Composite gel as well as preparation method and application thereof

Abstract

The invention relates to the field of vegetable protein gel products, in particular to rapeseed protein-soybean protein isolate composite gel and a preparation method thereof. The preparation method of the composite gel comprises the following steps: mixing rapeseed protein and soybean protein isolate, adjusting the pH value to 6-8, heating in a water bath at 80-100 DEG C, and immediately cooling to obtain the composite gel; the preparation method of the rapeseed protein comprises the following steps: crushing degreased rapeseed meal, dissolving the crushed degreased rapeseed meal in a sodium chloride solution according to a solid-to-liquid ratio of 1: (8-12), adjusting the pH value to 7.5-8.5, carrying out a reaction for 30-60 min, and carrying out centrifugation, membrane filtration, secondary centrifugation, dialysis and drying on the product to obtain the rapeseed protein. The hardness, elasticity, cohesiveness and chewiness of the compound protein gel prepared on the basis of the rapeseed protein are superior to those of soybean protein isolate gel. And the viscosity and modulus of the compound protein gel are superior to those of SP protein, and the compound protein gel has better external force resistance.

Description

Technical Field

[0001] The invention relates to the field of plant protein gel products, in particular to a rapeseed protein composite gel and a preparation method and application thereof. Background Art

[0002] Rapeseed protein, as a byproduct of rapeseed meal, has a rich and comprehensive amino acid composition. However, rapeseed meal contains a lot of anti-nutritional substances, such as phytate, some cellulose and glucosinolates. The traditional alkali dissolution and acid precipitation process will reduce the solubility of rapeseed protein and deteriorate its gel properties, making it unsuitable for use in food industry production. Single rapeseed protein gel is difficult to be used in food formulations, limiting the research and development of rapeseed protein foods.

[0003] Prior art CN112753846A discloses a super water-holding soy protein isolate gel and a preparation method thereof. The preparation method of the super water-holding soy protein isolate gel is as follows: at room temperature, the soy protein isolate powder is subjected to a high pressure field pretreatment to obtain a modified pretreated soy protein isolate solution, which is freeze-dried to obtain freeze-dried SPI; the freeze-dried SPI is added to distilled water to obtain a freeze-dried SPI solution; then the enzyme solution is added and stirred evenly, the uniform mixture is placed at 35-45°C for reaction for 0.5-3h, then placed at 90-110°C for inactivation for 10-15min, and then placed at 4-5°C for more than 24h to obtain a super water-holding soy protein isolate gel. The method is pretreated by a high pressure field, freeze-dried, and then cross-linked with an enzyme to obtain a super water-holding soy protein isolate gel. The super water-holding soy protein isolate gel has an increased water holding capacity, a reduced free water content, and a good improvement in the texture characteristics of the gel. However, from the perspective of modifying the finished soy protein powder, this method adds processes such as high pressure treatment, which is not only energy-intensive, but also difficult to industrialize.

[0004] Prior art CN106417887A discloses a method for producing a high water-retaining type soy protein isolate, comprising the following steps: (1) extraction: adding the raw material defatted soybean meal to water to obtain an extract; (2) centrifugal separation: washing the obtained solid phase 1 and liquid phase 1 with water to obtain solid phase 2 and liquid phase 2; (3) acid precipitation: adjusting the pH value of liquid phase 2 and then settling to obtain an acid precipitation liquid; (4) centrifugal separation of the acid precipitation liquid; (5) neutralization: obtaining a neutralized feed liquid; (6) adding a modified enzyme; (7) drying: spray drying to obtain a solid phase 3; and recovering the soy protein isolate. The advantages of this invention are that it reduces small molecule proteins, so that small molecule proteins are formed into large molecule proteins through cross-linking, and at the same time, it can also improve the sol structure formed after soy protein is dissolved in water, improve the water retention capacity and gel strength of the protein, thereby obtaining a soy protein product with excellent product performance and improving its economic value. However, enzymatic production will bring bitterness to the product and the application effect is not good.

[0005] Prior art CN 118402612 A provides a method for preparing soybean oil body-soy protein isolate gel. The present invention prepares SPI gel by ultrasonic treatment of SPI emulsions with different oil body contents, under the action of a coagulant of glucono-δ-lactone, and then by heating. Studies have found that soybean protein and soybean oil bodies, the main components of soybeans, interact with each other in the formation of gel, and soybean oil bodies can be evenly distributed in the SPI gel network structure. As a filling material, the formed gel has good texture and strong water retention, which is significantly better than the gel without ultrasonic treatment and can meet the needs of the food industry. However, due to the problem of the coagulant, the latex gel has insufficient improvement in texture characteristics and elastic properties, and is not stable enough. Summary of the invention

[0006] The purpose of the present invention is to provide a composite gel with significantly improved gel properties and using rapeseed protein as a raw material, as well as a preparation method and application thereof.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A method for preparing a composite gel comprises: mixing rapeseed protein and soy protein isolate, adjusting the pH to 6-8, heating in a water bath at 80-100° C., and then immediately cooling to obtain a composite gel;

[0009] The method for preparing rapeseed protein comprises the following steps: crushing defatted rapeseed meal, dissolving it in a sodium chloride solution at a solid-liquid ratio (g / mL) of 1:8-12, adjusting the pH to 7.5-8.5 and reacting for 30-60 minutes, centrifuging and membrane filtering the product, centrifuging again, dialyzing, and drying to obtain the rapeseed protein.

[0010] In a preferred embodiment, the concentration of the sodium chloride solution is 0.2-1.0 mol / L.

[0011] In one preferred embodiment, the molecular weight cut-off of the membrane filtration is no greater than 50 kDa.

[0012] In one preferred embodiment, the centrifugal rotation speed is 7000-9000r / min.

[0013] In one preferred embodiment, the rotation speed of the secondary centrifugation is 3000-4000 r / min.

[0014] In one preferred embodiment, the pH is adjusted using a base.

[0015] In one preferred embodiment, the concentration of the base is 1-2 mol / L.

[0016] In a preferred embodiment, the drying is vacuum freeze drying.

