Anti-freezing soybean protein isolate as well as preparation method and application thereof

Abstract

The invention relates to the technical field of soybean protein processing, and particularly discloses an anti-freezing soybean protein isolate as well as a preparation method and application thereof. Comprising the following steps: mixing soybean meal with water, filtering, adjusting acidity, standing and centrifuging to obtain soybean protein isolate; redissolving and homogenizing to obtain a soybean protein isolate solution; the method comprises the following steps: adding papain into a soybean protein isolate solution for enzymolysis and enzyme inactivation, then adding transglutaminase for enzyme crosslinking, and drying after enzyme inactivation to obtain the anti-freezing soybean protein isolate. The modified anti-freezing soybean protein isolate is prepared by a method of firstly hydrolyzing the soybean protein isolate by enzyme and then carrying out enzyme crosslinking, and the formed gel can still maintain the water holding rate of 85% or above within the freezing storage period of 20 days, so that the texture deterioration of the gel is delayed, and meanwhile, the viscosity of the anti-freezing soybean protein isolate is reduced; the pure plant source characteristic of the anti-freezing soybean protein isolate is maintained.

Description

Technical Field

[0001] The invention relates to the technical field of soybean protein processing, and in particular to an antifreeze type soybean protein isolate and a preparation method and application thereof. Background Art

[0002] Soy protein isolate is widely used in food production due to its good gel properties. In recent years, some frozen foods have been processed by adding soy protein isolate to improve their gel quality. However, in a frozen environment, the gel-forming ability of soy protein isolate and the quality characteristics of the gel after formation will change significantly. This undoubtedly has a great impact on the quality of soy gel and the gel added to the food system. The most obvious change is that the water retention capacity of the protein gel decreases after frozen storage, resulting in a large amount of water seeping out of the food after thawing. At the same time, the hardness of the gel after freezing increases significantly, which greatly affects the quality of the food. Therefore, how to modify the soy protein isolate in an appropriate manner so that its gel water retention performance is relatively stable during the frozen storage period has become an urgent problem facing the frozen food industry and soy protein isolate processing companies.

[0003] There have been related studies to improve the gel properties of soy protein isolate by modification. For example, by repeatedly freezing and thawing the soy protein isolate solution, the gel hardness and gel elasticity are improved; deamidated soy protein isolate is prepared by alkaline protease hydrolysis, and the modified soy protein isolate has improved gel properties and can form a good co-gel with muscle salt-soluble protein in meat; soy protein isolate is treated by combining high-pressure homogenization with heating. However, the soy protein isolate obtained by the above preparation methods has a decreased water retention capacity and increased gel hardness in a frozen environment, resulting in quality deterioration.

[0004] The application publication number is "CN104256054A", and the name of the patent application is: A kind of antifreeze soy protein isolate and its preparation method. In this patent application, gelatin is added, and the added gelatin is cross-linked with the whole soy protein molecule by transglutaminase, and the gelatin molecule is cross-linked to the soy protein molecule, thereby increasing the antifreeze property of the soy protein molecule, and further achieving the purpose of maintaining the moisture of the soy protein gel and improving the texture characteristics of the soy protein gel after freezing. However, the additional addition of gelatin molecules increases the production cost. At the same time, since gelatin comes from animals, the cross-linking of gelatin molecules and soy protein molecules changes the pure plant source characteristics of soy protein, and the viscosity of the prepared cross-linked soy protein is too large, which greatly limits its spray drying production process in the industrial process.

[0005] Therefore, it is necessary to develop a method for preparing a pure plant-based, low-viscosity, antifreeze-resistant soy protein isolate. Summary of the invention

[0006] In order to develop a method for preparing an antifreeze type soy protein isolate, the present invention provides an antifreeze type soy protein isolate and its preparation method and application. The preparation method provided by the present invention performs enzyme cross-linking modification on the soy protein isolate, and the water holding rate of the obtained antifreeze soy protein isolate gel can still be maintained at more than 85% during the 20-day frozen storage period, which better solves the outstanding practical problem that when soy protein isolate is currently added to frozen food, the water holding capacity of the protein gel decreases under extreme low temperature conditions, resulting in the deterioration of the quality of related foods.

