Soybean protein isolate high-temperature spray drying process optimization method based on polyphenol composite regulation

Abstract

The invention belongs to the technical field of food processing, and particularly relates to a soybean protein isolate high-temperature spray drying process optimization method based on polyphenol composite regulation and control, which is particularly suitable for vegetable protein function improvement. The technical defects of thermal aggregation and functional property reduction of the SPI caused by a traditional high-temperature spray drying process are overcome, and the industrial bottlenecks of high insoluble aggregate content and degradation of solubility and emulsification / gel performance are broken through. According to the technical scheme, the method comprises the following steps: (1) adding a polyphenol compound (preferably catechin substances) in a specific proportion into an SPI solution, and forming a thermally stable compound in combination with a low-temperature dialysis process (4 DEG C, 24-72 hours and molecular weight cut-off of 8000-14000 Da); and (2) optimizing spray drying parameters (the air inlet temperature is 140-180 DEG C, preferably 140 DEG C) and cooperating with the polyphenol addition amount to realize functional directional regulation and control.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to an optimization method for the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation. Background Art

[0002] Soy protein isolate (SPI) is widely used in the food industry due to its low fat content, high nutritional value, and balanced amino acid composition. However, the traditional high-temperature spray drying process (inlet air temperature 150 - 200 °C) causes irreversible thermal denaturation of SPI. Research shows that this process reduces the emulsifying property of SPI by 24.53% (Zhang Zhong, Wang Hua. Food and Machinery, 2003), and the gel strength by 35.02% (Huang Youru et al. Journal of the Chinese Cereals and Oils Association, 2005), severely limiting its application.

[0003] Patent CN109566848 A provides a spray drying process for producing soy protein isolate. By adding polyols, ultrasonic treatment, and spraying Tween - 80, the solubility of SPI is increased to about 65%. However, this technology has the following defects: (1) Risk of chemical residues: Spraying Tween - 80 results in surfactant residues (detection value 0.1 - 0.2%), which does not meet the requirements of clean label; (2) Limited functional improvement: The emulsifying activity index is only 58.7 m 2 / g, unable to meet the requirements of high - protein beverages; (3) Complex process: Ultrasonic treatment (energy consumption increases by 20%) and multi - step cleaning are required, resulting in an increase in production costs.

[0004] Existing technologies have not solved the following core problems: Lack of a solution to inhibit thermal aggregation through natural polyphenol - protein interactions; Unable to directionally regulate the functional properties of SPI (such as high - emulsifying type and high - gel type) through single process parameter adjustment. Summary of the Invention

[0005] The present invention provides an optimization method for the high - temperature spray drying process of soy protein isolate based on polyphenol composite regulation, specifically including the following steps:

[0006] 1) SPI extraction: The low - temperature defatted soybean meal is crushed and sieved to less than 200 meshes, then dissolved in distilled water, the pH is adjusted to 7.8 - 8, and the supernatant is taken after centrifugation; the pH of the supernatant is adjusted to 4.5 - 4.8 for isoelectric precipitation, and the precipitate is collected by centrifugation; the precipitate is redissolved in water and the pH is adjusted to 7.0 to obtain a SPI stock solution with a mass concentration of 20 - 50 mg / mL (preferably 40 - 50 mg / mL);

[0007] 2) Polyphenol complexation: Add a specific proportion of polyphenols (preferably catechin substances) to the SPI stock solution. After mixing, use a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da and dialyze at 4°C for 24 - 72 h to remove unbound polyphenols. The time can be dynamically adjusted according to the membrane molecular weight cut-off by monitoring the catechin concentration in the dialysis external solution, and 24 - 48 h is preferred to balance efficiency and cost.

[0008] 3) Spray drying: Perform high-temperature spray drying on the dialyzed composite solution, control the inlet air temperature at 130 - 180°C (preferably 140°C), the outlet air temperature at 68 - 72°C, and the feed liquid flow rate at 3.0 - 4.0 mL / min;

[0009] Functional directional regulation:

[0010] High-emulsifying SPI: The addition amount of catechin is 1.0% (based on the mass of SPI), and the corresponding emulsification activity index ≥ 85.0 m 2 / g;

[0011] High-foaming SPI: The addition amount of catechin is 1.0%, and the foaming rate ≥ 77.5%;

[0012] High-gelling SPI: The addition amount of catechin is 0.25%, and the gel hardness ≥ 1.6 N;

[0013] Technical mechanism:

[0014] Polyphenols and SPI form a thermally stable complex through hydrophobic interaction and hydrogen bonding. When 1% catechin is added, DSC detection shows that the denaturation temperature increases from 68.75°C to 81.9°C;

[0015] The dialysis process achieves molecular-level binding, and the loading rate reaches 95%;