[0017] In one preferred embodiment, the drying time is 10-14 hours.

[0018] In a preferred embodiment, the relative molecular weight of the rapeseed protein is no more than 50 kDa.

[0019] In a preferred embodiment, the rapeseed protein comprises relative molecular weights of 26-38 kDa, 18-24 kDa and 10-15 kDa.

[0020] In one preferred embodiment, the pH is 7-8.

[0021] When the pH is too low, both rapeseed protein and soy protein isolate show low solubility, and the solubility of rapeseed protein increases with the increase of pH value. However, when the pH is too high, the apparent color of the composite gel deepens and has a distinct alkaline odor, which has a negative effect on the sensory evaluation of the gel and significantly reduces the hardness of the composite gel.

[0022] When the pH is too low, the hardness, elasticity, cohesion and chewiness of the composite gel are poor. It can be inferred that the gel structure formed at a low pH is weak and there is no obvious interaction between soy protein isolate and rapeseed protein. On the contrary, when the pH is 7-8, the composite gel exhibits better texture characteristics, which is closely related to the solubility results.

[0023] When the pH is too low, the gel structure of the composite gel is poor. When the pH is 7-8, the water holding capacity of the composite gel is significantly improved. When the pH value is greater than 8, the taste has obvious astringency and bitterness in the sensory evaluation, and food additives are needed to cover up the bitterness and astringency in the design of food formulas.

[0024] In one preferred embodiment, the mass ratio of soy protein isolate to rapeseed protein is (5-9):(1-5).

[0025] During the solubility test, the addition of rapeseed protein in the aqueous solution changed the hydrophilic-hydrophobic balance in the system, increased the soluble protein aggregates, and improved the solubility of the composite gel. Soy protein isolate is the main body of the gel network structure. The reduction of soy protein isolate content will make the gel properties of the composite protein worse, which is not conducive to the improvement of gel performance.

[0026] In one preferred embodiment, the mass ratio of soy protein isolate to rapeseed protein is (8-9):(1-2).

[0027] When the mass ratio of soy protein to rapeseed protein is (8-9):(1-2), the hardness is significantly greater than that of pure soy protein gel, and the cohesion of the composite gel is significantly greater than that of pure soy protein gel. At this time, the rapeseed protein and soy protein in the composite gel system have a specific interaction, which strengthens the composite gel structure and makes the gel structure more stable. As the proportion of rapeseed protein increases, the hardness of the composite gel gradually decreases. Below this range, the composite gel behaves as a weak gel with a loose gel structure, no rigid structure, low hardness, and decreased cohesion.

[0028] In a preferred embodiment, the rapid cooling is performed by using an ice water bath for rapid cooling.

[0029] In the process of heat-induced protein gel formation, the heated gel needs to be cooled quickly to "anneal", which is conducive to the rapid formation of the protein gel network structure. When cooled at room temperature, the water in the gel will evaporate excessively, causing the gel surface to crack and the gel's texture properties to deteriorate.

[0030] In a preferred embodiment, the rapeseed protein has a protein content of 75-85%.

[0031] In a preferred embodiment, the protein content of the soy protein isolate is 75-85%.

[0032] The protein content in rapeseed protein and soy protein isolate is too low, and the solubility of rapeseed crude protein is extremely poor, which is not conducive to the preparation of rapeseed protein gel and the development of rapeseed protein products; the protein content is 75-85%, the solubility is moderate, and it can be used for the development of food gel products.

[0033] Based on the same inventive concept, the present invention also claims protection for the composite gel prepared by the preparation method.

[0034] Based on the same inventive concept, the present invention also claims to protect the use of the composite gel in the preparation of sausages, jellies, yogurt and ice cream.

[0035] The present invention uses rapeseed protein extracted from rapeseed meal and soy protein isolate as raw materials to prepare a composite protein gel product, and conducts a preliminary exploration of the gel properties and formation mechanism, providing a new idea for the development of new plant protein gel products.

[0036] The present invention uses soy protein isolate (SP) and rapeseed protein (RP) as raw materials, and finds that within a specific ratio range, the hardness, elasticity, cohesion and chewiness of the composite protein gel are better than those of the soy protein isolate gel, and its water holding capacity is equivalent to that of the soy protein isolate gel. In addition, the viscosity and modulus of the composite protein gel are better than those of the SP protein, and under strong shear frequency, the composite protein gel has better resistance to external forces than the SP gel; the addition of rapeseed protein has an improving effect on the gel properties of the composite protein.

[0037] Through the study of protein molecular weight, it was found that the subunit structure of rapeseed protein in the composite protein system produced a covalent effect with the subunit structure of soy protein isolate, forming a new protein aggregate; the thermal transition characteristic analysis found that the denaturation temperature of the composite gel was increased compared with the SP protein gel, and the thermal stability of the composite protein gel was improved; the Fourier infrared spectroscopy results found that there was a change in the secondary structure of soy protein isolate and rapeseed protein in the composite protein gel system, which was reflected in the transformation of β-turn to β-fold and α-helix structure, and this structural change was conducive to the formation of gel structure; the microstructure results showed that rapeseed protein could interact with soy protein isolate as a dispersed phase, making the gel surface smoother and denser. After the optimization of the present invention, the performance of the composite protein system was significantly improved compared with the separate soy protein isolate (SP) and rapeseed protein (RP), and it can be used for foods with good gel performance such as sausages, jellies, and yogurt. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The effects of pH and ratio of complex proteins on the dynamic rheology of gels;

[0039] Figure a1-a2: Changes of the composite protein gel modulus with frequency when pH = 6; Figure b1-b2: Changes of the composite protein gel modulus with frequency when pH = 7; Figure c1-c2: Changes of the composite protein gel modulus with frequency when pH = 8;

[0040] Figure 2 The SDS-PAGE electrophoresis patterns of complex proteins at different ratios;

[0041] Sample 1: SP protein; Sample 2: SR 9:1 complex protein; Sample 3: SR 8:2 complex protein; Sample 4: SR 7:3 complex protein; Sample 5: SR 6:4 complex protein; Sample 6: SR 5:5 complex protein; Sample 7: RP protein;

[0042] Figure 3 is the DSC spectrum of the composite gel;

[0043] Figure 4 This is the Fourier infrared spectrum scanning diagram of the composite gel;

[0044] Figure 5 is the secondary structure ratio of different composite gels;

[0045] Figure 6 is the microstructure of the composite gel;

[0046] a: SP gel; b: SR 9:1 complex protein gel; c: SR 8:2 complex protein gel; d: SR 7:3 complex protein gel; e: SR 6:4 complex protein gel; f: SR 5:5 complex protein gel; g: RP protein. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0048] All test data in the embodiment were processed using Excel software, and the results were expressed in the form of mean ± standard deviation; IBM SPSS Statistics 25 software was used to perform univariate analysis on the data. The test results were plotted using Origin 2024 software.