[0007] The present invention provides a method for preparing an antifreeze type soybean protein isolate, comprising the following steps:

[0008] Alkali dissolution and acid precipitation: soybean meal is mixed with water, filtered, acidified, allowed to stand, and centrifuged to obtain isolated soy protein; the isolated soy protein includes 7S component and 11S component;

[0009] Redissolution: re-dissolve the soy protein isolate in water, adjust the pH to neutral, and shear and homogenize to obtain a soy protein isolate solution;

[0010] Enzymatic hydrolysis: adding papain to the soy protein isolate solution for hydrolysis, hydrolyzing the 7S component into 7S peptide segments, performing the first enzyme inactivation, and obtaining a soy protein isolate hydrolyzate containing 7S peptide segments;

[0011] The mass ratio of papain to soy protein isolate solution is 1:1000-10000;

[0012] Enzyme cross-linking: Add transglutaminase to the soy protein isolate hydrolysate and mix well, adjust the pH to neutral, and perform a cross-linking reaction to cross-link the 7S peptide segment with the 11S component, then perform a second enzyme inactivation, and obtain the antifreeze type soy protein isolate after drying.

[0013] The present invention hydrolyzes the high-viscosity 7S component in the soy protein isolate with papain into a low-viscosity and highly hydrophilic 7S peptide segment, and then crosslinks the low-viscosity and highly hydrophilic 7S peptide segment with 11S by adding transglutaminase to the hydrolyzate, which not only effectively improves the antifreeze property of the gel of the 11S component, but also reduces the high viscosity of the 7S after crosslinking, so that the obtained antifreeze type soy protein isolate has low viscosity and is closer to the natural soy protein isolate, and can be directly used for industrial spray drying processing, reducing costs. The present invention not only avoids the change of the pure plant source characteristics of the soy protein isolate gel due to the addition of exogenous substances (such as gelatin), but also maintains the moisture and texture characteristics of the soy protein isolate gel after thawing at -18°C for 20 days by self-crosslinking the highly hydrophilic 7S peptide segment with 11S, thereby improving the water holding capacity of the gel after frozen storage and improving the antifreeze property of the soy protein isolate.

[0014] Furthermore, the mass ratio of the soybean meal to water is 1:15-20.

[0015] Furthermore, the mass ratio of the papain to the soy protein isolate solution is 1:1000-5000.

[0016] Furthermore, the first enzyme inactivation process is: treating at a pressure of 100MPa to 600MPa for 5min to 20min. Using ultrahigh pressure instead of the traditional boiling water bath for enzyme inactivation further reduces the problem of increased viscosity caused by protein aggregation during the boiling water enzyme inactivation process.

[0017] Furthermore, the mass ratio of the transglutaminase to the soy protein isolate hydrolyzate is 1:500-1000.

[0018] Furthermore, the second enzyme inactivation process is: treating at a pressure of 100MPa to 600MPa for 10min to 30min.

[0019] Furthermore, the drying method is spray drying.

[0020] Furthermore, the spray drying parameters are set as follows: air inlet temperature is 170°C to 180°C, air outlet temperature is 65°C to 75°C, compressor air pressure is 0.1 MPa, and peristaltic pump injection volume is 20ml / min to 30ml / min.

[0021] The present invention also provides an antifreeze type soybean protein isolate prepared by the above-mentioned preparation method.

[0022] The present invention also provides an application of the antifreeze type soy protein isolate in the preparation of soy protein gel. The soy protein gel maintains the pure plant source characteristics, can still maintain a water retention rate of more than 85% after being thawed at -18°C for 20 days, has low hardness, stable texture and low viscosity.

