of the glycosylated codfish protein-low-calorie sugar complex obtained by the method according to claim 1 in the preparation of a high internal phase emulsion, characterized in that: Specifically include: S1, mixing the glycosylated cod protein-low-calorie sugar complex with deionized water, adjusting the pH value to 7-10.5, and obtaining a composite solution; S2. Add edible vegetable oil into the composite solution and homogenize to obtain a high internal phase emulsion.

4. The use according to claim 3, characterized in that: The concentration of glycosylated codfish protein in the composite solution obtained in S1 is 30-70 mg / mL.

5. The use according to claim 3, characterized in that: The edible vegetable oil in S2 is soybean oil, the homogenization speed is 5k-16k rpm, the time is 30-180s, and the concentration of the edible vegetable oil in the high internal phase emulsion is 76-88vol%.

6. The high internal phase emulsion obtained by the application of claim 3.

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