[0049] Example 1

[0050] Preparation of Rapeseed Protein-Soybean Protein Composite Gel

[0051] 1 Materials and reagents

[0052] Defatted rapeseed meal; soy protein isolate (protein content of about 80.1%) was purchased from Shaanxi Haibo Biotechnology Co., Ltd.; hydrochloric acid and sodium hydroxide were purchased from Sinopharm Group; bovine serum albumin (BSA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; biuret reagent AB liquid was purchased from Shandong Puhuifen Chemical Technology Co., Ltd.;

[0053] 2. Instruments and Equipment

[0054] Magnetic stirrer (ZNCL-B140×140): Gongyi Zhongtian Instrument Technology Co., Ltd.; Electric constant temperature water bath (DK-98-Ⅱ): Tianjin Test Instrument Co., Ltd. Food physical property tester (Texture Analyser): British Stable Micro Systems; Refrigerated high-speed centrifuge (GL-10MD): Hunan Xiangyi Instrument Co., Ltd.; pH meter (PHS-2F): Shanghai Yidian Scientific Instrument Co., Ltd.; Microplate reader (Varioskan Flash): Finland Thermo Fisher Scientific; Analytical balance (AE2204): Xiangyi Balance Instrument Equipment Co., Ltd.

[0055] 3 Test methods

[0056] 3.1 Preparation of rapeseed protein isolate

[0057] Method 1: Defatted rapeseed meal was crushed and passed through an 80-mesh sieve, and the defatted rapeseed meal powder was dissolved in a 0.2 mol / L sodium chloride solution at a solid-liquid ratio of 1:10. The pH of the mixture was adjusted to 8 with 1 mol / L sodium hydroxide solution at room temperature and then reacted for 60 minutes. The mixture was then centrifuged at 8000 g for 10 minutes, and the supernatant was collected and filtered through a membrane filtration system with a molecular weight cutoff of 50 kDa to obtain the supernatant obtained by centrifuging the membrane retentate at a speed of 4000 g. The supernatant was then dialyzed for 72 hours and concentrated, and the rapeseed protein component (protein content was about 80.9%) was obtained by vacuum freeze drying for 12 hours, and stored in a refrigerator at 4°C.

[0058] Method 2: Defatted rapeseed meal was crushed and passed through a 80-mesh sieve. A 0.17 mol / L NaCl solution was prepared and mixed evenly with the sample at a solid-liquid ratio of 1:20. The pH was adjusted to 9.5 with 0.1 mol / L NaOH. The mixture was in a 50°C water bath for 60 min and then centrifuged at 8000 r / min for 10 min. The supernatant was dialyzed for 48 h and then concentrated. The protein (protein content was about 80.9%) was obtained by vacuum freeze-drying for 12 h.

[0059] Method 3: Grind the defatted rapeseed meal and pass it through an 80-mesh sieve. Dissolve the defatted rapeseed meal powder in 0.2 mol / L sodium chloride solution at a solid-liquid ratio of 1:10. Adjust the pH of the mixture to 8 with 1 mol / L sodium hydroxide solution at room temperature and react for 60 minutes. Then, centrifuge at 8000g for 10 minutes, collect the supernatant, centrifuge at 4000g, and then dialyze for 72 hours to concentrate. Vacuum freeze-dry for 12 hours to obtain the rapeseed protein component (protein content is about 80.9%), and store it at 4°C.

[0060] 3.2 Preparation of composite gel

[0061] A certain amount of rapeseed protein (RP) and soybean protein (SP) obtained by three methods were weighed to prepare a composite protein solution with a mass fraction of 16% (w / w), wherein the mass ratio of soybean protein isolate to rapeseed protein was 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5, respectively. Letters and numbers are used to represent the soy protein-rapeseed protein composite gel, such as soy protein isolate and rapeseed protein mixed in a ratio of 9:1 is recorded as SR 9:1. The pH value of the composite protein solution was adjusted to 6, 7, 8, and 9 with 1 mol / L sodium hydroxide or hydrochloric acid solution. The prepared protein solution was stirred evenly with a magnetic stirrer, sealed with plastic wrap, and heated in a constant temperature water bath at 95°C for 30 minutes. After heating, it was immediately cooled with an ice water bath, and the prepared gel was placed in a refrigerator at 4°C and stored overnight. Since rapeseed protein at a concentration of 16% (w / w) cannot form a gel with a specific structure, the gel structure is loose and the strength is very poor, the pure rapeseed protein gel is not studied here.

[0062] The following performance analysis was performed on the above gel:

[0063] 3.1 Solubility determination of complex protein solution

[0064] Refer to the method of Zhang Mengqi (Zhang Mengqi. Study on the interaction between egg protein and soy protein isolate and its gel properties [D]. Jiangnan University, 2021.) and make slight modifications. Dilute the prepared composite protein solution to a concentration of 1% (w / w), take 10 mL of composite protein solutions of different pH or different proportions, centrifuge at room temperature and 5000 rpm for 10 minutes, take the supernatant, and use the biuret method to determine the protein content before and after centrifugation, and use BSA as the standard to make a standard curve. Each group of samples was measured three times and the average value was taken. The solubility (%) is the ratio of the protein content in the supernatant after centrifugation to the protein content in the solution before centrifugation. The solubility results of the soy protein-rapeseed protein composite solution prepared by method 1 are shown in Table 1.