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

[0024] The soy protein isolate gel prepared by the present invention maintains its pure plant source characteristics. The 7S component in the soy protein isolate is hydrolyzed into 7S peptide segments by papain, and then transglutaminase is added to the hydrolyzate to crosslink the 7S peptide segments with low viscosity and high hydrophilicity with 11S, which effectively improves the antifreeze property of the gel of the 11S component and reduces the high viscosity of the 7S after crosslinking. The method provided by the present invention uses enzyme hydrolysis combined with enzyme crosslinking to modify the soy protein isolate to obtain antifreeze soy protein isolate. The soy protein isolate gel water holding rate formed by the modified antifreeze soy protein isolate can still maintain more than 85% during the 20-day frozen storage period at -18°C, which better solves the outstanding practical problem that when the soy protein isolate is currently added to frozen food, the water holding property of the protein gel decreases under freezing conditions below -18°C, resulting in the deterioration of the quality of related foods. At the same time, the antifreeze type soybean protein isolate prepared by the present invention as a raw material has a small increase in the hardness of the antifreeze soybean protein isolate gel, which effectively prevents a large amount of water loss of the gel caused by freezing, and at the same time inhibits the increase in the hardness of the gel during the frozen storage period, delaying the deterioration of the texture of the gel.

[0025] Compared with the traditional soy protein isolate modification method (such as enzymatic cross-linking modification of soy protein isolate alone), the method provided by the present invention significantly reduces the viscosity of the soy protein isolate gel formed. The traditional method makes the viscosity of the soy protein isolate gel too high, and it is necessary to reduce the concentration of the protein solution before spray drying in the factory production process during industrial production, which greatly increases the factory production cost. The viscosity of the soy protein isolate gel prepared by the method of the present invention is similar to the viscosity of the gel formed by the unmodified soy protein isolate, and there is no significant difference, so the cost increase is greatly avoided. The preparation method of the present invention has mild modification conditions, simple process, easy operation, short production time, and the soy protein isolate gel after the modification not only has significantly improved antifreeze properties, but also has no significant difference in viscosity from the original soy protein isolate, and has broad industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The figure shows the change of gel water holding capacity of the soy protein isolate obtained in the present invention during frozen storage. In the figure, the modified soy protein isolate is the product prepared in Example 1, and the soy protein isolate is the product prepared in the comparative example.

[0028] Figure 2The figure shows the change of gel hardness of the soy protein isolate obtained by the present invention during frozen storage. In the figure, the modified soy protein isolate is the product prepared in Example 1, and the soy protein isolate is the product prepared in the comparative example.

[0029] Figure 3 The viscosity of soy protein isolate gels of different concentrations prepared from the soy protein isolate obtained in Example 1 and Comparative Examples 1-2 of the present invention is shown. In the figure, MMSPI represents the viscosity change of soy protein isolate gels of different concentrations prepared from the antifreeze type soy protein isolate obtained in Example 1; SPI represents the viscosity change of soy protein isolate gels of different concentrations prepared from the soy protein isolate obtained in Comparative Example 1; and MSPI represents the viscosity change of soy protein isolate gels of different concentrations prepared from the soy protein isolate obtained in Comparative Example 2.

[0030] Figure 4 To study the effect of papain addition amount on the hardness of prepared soy protein gel during frozen storage.

[0031] Figure 5 To study the effect of papain addition on the water holding capacity of prepared soy protein gel during frozen storage.

[0032] Figure 6 The electrophoresis patterns of the soy protein isolates prepared in Example 1 and Comparative Examples 2-3 of the present invention are shown. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0034] Example 1: A method for preparing antifreeze type soy protein isolate.

[0035] Soybeans and water were mixed in a mass ratio of 1:15 between soybean meal (purchased from Jinyuan Grain and Oil Industry Co., Ltd.) and water, and stirred at 50°C and pH 8.0 for 1.5 hours. The soybean meal aqueous solution was then filtered through gauze, the filtrate was centrifuged at 4000rpm for 20 minutes, the precipitate was discarded, the supernatant was adjusted to pH 4.5 and allowed to settle for 30 minutes, the precipitate was collected by centrifugation and redissolved in water to pH 7.0, and sheared and homogenized at 15000rpm for 5 minutes to obtain a soy protein isolate solution. Papain is added to the soy protein isolate solution in an amount of 1:1000 in a mass ratio of the enzyme to the soy protein isolate solution, and after enzymatic hydrolysis at 50°C for 30 minutes, the solution is treated at an ultra-high pressure of 100 MPa for 20 minutes (enzyme inactivation) to obtain a soy protein isolate hydrolysate, and then transglutaminase is added in a weight ratio of the enzyme to the soy protein isolate hydrolysate of 1:500, and after stirring and mixing, the solution is cross-linked at 50°C for 2 hours; after the reaction stops, the solution is treated at an ultra-high pressure of 100 MPa for 10 minutes, and spray-dried to obtain a finished product, namely, an antifreeze type soy protein isolate (MMSPI), also known as modified soy protein isolate.