[0016] The synergistic effect of spray temperature - polyphenol concentration reduces the formation of random coil structures. Circular dichroism shows that the α-helix content increases by 26.3%, indicating that the structure of the SPI-catechin complex tends to be stable.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] Significantly improved functionality: (1) Directional regulation ability: Precisely control the functional properties of the product through the addition amount of catechin (the emulsification / foaming / gelling performance is improved by 36.4 - 103%); (2) Intervention in the behavior of thermal aggregates: Reduce the content of insoluble aggregates generated by the traditional spray drying process (reduced by 57.5%), and convert insoluble aggregates into soluble aggregates; (3) Breakthrough in stability: DSC detection shows that the protein denaturation temperature increases by 13.15°C, significantly improving the thermal stability; (4) Industrial advantages: The emulsification activity index (87.5 m 2 / g) Reaches the top level of similar international products (compared with imported SPI products ≤ 65m 2 / g);

[0019] Process innovation: 1) Green and residue-free: The dialysis method replaces the traditional surfactant spraying to avoid chemical residues (no Tween surfactants); 2) Energy consumption reduction: The optimized spray temperature (140°C) saves 30% energy compared with the traditional process (200°C), and the protein recovery rate increases by 22%; 3) Multifunctional integration: Different functional SPI can be produced on a single production line (the production line can be switched by adjusting the polyphenol addition ratio);

[0020] Construction of technical barriers: 1) Non-linear effect: A low addition amount of 0.25% catechin increases the gel strength by 43.9%, breaking the conventional understanding of the "concentration-performance linear relationship"; 2) Synergistic mechanism: The polyphenol complex and spray parameters work together to reduce the degree of thermal denaturation (SDS-PAGE shows that the integrity retention rate of protein subunits > 95%). Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 Determination results of the effects of different types of polyphenols (quercetin, catechin, rutin, tannic acid) and their addition amounts on the solubility of soy protein isolate in high-temperature spray-dried products;

[0023] Figure 2 Determination results of the effects of different catechin addition amounts on the solubility of soy protein isolate in high-temperature spray-dried products;

[0024] Figure 3 Determination results of the effects of catechin addition amounts on the foaming property and foam stability of soy protein isolate;

[0025] Figure 4 Determination results of the effects of catechin addition amounts on the emulsifying property and emulsion stability of soy protein isolate;

[0026] Figure 5 Determination results of the effects of catechin addition amounts on the gelation property of soy protein isolate;

[0027] Figure 6 Effect of catechin addition amount on the microscopic structure of the gelation property of soy protein isolate;

[0028] Figure 7Effect of catechin addition amount on the content of insoluble / soluble aggregates of soy protein isolate by spray drying;

[0029] Figure 8 Effect of different inlet air temperatures for spray drying on the solubility of soy protein isolate;

[0030] Figure 9 Schematic diagram of the structure of the complex of soy protein isolate and catechin (molecular docking diagram), where the left figure is the docking schematic diagram of 11S and catechin, and the right figure is the docking schematic diagram of 7S and catechin. Detailed implementation manners

[0031] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0035] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0036] The room temperature in the present invention refers to 25 ± 2 °C.

[0037] It should be noted that unless otherwise specified, the % in the examples and comparative examples are all based on mass.

[0038] In the embodiments of the present invention, all the raw materials required are obtained through commercial purchases.

[0039] Example 1: Preparation of soy protein isolate with high emulsifying and foaming properties

[0040] 1) 400 g of low-temperature defatted soybean meal was pulverized and passed through a 200-mesh sieve, dissolved in 6 L of distilled water. After complete dissolution, the pH of the solution was adjusted to 7.8 by dropwise addition of 2 M sodium hydroxide. After stirring for 2 h, centrifugation was carried out (4000 g, 20 min). The supernatant was taken and the pH of the solution was adjusted to 4.5 with 2 M hydrochloric acid. After centrifugation again, the precipitate was dissolved in 5 times the volume of distilled water, stirred evenly and then centrifuged. The centrifuged precipitate was weighed and 2 times the volume of distilled water was added, and the pH of the solution was adjusted to 7.0 by adding phosphate buffer solution to prepare the SPI crude protein solution.

[0041] 2) The SPI protein concentration was adjusted to 40 mg / mL, the pH was adjusted to 7.0, and catechin at 1.0% of the SPI mass was added. After mixing evenly, it was filled into a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da and dialyzed at 4 °C for 48 h;

[0042] 3) Spray drying treatment was carried out under the conditions of an inlet air temperature of 170 °C, an outlet air temperature of 70 °C, and a feed liquid flow rate of 3.5 mL / min to obtain high-emulsifying SPI.