[0065] Table 1 Solubility of soy protein-rapeseed protein composite solutions prepared by method 1 at different pH values ​​or different ratios

[0066]

[0067] Note: Different lowercase letters in the same column indicate significant differences (P<0.05), the same below; the solubility unit is %.

[0068] Protein solubility plays a crucial role in influencing protein gel properties and food processing, and is a prerequisite for other excellent functional properties. However, due to protein denaturation and aggregation during industrial extraction, industrially produced plant proteins usually have lower solubility and poorer functional properties than those produced in the laboratory. Therefore, a deeper understanding of protein solubility may help to better utilize plant proteins.

[0069] As shown in Table 1, under the condition of pH = 6, both soy protein and rapeseed protein showed the lowest solubility, which were 24.33% and 31.63% respectively; when the pH rose to 8, rapeseed protein showed the best solubility (87.40%), which was significantly greater than the solubility of soy protein. This is because both rapeseed globulin and albumin have good solubility in an alkaline environment. When pH = 9, the apparent color of the composite gel of all proportions deepened, and there was a distinct alkaline odor, which had a negative effect on the sensory evaluation of the gel, and the hardness of the composite gel decreased significantly. Under the same pH conditions, the solubility of the composite protein solution showed a gradual upward trend with the increase of rapeseed protein content. It can be inferred that the addition of rapeseed protein changed the hydrophilic and hydrophobic balance in the system, increased the soluble protein aggregates, and had a positive effect on the solubility of the composite protein system.

[0070] At pH 7, the solubility data of the soy protein-rapeseed protein composite solution prepared by method 2 and the soy protein-rapeseed protein composite solution prepared by method 3 are as follows:

[0071] Table 2 Solubility of soy protein-rapeseed protein composite solutions prepared by different methods

[0072]

[0073] Note: Different lowercase letters in the same row indicate significant differences (P<0.05), the same below; the solubility unit is %.

[0074] As can be seen from Table 2, the solubility of the rapeseed protein composite solutions prepared by different preparation methods is significantly different. Compared with method 1, the lower solubility of method 2 may be due to the denaturation of the protein sample under strong alkali (pH to 9.5), which loses solubility and becomes loose in structure, exposing more hydrophobic groups, resulting in reduced sample solubility. Compared with method 1, the reduced solubility of method 3 may be due to the difference in the amino acid composition of the obtained protein, which affects its solubility characteristics.

[0075] 3.2 Determination of texture properties of composite protein gel

[0076] Refer to the method of Jia Changxiu (Jia Changxiu. Research on the construction and main properties of a mixed system based on soy protein isolate and whey protein [D]. Shenyang Agricultural University, 2022.) and make slight modifications. The composite protein gel was cut into small cubes of uniform size, and the texture properties of the composite gel were determined using a food physical property tester. The parameters of the physical property tester were set as follows: P / 36 probe was selected, the speed before the test was 2mm / s, the speed after the test was 1mm / s, the interval time was 5s, the compression deformation was 50%, and the trigger force was 5g. The texture properties of the soy protein-rapeseed protein composite solution prepared by method 1 are shown in Table 3.

[0077] Table 3 Hardness values ​​of composite gels at different pH

[0078]

[0079] At pH 7, the hardness data of the soy protein-rapeseed protein composite solution prepared by method 2 and the soy protein-rapeseed protein gel prepared by method 3 are as follows:

[0080] Table 4 Hardness value of soybean protein-rapeseed protein prepared by different methods under pH=7 condition

[0081] Group Method 1 Method 2 Method 3 SP <![CDATA[982.03±5.06 b ]]> <![CDATA[982.03±5.06 b ]]> <![CDATA[982.03±5.06 b ]]> SR 9:1 <![CDATA[1079.93±40.40 a ]]> <![CDATA[452.66±21.09 c ]]> <![CDATA[543.32±18.97 b ]]> SR 8:2 <![CDATA[600.96±35.72 a ]]> <![CDATA[385.66±11.22 c ]]> <![CDATA[419.76±23.21 b ]]> SR 7:3 <![CDATA[573.92±6.95 a ]]> <![CDATA[263.66±32.18 c ]]> <![CDATA[369.12±34.31 b ]]> SR 6:4 <![CDATA[217.66±7.20 a ]]> <![CDATA[125.66±20.54 b ]]> <![CDATA[112.68±35.17 b ]]> SR5:5 <![CDATA[202.06±0.49 a ]]> <![CDATA[109.66±32.54 b ]]> <![CDATA[107.54±24.31 b ]]>

[0082] Note: Different lowercase letters in the same line indicate significant differences (P<0.05), the same below; the unit of solubility is %. Hardness is the force required to deform an object. In the food field, it is a physical indicator that describes the hardness of food and the strength required for chewing. As shown in Table 3, at pH = 7, the hardness of the composite gel first increased and then decreased with the addition of rapeseed protein. When the mass ratio of soy protein: rapeseed protein was 9:1, the hardness was 1079.93g, which was significantly greater than the hardness of pure soy protein gel. It can be inferred that at this time, rapeseed protein and soy protein in the composite gel system had a specific interaction, which enhanced the structure of the composite gel. Because pure rapeseed protein cannot form a structural gel under this condition, as the proportion of rapeseed protein continues to increase, the hardness of the composite gel gradually decreases. SR 6:4 and SR 5:5 appear as weak gels with loose gel structures and no rigid structure. This is because soybean protein is the main component of the gel network in the soybean protein-rapeseed protein composite gel system. When pH = 6, soybean protein isolate forms a gel network structure due to self-aggregation. The addition of rapeseed protein leads to a decrease in the degree of cross-linking between protein molecules and a decrease in gel strength. When pH = 8, the hardness of the composite gel shows a general increase trend, and the alkaline environment has a significant promoting effect on the formation of the composite gel. This is because the composite protein network structure becomes dense and porous under high temperature and alkaline conditions, and the hardness and strength of the protein gel increase. The high temperature and alkaline environment break the disulfide bonds between and within the protein molecules, unfold the protein structure, and promote the aggregation between protein molecules. At the same time, in an alkaline environment, the solubility of the composite protein solution increases significantly, which strengthens the cross-linking between protein molecules, which corresponds to the results in Table 1. In contrast, at pH = 6, the solubility of the composite protein solution decreases significantly, the insoluble protein content increases and precipitates in the form of precipitation, and no chemical bond changes occur between protein molecules, so a randomly aggregated protein network structure can only be formed under hydrophobic interactions.