[0036] Spray drying parameters: air inlet temperature is 180°C, air outlet temperature is 70°C, compressor air pressure is 0.1MPa, and peristaltic pump injection volume is 20ml.

[0037] Example 2: A method for preparing antifreeze type soy protein isolate.

[0038] Soybeans and water were mixed in a mass ratio of 1:15, and stirred at 50°C and pH8.0 for 1.5h. Then the soybean meal aqueous solution was filtered through gauze, the filtrate was centrifuged at 4000rpm for 20min, the precipitate was discarded, the supernatant was adjusted to pH4.5 and allowed to settle for 30min, the precipitate was collected by centrifugation and redissolved in water to pH7.0, and sheared and homogenized at 15000rmp for 5min to obtain a soy protein isolate solution. Papain was added to the soy protein isolate solution in a mass ratio of 1:2000 of the enzyme to the soy protein isolate solution, and after enzymolysis at 40°C for 20min, the soy protein isolate hydrolyzate was obtained by ultrahigh pressure treatment at 300MPa for 15min, and then transglutaminase was added in a weight ratio of 1:600 ​​of the enzyme to the soy protein isolate hydrolyzate, and after stirring and mixing, the reaction was carried out at 50°C for 2h; after the reaction stopped, the soy protein isolate was treated at 300MPa for 15min, and the finished product was obtained by spray drying, i.e., antifreeze type soy protein isolate.

[0039] Spray drying parameters: air inlet temperature is 180°C, air outlet temperature is 70°C, compressor air pressure is 0.1MPa, and peristaltic pump injection volume is 20ml.

[0040] Example 3: A method for preparing antifreeze type soy protein isolate.

[0041] Soybeans and water were mixed in a mass ratio of 1:15, and stirred at 50°C and pH8.0 for 1.5h. Then the soybean meal aqueous solution was filtered through gauze, the filtrate was centrifuged at 4000rpm for 20min, the precipitate was discarded, the supernatant was adjusted to pH4.5 and allowed to settle for 30min, the precipitate was collected by centrifugation and redissolved in water to pH7.0, and sheared and homogenized at 15000rmp for 5min to obtain a soy protein isolate solution. Papain was added to the soy protein isolate solution in a mass ratio of 1:5000 for enzyme to soy protein isolate, and after enzymolysis at 45°C for 10min, 600MPa ultrahigh pressure treatment was performed for 10min to obtain a soy protein isolate hydrolyzate, and then transglutaminase was added in a weight ratio of 1:700 for enzyme to soy protein isolate hydrolyzate, and after stirring and mixing, the reaction was carried out at 40°C for 3h; 600MPa ultrahigh pressure treatment was performed for 10min, and the finished product was obtained by spray drying, i.e., an antifreeze type soy protein isolate.

[0042] Spray drying parameters: air inlet temperature is 180°C, air outlet temperature is 70°C, compressor air pressure is 0.1MPa, and peristaltic pump injection volume is 20ml.

[0043] Example 4: A method for preparing antifreeze type soy protein isolate.

[0044] Soybeans and water were mixed in a mass ratio of 1:15, and stirred at 50°C and pH8.0 for 1.5h. Then the soybean meal aqueous solution was filtered with gauze, the filtrate was centrifuged at 4000rpm for 20min, the precipitate was discarded, the supernatant was adjusted to pH4.5 and allowed to stand for 30min, the precipitate was collected by centrifugation and redissolved with water to pH7.0, and sheared and homogenized at 15000rmp for 5min to obtain a soy protein isolate solution. Papain was added to the soy protein isolate solution in a mass ratio of 1:10000 for enzyme to soy protein isolate, and after enzymolysis at 50°C for 5min, 600MPa ultrahigh pressure treatment was performed for 5min to obtain a soy protein isolate hydrolyzate, and then transglutaminase was added in a mass ratio of 1:1000 for enzyme to soy protein isolate hydrolyzate, and after stirring and mixing, the reaction was carried out at 40°C for 3h; 600MPa ultrahigh pressure treatment was performed for 10min, and the finished product was obtained by spray drying, i.e., an antifreeze type soy protein isolate.