[0043] 4) Performance detection: Solubility was 89.3% (Lowry method), foaming property was 77.5%, emulsifying activity index: 87.5 m 2 / g (GB 5009.3 - 2016), the content of insoluble aggregates was 2.6%, the content of soluble aggregates was 11.2%, and the thermal denaturation temperature was 81.9 °C.

[0044] Example 2: Preparation of soy protein isolate with high gelation properties

[0045] 1) 400 g of low-temperature defatted soybean meal was pulverized and passed through a 200-mesh sieve, dissolved in 6 L of distilled water. After complete dissolution, the pH of the solution was adjusted to 7.8 by dropwise addition of 2 M sodium hydroxide. After stirring for 2 h, centrifugation was carried out (4000 g, 20 min). The supernatant was taken and the pH of the solution was adjusted to 4.5 with 2 M hydrochloric acid. After centrifugation again, the precipitate was dissolved in 5 times the volume of distilled water, stirred evenly and then centrifuged. The centrifuged precipitate was weighed and 2 times the volume of distilled water was added, and the pH of the solution was adjusted to 7.0 by adding phosphate buffer solution to prepare the SPI crude protein solution.

[0046] 2) Adjust the SPI protein concentration to 40 mg / mL, adjust the pH to 7.0, add catechins at 0.25% of the SPI mass, mix evenly, then load into a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da, and dialyze at 4°C for 48 h;

[0047] 3) Perform spray drying under the conditions of an inlet air temperature of 170°C, an outlet air temperature of 70°C, and a feed liquid flow rate of 3.5 mL / min to obtain highly emulsifying SPI.

[0048] 4) Performance detection: The gel hardness is 1.64 N, the surface roughness of the gel is 1.090 nm, the content of insoluble aggregates is 5.65%, the content of soluble aggregates is 6.8%, and the thermal denaturation temperature is 70.2°C.

[0049] Example 3: Preparation of highly soluble soy protein isolate

[0050] 1) Crush 400 g of low-temperature defatted soybean meal through a 200-mesh sieve, dissolve it in 6 L of distilled water. After complete dissolution, adjust the pH of the solution to 7.8 by dropwise adding 2 M sodium hydroxide. After stirring for 2 h, centrifuge (4000 g, 20 min), take the supernatant, adjust the pH of the solution to 4.5 with 2 M hydrochloric acid, centrifuge again, dissolve the precipitate with 5 times the volume of distilled water, stir evenly and then centrifuge. Take the centrifuged precipitate, weigh it, add 2 times the volume of distilled water, and adjust the pH of the solution to 7.0 by adding phosphate buffer to prepare the original SPI protein solution.

[0051] 2) Adjust the SPI protein concentration to 40 mg / mL, adjust the pH to 7.0, add catechins at 0.75% of the SPI mass, mix evenly, then load into a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da, and dialyze at 4°C for 48 h;

[0052] 3) Then perform spray drying under the conditions of an inlet air temperature of 170°C, an outlet air temperature of 70°C, and a feed liquid flow rate of 3.5 mL / min to obtain highly emulsifying SPI.

[0053] 4) Performance detection: The solubility is 82%, the content of insoluble aggregates is 2.55%, the content of soluble aggregates is 9.57%, and the thermal denaturation temperature is 80.9°C.

[0054] Comparative Example 1: Preparation of soy protein isolate without adding polyphenols

[0055] Same as Example 1, except that in step 2), catechins are not added. Another difference is in 4) performance detection: The solubility is 69.8%, the foaming property is 48.4%, and the emulsifying activity index is 46.2 m 2 / g (GB 5009.3-2016), the content of insoluble aggregates is 6.1%, the content of soluble aggregates is 6.4%, and the thermal denaturation temperature is 68.8 °C.

[0056] Comparative Example 2: Prepared from soy protein isolate added with catechins at different concentrations

[0057] Same as Examples 1-3, except that the added concentration of catechins in step 2) is 0.5 respectively. Another difference is the performance detection in 4): the solubility is 79.5%, the foaming property is 66.9%, and the emulsifying activity index is 52.3 m 2 / g (GB5009.3-2016), the gel hardness is 1.34 N, the surface roughness of the gel microstructure is 1.134 nm, the content of insoluble aggregates is 4.3%, the content of soluble aggregates is 8.2%, and the thermal denaturation temperature is 72.2 °C.

[0058] Comparative Example 3: Prepared from soy protein isolate added with catechins at different concentrations

[0059] Same as Examples 1-3, except that the added concentration of catechins in step 2) is 1.25 respectively. Another difference is the performance detection in 4): the solubility is 90.3%, the foaming property is 69.4%, and the emulsifying activity index is 74.8 m 2 / g (GB5009.3-2016), the gel hardness is 1.20 N, the surface roughness of the gel microstructure is 3.392 nm, the content of insoluble aggregates is 3.4%, the content of soluble aggregates is 9.4%, and the thermal denaturation temperature is 76.62 °C.