[0083] Other texture properties of the soy protein-rapeseed protein composite solution prepared by method 1 are shown in Table 5.

[0084] Table 5 Other texture characteristics of composite gels at different pH

[0085]

[0086] Elasticity refers to the ability of an object to deform under the action of an external force and to return to its original state when the external force is removed. As shown in Table 2, under the condition of pH = 6 or 7, the composite protein gels with pure soy protein and rapeseed protein added less than 30% showed poor elasticity. However, within this range, as the addition of rapeseed protein reached 30%, that is, when SR was 7:3, the elasticity of the composite gel showed a significant increase trend. It is worth noting that when the addition of rapeseed protein was 30%-50%, that is, the three composite protein gels of SR 7:3, SR 6:4 and SR 5:5 were weak gel structures, with poor gel structures and low hardness, while the elasticity test results of the physical property tester showed good elasticity values, which were significantly greater than other composite protein gels. This is because the weak gel structure did not undergo obvious deformation when squeezed, and the deformation time required for the second extrusion did not change much, and the elasticity results obtained were close to 1, which cannot indicate that these three composite protein gels have good gel structures and elastic properties. When pH = 8, the elastic properties of soy protein gel and composite gel increased significantly. The SR 9:1 and 8:2 composite gels had comparable elastic properties to pure soy protein while significantly improving their hardness properties, indicating that the alkaline environment significantly promoted the formation of a new gel structure between rapeseed protein and soy protein.

[0087] Cohesion is used to characterize the internal contraction force of an object. It is a manifestation of the object's ability to resist deformation and can also reflect the strength of the intermolecular bonding within the object. The calculation formula for the cohesion index of the physical property analyzer is the ratio of the two compression force areas. Since the weak gel structure will not have a significant impact on the gel force area when the probe is compressed, the cohesion result of the gel at this time cannot prove the degree of mutual binding of proteins within the gel molecules, so no excessive analysis is performed. As can be seen from Table 2, under neutral and alkaline conditions, the effect of rapeseed protein addition on the cohesive properties of the composite gel generally shows a trend of first increasing and then decreasing. At pH = 7, the cohesion of the SR 9:1 composite gel (0.454) is significantly greater than that of the pure soy protein gel (0.311). Combined with the hardness and elasticity results, it shows that the soy protein and rapeseed protein in the SR 9:1 composite gel have a certain interaction, which makes the gel structure more stable. When pH = 8, the cohesion of soy protein and composite protein gels increased significantly. On the one hand, it shows that the alkaline environment promotes the formation of protein gel and there is interaction between soy protein and rapeseed protein. On the other hand, when the addition of rapeseed protein exceeds 30%, the cohesion of the gel decreases, the structure of the composite gel weakens, and excessive rapeseed protein weakens the composite gel.

[0088] Chewing degree can be interpreted as the energy required to chew solid food. It is a measure of the ability of gel to withstand external forces and a comprehensive reflection of hardness, elasticity and cohesion. From Table 2, it can be seen that the chewiness of the gel at pH = 8 is much greater than that of the gel at pH = 6 and 7. At the same time, the chewiness of SR 9:1 and SR 8:2 composite gels is significantly greater than that of other protein gels, which is consistent with the results of hardness and elasticity. This shows that in the SR 9:1 composite gel, there is a strong interaction between soy protein and rapeseed protein, which is conducive to the improvement of the performance of soy protein gel.

[0089] 3.3 Determination of water holding capacity of composite protein gel

[0090] The water retention rate of gel was determined by centrifugation. A certain amount of composite protein gel was weighed and recorded as m1, placed in a 50mL centrifuge tube, and centrifuged at 10000g for 30 minutes at room temperature. After centrifugation, the supernatant in the centrifuge tube was poured out, and the water on the surface of the gel was carefully wiped with filter paper, and the mass of the gel after centrifugation was recorded as m2. The formula for the water retention rate of gel is as follows:

[0091]

[0092] The gel water holding rate results of the soy protein-rapeseed protein composite solution prepared by method 1 are shown in Table 6.

[0093] Table 6 Water holding capacity of composite gel at different pH values ​​and different proportions (P<0.05)

[0094]

[0095] Water holding capacity is one of the most important indicators for evaluating the quality of protein gel, which reflects the water-locking capacity of the protein gel network structure. As shown in Table 6, the soy protein gel and composite gel at pH = 6 have certain water holding capacity, and rapeseed protein has no significant effect on the water holding capacity of the composite gel. When pH = 7 or 8, the water holding capacity of the protein gel is significantly increased compared with that at pH = 6; as the addition of rapeseed protein exceeds 30%, the water holding capacity of the composite protein gel shows a gradual downward trend, indicating that the structure of the composite protein gel becomes weaker at this time, and the composite gel may have a loose structure, larger gel pores, and a decreased water-locking capacity. Compared with SR 9:1 and SR 8:2 composite protein gels, the water holding capacity of the three SP gels is not sensitive to the change of pH from 6 to 7 and is maintained at a level close to 100%, indicating that the water-locking capacity of the three protein gels has reached a threshold, and the addition of rapeseed protein (0-20%) at this time does not destroy the gel structure of the composite protein, which is equivalent to the structure of SP gel.

[0096] The following studies are all conducted on the soy protein-rapeseed protein composite solution prepared by method 1, and the rapeseed protein prepared by other methods is not included in the following studies.