[0045] Spray drying parameters: air inlet temperature is 180°C, air outlet temperature is 70°C, compressor air pressure is 0.1MPa, and peristaltic pump injection volume is 20ml.

[0046] Comparative Example 1: A method for preparing soy protein isolate.

[0047] Soybeans and water were mixed in a mass ratio of 1:15, and stirred at 50°C and pH 8.0 for 1.5 hours. The soybean meal aqueous solution was then filtered through gauze, the filtrate was centrifuged at 4000rpm for 20 minutes, the precipitate was discarded, the supernatant was adjusted to pH 4.5 and allowed to settle for 30 minutes, the precipitate was collected by centrifugation and water was added to adjust the pH to 7.0, sheared and homogenized at 15000rmp for 5 minutes, and then spray-dried to obtain the finished product, i.e., soy protein isolate (SPI).

[0048] Spray drying parameters: air inlet temperature is 180°C, air outlet temperature is 70°C, compressor air pressure is 0.1MPa, and peristaltic pump injection volume is 20ml.

[0049] Comparative Example 2: A method for preparing antifreeze-resistant soy protein isolate.

[0050] Soybeans and water were mixed in a mass ratio of 1:15, and stirred at 50°C and pH 8.0 for 1.5 hours. Then the soybean meal aqueous solution was filtered through gauze, the filtrate was centrifuged at 4000rpm for 20 minutes, the precipitate was discarded, the supernatant was adjusted to pH 4.5 and allowed to settle for 30 minutes, the precipitate was collected by centrifugation and water was added to adjust the pH to 7.0, and the soy protein isolate solution was obtained after shearing and homogenization at 15000rpm for 5 minutes; pork bone gelatin was added to the soy protein isolate solution in a mass ratio of 1:10, and stirred evenly, and transglutaminase was added in a weight ratio of 1:1000 to the soy protein isolate, and stirred and mixed, and reacted at 40°C for 3 hours; the enzyme was inactivated in a boiling water bath for 5 minutes, and then spray-dried to obtain the finished product, i.e., enzyme-crosslinked soy protein isolate (MSPI).

[0051] Spray drying parameters: air inlet temperature is 180°C, air outlet temperature is 70°C, compressor air pressure is 0.1MPa, and peristaltic pump injection volume is 20ml.

[0052] In Comparative Example 2, gelatin was added to the soy protein isolate molecules by transglutaminase, and the cross-linking modification treatment and the boiling water bath treatment during the enzyme inactivation process greatly increased the viscosity of the enzyme cross-linked soy protein isolate. In this way, the concentration of the protein solution before spray drying in the factory production process must be reduced during industrial production, which greatly increases the factory production cost.

[0053] The present invention hydrolyzes the 7S component in the soy protein isolate by specific enzymes, so that the high-viscosity 7S component is hydrolyzed into low-viscosity 7S peptide segments, and adds transglutaminase to the hydrolyzate to cross-link the low-viscosity and highly hydrophilic 7S peptide segments with the main 11S molecules of the soy protein isolate, thereby effectively improving the gel antifreeze property of the 11S component through the 7S peptide segments and reducing the high viscosity of the 7S after cross-linking.

[0054] Comparative Example 3: A method for preparing antifreeze-resistant soy protein isolate.

[0055] The MSPI obtained in Comparative Example 2 was prepared into an aqueous solution at a rate of 4% (w / v), the pH was adjusted to 7.0 with 1 mol / L NaOH, the solution was enzymatically hydrolyzed with papain at 50°C, the enzyme / protein (W / W) was 0.05 / 100, the enzymatic hydrolysis lasted for 10 minutes, and the enzyme was inactivated in a boiling water bath for 5 minutes; after the enzyme was inactivated, the solution was placed in an ice water bath to rapidly reduce the solution temperature to room temperature, and the solution was spray dried to obtain a finished product, namely, an enzyme-crosslinked soybean protein isolate hydrolysate (EMSPI).

[0056] In order to study the influence of different processes on the water holding capacity, hardness and viscosity of the prepared soy protein isolate gel, the present invention uses the antifreeze type soy protein isolate prepared in Example 1 as a representative to prepare the soy protein isolate gel. At the same time, the soy protein isolates obtained in Comparative Examples 1 to 3 are also prepared into soy protein isolate gels. The water holding capacity and hardness of the gels at different refrigeration days and different treatment methods are tested respectively. The specific methods and results are as follows:

[0057] 1. Experimental methods

[0058] The antifreeze-resistant soy protein isolate gel prepared in Example 1 was prepared into solutions with concentrations of 4%, 6%, 8%, 10%, and 12% (w / v) using distilled water, stirred at room temperature for 2 h, centrifuged at 1000 r for 5 min to remove bubbles, poured into a gel tube and sealed, and placed in a 90°C water bath for 1 h. After being taken out, it was immediately placed in an ice water bath for 15 min, and then placed in a 4°C refrigerator for 12 h to obtain a gel of the corresponding concentration.

[0059] The soy protein isolate prepared in Comparative Example 1 was prepared into solutions with concentrations of 4%, 6%, 8%, 10%, and 12% (w / v) with distilled water, stirred at room temperature for 2 h, centrifuged at 1000 r for 5 min to remove bubbles, poured into a gel tube and sealed, and placed in a 90°C water bath for 1 h. After being taken out, it was immediately iced in water for 15 min, and then placed in a 4°C refrigerator for 12 h to obtain a gel of corresponding concentration.

[0060] The soy protein isolates prepared in Comparative Examples 2-3 were prepared into solutions with concentrations of 4%, 6%, 8%, 10%, and 12% (w / v) with distilled water, stirred at room temperature for 2 h, centrifuged at 1000 r for 5 min to remove bubbles, poured into gel tubes and sealed, and placed in a 90°C water bath for 1 h. After being taken out, they were immediately iced in water for 15 min, and then placed in a 4°C refrigerator for 12 h to obtain gels of corresponding concentrations.

[0061] The water holding capacity of gel products of different concentrations prepared by the antifreeze-resistant soy protein isolate prepared in Example 1 and the soy protein isolate prepared in Comparative Examples 1 to 3 was tested respectively, and the gel texture analysis and viscosity analysis were performed. The details are as follows:

[0062] (1) Gel water retention: Place the gel block in a centrifuge tube with a filter in the middle and a mass of m1. Weigh the centrifuge tube and gel together as m2. Centrifuge at 4000 r / min for 20 min. After centrifugation, the total mass of the centrifuge tube and gel is m3.

[0063] Water retention % = (m3-m1) / (m2-m1).

[0064] (2) Gel texture analysis: The test was conducted using a Brookfield CT3 texture analyzer. A cylindrical gel block with a height of 10 mm and a diameter of 20 mm was cut and analyzed using the TPA texture mode, with the probe model TA41, a trigger point of 5 g, and a downward pressure distance of 4 mm.

[0065] (3) Viscosity detection:

[0066] Prepare a protein solution of a certain concentration, stir it at room temperature for 2 hours, and then measure the viscosity of the solution with an Ubbelohde viscometer. The calculation formula for the viscosity coefficient of the SPI solution is:

[0067]

[0068] Where η1: viscosity coefficient of distilled water, 0.839 mPa·s; η2: viscosity coefficient of the measured fluid; ρ1: density of distilled water, 1 g / cm 3 ; ρ2: measure the density of the fluid.

[0069] (4) Polyacrylamide gel electrophoresis (SDS-PAGE)

[0070] Bio Craft Model BE-210N vertical plate electrophoresis apparatus was used. The mass fraction of separation gel was 12.5%, and the mass fraction of concentrated gel was 5%. During the electrophoresis, the current of the concentrated gel part was maintained at 15mA, and the current of the separation gel part was maintained at 25mA. After the electrophoresis, the electrophoresis film was fixed with a fixative for 4h, and then stained with Coomassie Brilliant Blue G-250 staining solution for 3h. After the staining was completed, the film was decolorized with distilled water until the background color was basically removed, and then scanned for imaging.