[0097] 3.4 Dynamic rheological properties of composite gels

[0098] Dynamic rheology test refers to an oscillatory shear test under periodic stress or strain conditions, also known as dynamic vibration frequency sweep measurements. The test conditions are as follows: prepare a soy protein-rapeseed protein composite gel with a mass fraction of 16% (w / w) and different proportions at three pH values. Rotational rheometer parameter settings: Use a 40mm plate with a gap of 1mm, place the composite protein gel between two parallel plates, adjust the gap between the two parallel plates to 1mm, remove excess protein gel, and add an appropriate amount of silicone oil to the gap of the plate to prevent moisture loss. The temperature is set to 25°C, the scanning strain is 1%, the frequency range is 0.1Hz-100 Hz, and the elastic modulus (G') and viscous modulus (G") are recorded. The results are as follows Figure 1 As shown in the figure, Figure a1-a2: the change of the composite protein gel modulus with frequency when pH = 6; Figure b1-b2: the change of the composite protein gel modulus with frequency when pH = 7; Figure c1-c2: the change of the composite protein gel modulus with frequency when pH = 8.

[0099] The shear rate-viscosity change curve shows that the SR composite protein system with different proportions shows an obvious viscosity change trend. According to the change curves of the elastic modulus and viscous modulus of SP gel and SR composite protein gel with frequency, the effect of rapeseed protein addition on the viscoelasticity of SR composite protein gel is further analyzed. The elastic modulus (G') mainly reflects the elasticity of the gel and the ability of the gel to recover deformation under the action of external force. The viscous modulus (G") is the viscosity response value of the gel, which reflects the fluidity of the gel under the action of external force. Overall, the modulus of SP protein gel and SR composite protein gel increases with the increase of frequency, indicating that the soy protein isolate-rapeseed protein composite gel is a weak gel structure in a solid-like state with a certain gel network structure and obvious viscoelastic characteristics. At this time, the protein gel structure is a physical gel supported by non-covalent bonds. The non-covalent bond energy is small and is greatly affected by the frequency. Figure 1 From a1 and a2, it can be seen that the elastic modulus of SP protein gel is significantly greater than that of SR composite protein gel at pH = 6; when the frequency is greater than 50 Hz, SR 6:4 and SR 5:5 composite protein gels show a G' and G" crossover phenomenon (G">G'), at which time the composite protein gel is dispersed and viscous, and the gel strength decreases. This indicates that the interaction between rapeseed protein and soy protein isolate is hindered under the condition of pH = 6, and the addition of rapeseed protein weakens the strength of SR composite protein gel, showing a weak gel structure with high viscosity.

[0100] In the low-medium frequency band (0-50Hz) under neutral conditions, the elastic modulus of SR 8:2 and SR 7:3 composite protein gels increased significantly compared with pH=6. SR 9:1 composite protein gel and SP gel had comparable elastic modulus. Protein molecules were arranged regularly under the action of external force. Rapeseed protein and soy protein isolate interacted with each other and did not weaken the strength of soy protein isolate gel. When the action frequency was greater than 80Hz, the elastic modulus of SP gel and SR composite protein gel showed a downward trend. The SR composite protein gel showed a crossover phenomenon of G' and G", indicating that under the action of high-frequency external force, the gel molecular structure became chaotic and the gel strength decreased, and the gel network structure became unstable.

[0101] Depend on Figure 1 From c1 and c2, we can see that under the condition of pH = 8, the elastic modulus of the three composite protein gels SR 9:1, SR 8:2 and SR 7:3 maintain an upward trend at the entire frequency range, and are significantly greater than the elastic modulus of the SP protein gel, indicating that the alkaline environment promotes the interaction between rapeseed protein and soy protein isolate. This may be related to the denaturation of soy protein isolate under alkaline conditions, the unfolding of protein molecular chains, the high solubility of rapeseed protein, and the interaction between it and the soy protein isolate gel as a dispersed phase, thereby improving the viscoelasticity of the composite protein.

[0102] 3.4 Determination of relative molecular weight of composite gel

[0103] Refer to the method of Geng Rui (Geng Rui. Effects of pH shift combined with mild heat treatment on the structure and functionality of soy protein isolate and its interaction with myofibrillar protein [D]. Northeast Agricultural University, 2015.) and make slight modifications. 4 mg / mL protein sample was mixed with 0.01 mol / LTris-HCl buffer at pH 8.0 containing 0.05% (w / v) bromophenol blue, 2% (w / v) SDS and 10% (w / v) glycerol in a 1:1 ratio to make the protein concentration in the sample 2 mg / mL. The sample was electrophoresed after boiling water bath for 5 minutes. The electrophoresis used 40% concentration of stacking gel, 12% concentration of separation gel, 1mm gel plate, and the sample volume was 10μL; the voltage of the stacking gel was 80V, and the voltage of the separation gel was 120V. After the electrophoresis, it was stained with Coomassie Brilliant Blue Rapid Stain for 30 minutes, and decolorized with deionized water several times until the background color faded. A total of 7 groups of samples. The results are as follows Figure 2 As shown in the figure, sample 1: SP protein; sample 2: SR 9:1 complex protein; sample 3: SR 8:2 complex protein; sample 4: SR 7:3 complex protein; sample 5: SR 6:4 complex protein; sample 6: SR 5:5 complex protein; sample 7: RP protein.

[0104] Soy protein isolate is mainly composed of β-conglycinin (α′, α and β) and soybean globulin (A and B) subunits, and the bands are mainly concentrated around 72kDa, 46kDa, 37kDa, 30kDa and 20kDa. Rapeseed protein is composed of several lower peptides with relative molecular weights of 33kDa, 23kDa, 20kDa and 13kDa, mainly α-peptide (26.7-37.1kDa), β-peptide (18.3-22.9kDa) and rapeseed albumin (10-15kDa) in rapeseed globulin. With the increase of the addition ratio of rapeseed protein, the color of the subunit bands of soy protein isolate in the composite protein gradually becomes lighter, and the color of the bands of globulin and albumin in rapeseed protein also becomes lighter, especially in the two composite proteins SR 9:1 and SR 8:2. The subunit band of rapeseed protein in the composite protein decreased, indicating that part of the rapeseed protein and soybean protein isolate were cross-linked. Combined with the increase in the solubility of the composite protein and the change in the gel properties at pH = 7 in the previous article, it can be seen that the increase in the solubility of the composite protein provides conditions for the cross-linking between proteins. There is a covalent effect between rapeseed protein and soybean protein isolate in the SR 9:1 composite protein, which improves the hardness, elasticity and other texture properties of the SR 9:1 composite protein gel. In terms of rheological behavior, the SR 9:1 composite protein gel also has the largest elastic modulus. In addition, as the addition ratio of rapeseed protein continues to increase, the proportion of high molecular weight subunits in the composite protein decreases. Combined with the gel properties, soybean protein isolate is the main body of the gel network structure. The reduction in the content of soybean protein isolate will make the gel properties of the composite protein worse, which is not conducive to the improvement of gel performance.