[0071] 2. Experimental results

[0072] The results are as follows Figure 1As shown, compared with the soy protein isolate obtained in Comparative Examples 1 to 3, the antifreeze soy protein isolate gel prepared with the antifreeze soy protein isolate as the raw material in Example 1 has a water holding capacity of more than 85% during the process of being placed in a -18°C refrigerator for 1 to 20 days. The water holding capacity of the gel formed by the antifreeze soy protein isolate prepared in Example 1 after thawing after being frozen at -18°C for 20 days is significantly higher than that of the gel formed by the natural soy protein isolate without name change in Comparative Example 1; the water holding capacity of the gel formed by the antifreeze soy protein isolate prepared by enzymatic hydrolysis and enzyme cross-linking of soy protein isolate in Example 1 is significantly higher than that of the gel formed by the soy protein isolate obtained by gelatin cross-linking modification in Comparative Example 2; the water holding capacity of the gel formed by the antifreeze soy protein isolate prepared by the method of hydrolysis first and then cross-linking in Example 1 is significantly higher than that of the enzyme cross-linked soy protein isolate hydrolysate obtained by cross-linking with gelatin and then hydrolyzing with papain in Comparative Example 3.

[0073] like Figure 2 As shown, the antifreeze type soy protein isolate gel prepared using the antifreeze type soy protein isolate of Example 1 as the raw material has a small increase in hardness, while the soy protein isolate gel hardness increase obtained in Comparative Examples 1 to 3 is significantly higher than that of the present invention. It can be seen that the antifreeze type soy protein isolate prepared in Example 1 effectively prevents a large amount of water loss of the gel caused by freezing, and at the same time inhibits the increase in gel hardness during frozen storage, delaying the deterioration of the gel texture.

[0074] like Figure 3 As shown, the viscosity of the antifreeze type soy protein isolate (MMSPI) prepared in Example 1 is not significantly different from the viscosity of the natural soy protein isolate prepared in Comparative Example 1 (SPI), indicating that the modification method of the present invention does not change the viscosity of the soy protein isolate. The viscosity of the modified soy protein isolate obtained by adding gelatin and then performing enzyme crosslinking in Comparative Example 2 is significantly higher than that of Example 1 of the present invention. Comparative Example 3 was modified by first performing enzyme crosslinking and then hydrolyzing with papain, and it was found that the viscosity of the modified soy protein isolate obtained was also significantly higher than that of Example 1 of the present invention. It shows that the treatment order of enzyme hydrolysis and enzyme crosslinking has a significant effect on the viscosity of the gel formed by the modified soy protein isolate. The viscosity of the modified soy protein isolate obtained by first enzymatic hydrolysis with papain and then crosslinking with glutaminase in Example 1 of the present invention is closest to that of the natural soy protein isolate.

[0075] It can be seen that the method of the present invention can avoid the increase of additional costs caused by the excessively high viscosity of the soy protein isolate gel due to modification (if the viscosity is too high, the concentration of the protein solution before spray drying in the factory production process needs to be reduced during industrial production). At the same time, it can also avoid the addition of exogenous substances (such as gelatin) to change the pure plant source characteristics of the soy protein isolate gel.

[0076] like Figure 4As shown, the hardness of the soy protein gel prepared by the antifreeze type soy protein isolate obtained when the mass ratio of papain to soy protein isolate solution is 1:1000-5000 in the preparation process of the present invention is significantly lower than the hardness when the mass ratio of papain to soy protein isolate solution is 1:10000 in the preparation process. This indicates that the amount of papain added in the preparation process of soy protein isolate significantly affects the hardness of the prepared gel.

[0077] like Figure 5 As shown, the soy protein gel prepared by the antifreeze type soy protein isolate obtained when the mass ratio of papain to soy protein isolate solution is 1:1000-5000 in the preparation process of the present invention has significantly higher water holding capacity during refrigeration than the soy protein isolate gel prepared when the mass ratio of papain to soy protein isolate solution is 1:10000.

[0078] like Figure 6 As shown, in the electrophoresis pattern of the antifreeze type soy protein isolate (MMSPI) prepared in Example 1, the α′, α, and β subunits of the 7S component are almost invisible, which indicates that the 7S component in the soy protein isolate can be effectively hydrolyzed by the preparation method of Example 1, thereby reducing the viscosity to the greatest extent. The antifreeze type soy protein isolate (EMSPI) obtained in Comparative Example 3 still retains a large number of 7S components. At the same time, since this method adopts the method of first cross-linking and then hydrolyzing, the A subunits in some 11S components are also partially hydrolyzed, which greatly reduces the gel-forming ability of the protein. In short, the antifreeze type soy protein isolate (MMSPI) prepared in Example 1 can not only hydrolyze the 7S component to the greatest extent, thereby effectively reducing the viscosity of the protein, but also greatly improve the antifreeze property of the protein gel by means of the formed 7S glycopeptide, while completely retaining the 11S component of the soy protein and maintaining its gel-forming ability.

[0079] In summary, the antifreeze soy protein isolate prepared by the present invention is not only entirely derived from plant soy protein (without the addition of external molecules such as gelatin), but also the soy protein isolate gel formed by the modified antifreeze soy protein isolate can still maintain a water holding rate of more than 85% during a 20-day frozen storage period at -18°C, with a small increase in hardness, which effectively prevents a large amount of water loss of the soy protein isolate gel caused by freezing while ensuring pure plant-derived soy protein, and inhibits the increase in gel hardness during frozen storage, delaying the deterioration of the gel texture. At the same time, it also effectively solves the problem of excessive gel viscosity caused by enzyme cross-linking modification.

[0080] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0081] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an antifreeze type soy protein isolate, characterized in that: The steps include: Alkali dissolution and acid precipitation: soybean meal is mixed with water, filtered, acidified, allowed to stand, and centrifuged to obtain isolated soy protein; the isolated soy protein includes 7S component and 11S component; Redissolution: re-dissolve the soy protein isolate in water, adjust the pH to neutral, and shear and homogenize to obtain a soy protein isolate solution; Enzymatic hydrolysis: adding papain to the soy protein isolate solution for hydrolysis, hydrolyzing the 7S component into 7S peptide segments, performing the first enzyme inactivation, and obtaining a soy protein isolate hydrolyzate containing 7S peptide segments; The mass ratio of papain to soy protein isolate solution is 1:1000-10000; Enzyme cross-linking: Add transglutaminase to the soy protein isolate hydrolysate and mix well, adjust the pH to neutral, and perform a cross-linking reaction to cross-link the 7S peptide segment with the 11S component, then perform a second enzyme inactivation, and obtain the antifreeze type soy protein isolate after drying.

2. The method for preparing the antifreeze type soybean protein isolate according to claim 1, characterized in that: The mass ratio of the soybean meal to water is 1:15-20.

3. The method for preparing the antifreeze type soybean protein isolate according to claim 1, characterized in that: The mass ratio of the papain to the soy protein isolate solution is 1:1000-5000.

4. The method for preparing the antifreeze type soybean protein isolate according to claim 1, characterized in that: The first enzyme inactivation process is: treatment at a pressure of 100MPa to 600MPa for 5min to 20min.

5. The method for preparing the antifreeze type soybean protein isolate according to claim 1, characterized in that: The mass ratio of the transglutaminase to the soy protein isolate hydrolyzate is 1:500-1000.

6. The method for preparing the antifreeze type soybean protein isolate according to claim 1, characterized in that: The second enzyme inactivation process is: treating at a pressure of 100MPa to 600MPa for 10min to 30min.

7. The method for preparing the antifreeze type soy protein isolate according to claim 1, characterized in that: The drying method is spray drying.

8. The method for preparing the antifreeze type soybean protein isolate according to claim 7, characterized in that: The spray drying parameters are set as follows: air inlet temperature is 170°C to 180°C, air outlet temperature is 65°C to 75°C, compressor air pressure is 0.1 MPa, and peristaltic pump injection volume is 20ml / min to 30ml / min.

9. An antifreeze type soy protein isolate prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the antifreeze type soy protein isolate according to claim 9 in preparing soy protein gel, characterized in that: The soy protein gel maintains the pure plant-based characteristics, can still maintain a water holding rate of more than 85% after being frozen and thawed for 20 days at -18°C, has low hardness, stable texture and low viscosity.

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