[0105] 3.5 Determination of thermal transition characteristics of complex proteins (Differential scanning calorimetry, DSC)

[0106] The freeze-dried sample was manually ground and pulverized, passed through an 80-mesh sieve, and set aside. Weigh 8 to 10 mg of the sample and place it in a high-pressure aluminum pan and seal it. Differential scanning calorimeter (DSC) scanning conditions: scanning temperature range 25°C-120°C, heating rate 5°C / min, N2 protection, N2 flow rate 50mL / min. An empty aluminum pan was used as a reference, and the denaturation temperature (Td) was recorded. The results are as follows: Figure 3 The heat absorption peak in the curve indicates that the protein gel denatures at this temperature and the protein structure unfolds. Figure 3It can be seen that the SP protein gel has two thermal absorption peaks, and the denaturation temperatures are 78.75℃ and 93.09℃, corresponding to the 7S subunit protein and the 11S subunit protein; the RP protein gel has absorption peaks at 75.76℃ and 85.03℃, and it can be found that the thermal stability of the RP protein is weaker than that of the SP protein, which is related to the differences in the subunit composition and structural stability of the subunit proteins of the two proteins. With the addition of rapeseed protein, the denaturation temperature of the SR9:1 protein gel increases slightly, indicating that the thermal stability of the protein gel is improved and there are new protein cross-linking structures in the gel molecules. When the addition amount of rapeseed protein exceeds 30%, the denaturation temperature of the three composite proteins SR 7:3-SR 5:5 gradually decreases, which matches the decrease in the gel formation temperature of the composite proteins in the rheological behavior results, indicating that the gel strength formed by these three composite proteins is weak and the thermal stability is poor.

[0107] 3.6 Determination of amino acid composition of composite gel

[0108] Refer to the method of Zhang Mengqi (Zhang Mengqi. Study on the interaction between egg protein and soy protein isolate and its gel properties [D]. Jiangnan University, 2021.) and make slight modifications. First, weigh about 0.3g of gel sample in the hydrolysis tube, add 8mL of 6mol / L hydrochloric acid solution to soak it, and seal it with nitrogen for 3min. Secondly, all samples were hydrolyzed at 120℃ for 22h, poured into a 25mL volumetric flask after hydrolysis, neutralized with 4.8mL of 10mol / L sodium hydroxide solution, and then fixed to volume with distilled water. Then filter the solution with double-layer filter paper, and then measure 1mL of the filtrate sample into a centrifuge tube for centrifugation. Finally, take 400μL of the supernatant after centrifugation and place it in a liquid sample bottle, and determine the content of each amino acid by an amino acid-specific high-performance liquid chromatograph. The results are shown in Table 7.

[0109] Table 7 Amino acid composition of composite gel in different proportions

[0110]

[0111] The amino acid composition of protein mainly affects the nutritional value and functional properties of protein. For protein gel, the ratio of hydrophilic-hydrophobic amino acids directly affects the behavior of protein in aqueous solution, thereby affecting the quality characteristics of gel. Table 7 shows the content of each amino acid in composite protein gels of different proportions. The dominant amino acids in soy protein isolate are glutamic acid (176.8 mg / g) and aspartic acid (100.639 mg / g), methionine (11.488 mg / g) and cysteine ​​(the cysteine ​​content in SP protein is too low, exceeding the lower limit of instrument detection). Rapeseed protein also contains more glutamic acid (106.958 mg / g) and a certain amount of cysteine ​​(8.878 mg / g), of which cysteine ​​can participate in the formation of disulfide bonds, but the total amount of amino acids in soy protein isolate is much greater than that in rapeseed protein, which indicates that there will be great differences in functional properties between soy protein isolate and rapeseed protein.

[0112] The total content of hydrophilic amino acids in SP protein is 576.815 mg / g, the total content of hydrophobic amino acids is 297.519 mg / g, and the ratio of hydrophilic amino acids to hydrophobic amino acids is 1.939; the total content of hydrophilic amino acids in RP protein is 316.967 mg / g, the total content of hydrophobic amino acids is 144.942 mg / g, and the ratio of hydrophilic amino acids to hydrophobic amino acids is 2.187. According to the spatial structure of protein, hydrophilic amino acids are generally located on the surface of the protein structure, while hydrophobic amino acids are hidden inside the protein structure, indicating that rapeseed protein is more hydrophilic than soy protein isolate, which will affect the solubility and gel properties of both. When the amount of rapeseed protein added increases, the hydrophilicity ratio of the composite protein system also increases, and rapeseed protein and soy protein isolate can interact better in aqueous solution.

[0113] 3.7 Determination of protein secondary structure in composite gel (Fourier transform infrared spectroscopy, FTIR)

[0114] Refer to the method of Zhang Jinchuang (Zhang Jinchuang. Conformational changes and quality control of peanut protein during high-moisture extrusion [D]. Chinese Academy of Agricultural Sciences, 2019.) and make slight modifications. The gel sample was fully dried under an infrared lamp to remove moisture, and then a small amount of sample was mixed with potassium bromide, ground in an agate mortar for 15 minutes, and pressed into tablets under 10kg pressure. Fourier transform infrared spectrometer operating conditions: spectral range 500-4000cm-1, resolution 4cm, signal scanning accumulation 64 times, each sample was measured 3 times, and the average value was calculated. EZ OMNIC software and peak fit 4 were used to perform corresponding spectral processing and analysis on the amide I band (between 1600-1700cm-1), identify the corresponding relationship between the position of each sub-peak and the secondary structure component, and obtain the protein secondary structure ratio. The results are as follows Figure 4 and Figure 5 shown. Figure 4 The Fourier transform infrared spectra of the composite protein gels with different ratios are shown in Figure 2. The second-order derivative deconvolution of the FTIR data is performed using Peak fit 4 software to obtain the changes in the secondary structure of the protein, and the percentage of the peak area to the total area of ​​the amide I band is used to represent the proportion of the secondary structure of the protein, such as Figure 5 As shown. The amide I band (1600cm-1-1700cm-1) concentrates the information of protein secondary structure, mainly β-fold (1600-1640cm-1), random curl (1640-1650cm-1), α-helix (1650-1660cm-1) and β-turn (1660-1700cm-1). The β-fold structure accounts for the largest proportion in SP protein gel, the α-helix structure accounts for the lowest proportion, and the β-turn structure accounts for the largest proportion in RP protein. With the addition of rapeseed protein, the β-fold structure (42.00%-42.22%) and α-helix structure (15.14%-15.36%) in SR 9:1 composite gel increased slightly, accompanied by a decrease in the proportion of β-turn structure (25.288%-24.717%), which intuitively reflects that soy protein isolate and rapeseed protein can interact with each other, which is manifested in the mutual conversion of protein secondary structure. Both β-folds and α-helices are mainly maintained by hydrogen bonds, which can explain the change in the modulus of the complex protein during gel formation: that is, at high temperatures, the complex protein denatures, the spatial structure unfolds, and the internal hydrophobic groups are exposed. When the temperature drops, β-folds and α-helices are generated between rapeseed protein and soy protein isolate under the action of hydrogen bonds to form a gel network structure, and the gel modulus rises sharply. A larger proportion of β-folds and α-helical structures means better gel performance.

[0115] When the amount of rapeseed protein added is further increased, it is more obvious that the β-fold structure and the β-turn structure are constantly transforming. The β-turn structure in the composite protein gel is constantly increasing, and the β-fold structure is constantly decreasing, which is directly reflected in the functional properties of the composite protein gel, such as the deterioration of quality, reduced water holding capacity and reduced texture characteristics. The value of α-helix / β-fold increases with the increase of rapeseed protein addition, indicating that the molecular chain in the composite protein becomes flexible, which is reflected in the reduction of rigid structural characteristics such as gel hardness and chewiness and the increase of elasticity and gel viscosity modulus.

[0116] 3.8 Microstructure observation of composite gel

[0117] The freeze-dried gel sample was fixed on the sample stage, and after being gold-sprayed using a gold-spraying instrument, it was placed under a scanning electron microscope for magnified observation and photography. Figure 6As shown in the figure, a: SP gel; b: SR 9:1 composite protein gel; c: SR 8:2 composite protein gel; d: SR 7:3 composite protein gel; e: SR 6:4 composite protein gel; f: SR 5:5 composite protein gel; g: RP protein. It can be seen from the figure that composite protein gels with different proportions have significantly different gel network structures. The surface of SP protein gel is relatively rough and uneven, showing a layered and folded structure, which is a typical gel structure dominated by β-folding. Rapeseed protein presents an obvious globular protein structure. When rapeseed protein is added to the soy protein isolate system, such as Figure 6 As shown in figure b, the layered wrinkled structure on the surface of SR 9:1 composite protein gel is significantly reduced, and the gel surface tends to be smooth. This may be because rapeseed protein as a filling phase interacts with soy protein isolate based on hydrogen bonding. During the formation of protein gel, the proteins aggregate with each other, and rapeseed protein is filled into the uneven layered structure of soy protein isolate gel, making the gel structure more complete, increasing the rigidity of the gel, and improving the functional properties of the gel.

[0118] When the proportion of rapeseed protein added continues to increase, it can be found that the surface of the gel begins to have an uneven structure, gradually developing into a loose, porous and rough gel. At this time, the gel properties of the composite protein (such as SR 6:4 and SR 5:5) are poor, and the loose and porous network structure leads to a decrease in the water holding capacity of the gel. This is because rapeseed protein is a protein with poor gel properties and low protein molecular weight. When the concentration of soy protein isolate is reduced, rapeseed protein and soy protein isolate phase separation occur, resulting in an uneven gel structure and the inability to form a rigid gel network structure.

[0119] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. The replacement may be a replacement of a part of the structure, device, method step, or a complete technical solution. Any equivalent replacement or change according to the technical solution and the inventive concept of the present invention shall be covered within the protection scope of the present invention.

Claims

1. A method for preparing a composite gel, characterized in that: include: After mixing rapeseed protein and soy protein isolate, adjusting the pH to 6-8, heating in a water bath at 80-100° C., and then immediately cooling to obtain a composite gel; The mass ratio of soy protein isolate to rapeseed protein is (5-9):(1-5); the method for preparing rapeseed protein comprises the following steps: crushing defatted rapeseed meal, dissolving it in a sodium chloride solution at a solid-liquid ratio of 1:8-12, adjusting the pH to 7.5-8.5 and reacting for 30-60 minutes, centrifuging the product, membrane filtering, centrifuging it again, dialyzing it, and drying it to obtain rapeseed protein.

2. The preparation method according to claim 1, characterized in that: The molecular weight cut-off of membrane filtration is no more than 50 kDa.

3. The preparation method according to claim 1, characterized in that: The pH is adjusted using alkali; the concentration of the alkali is 1-2 mol / L.

4. The preparation method according to claim 1, characterized in that: The pH is 7-8.

5. The preparation method according to claim 1, characterized in that: The mass ratio of soy protein isolate to rapeseed protein is (8-9):(1-2).

6. The preparation method according to claim 1, characterized in that: The rapid cooling is to use an ice water bath to quickly cool down the temperature.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The protein content of the rapeseed protein is 75-85%.

8. The preparation method according to any one of claims 1 to 6, characterized in that: The protein content of the soy protein isolate is 75-85%.

9. The composite gel prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the composite gel according to claim 9 in the preparation of sausages, jelly, yogurt and ice cream.

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