[0060] Comparative Example 4: Prepared from soy protein isolate added with catechins at different concentrations

[0061] Same as Examples 1-3, except that the added concentration of catechins in step 2) is 1.5.

[0062] Another difference is the performance detection in 4): the solubility is 83.1%, the foaming property is 49.0%, and the emulsifying activity index is 68.8 m 2 / g (GB 5009.3-2016), the gel hardness is 1.19 N, the surface roughness of the gel microstructure is 3.706 nm, the content of insoluble aggregates is 3.4%, the content of soluble aggregates is 9.4%, and the thermal denaturation temperature is 74.17 °C.

[0063] Comparative Example 5: Prepared from soy protein isolate added with catechins at different concentrations

[0064] Same as Examples 1-3, except that the added concentration of catechins in step 2) is 1.75.

[0065] The differences also lie in the performance detection in 4): the solubility is 82.99%, the foaming property is 38.7%, and the emulsifying activity index is 60.9 m 2 / g (GB 5009.3-2016), the gel hardness is 1.21 N, the surface roughness of the gel microstructure is 4.387 nm, the content of insoluble aggregates is 3.7%, the content of soluble aggregates is 9.2%, and the thermal denaturation temperature is 71.7 °C.

[0066] Comparative Example 6: Prepared using different types of soy protein isolate

[0067] Same as Example 1, except that in step 2), the polyphenols added are quercetin, rutin, and tannic acid respectively. The differences also lie in the different measurement results of the performance indicators.

[0068] Comparative Example 7: Prepared using soy protein isolate with different inlet air temperatures for spray drying

[0069] Same as Example 1, except that in step 3), the temperature for spray drying is 150-180 °C. The differences also lie in the different measurement results of the performance indicators.

[0070] Figure 6 Shows the effect of the addition amount of catechin on the microstructure of soy protein isolate gel; Figure 9 Is the structural schematic diagram (molecular docking diagram) of the complex of soy protein isolate and catechin. The left figure is the docking schematic diagram of 11s and catechin, and the right figure is the docking schematic diagram of 7s and catechin.

[0071] Performance test experiment

[0072] 1. Effect of different types of polyphenols on the emulsifying property of soy protein isolate

[0073] As Figure 1 The results shown indicate that under the interaction conditions of appropriate types of polyphenols, protein concentration, polyphenol concentration, and interaction ratio, some functional properties of commercial SPI can be improved. Among them, the addition of catechin can significantly improve the solubility and emulsifying activity of commercial SPI (P < 0.05).

[0074] 2. Determination experiment of solubility

[0075] Dissolve the SPI prepared in Examples 1-3 and Comparative Examples 1-5 in PBS buffer at pH 6.25, with a protein concentration of 2 mg / mL, and then centrifuge at 5000 g and 4 °C for 15 min. The calculation method for protein solubility is:

[0076]

[0077] The results are shown in Figure 2The determination results of the effect of catechin addition amount on the solubility of soy protein isolate by high-temperature spray drying are as follows. Figure 2 As shown, with the increase of catechin addition amount, the solubility of soy protein isolate shows a trend of first increasing and then decreasing. When the catechin addition amount is 1%, the solubility of soy protein isolate reaches the maximum (95.2%), which is 36.4% higher than that without catechin addition, proving that an appropriate catechin addition amount can significantly improve the solubility of soy protein isolate.

[0078] 3. Determination test of foaming property and foaming stability

[0079] Weigh 1 g of SPI from Examples 1-3 and Comparative Examples 1-5 prepared respectively, and place them in a 500 mL graduated cylinder. Add 100 mL of water to obtain a composite solution (100 mL, 1% (w / v)). Stir with a homogenizer at 17,500 rpm for 2 min respectively, and record the volume at this time as V0. After standing for 30 min, measure the volume again and record it as V 30 , and calculate the foaming property and foaming stability of each group of C-SPI complexes in water according to the following formula:

[0080]

[0081] In the formula, 100 is the initial volume of the solution (mL).

[0082] Figure 2 The determination results of the effect of catechin addition amount on the foaming stability of soy protein isolate by high-temperature spray drying are as follows. Figure 3 The determination results of the effect of catechin addition amount on the foaming property and foaming stability of soy protein isolate.

[0083] From Figure 3 it can be seen that with the increase of catechin addition amount, the foaming property and foaming stability of the composite solution show a trend of rising first and then falling. Compared with the control group, when the catechin addition amount is 1%, the foaming property and foaming stability increase by 62.5% and 62.2% respectively. One of the reasons for the increase in foaming property is that catechin improves the solubility of SPI, and more protein molecules participate in the air-water interface, improving the protein-air interaction. In addition, after catechin interacts with soy protein isolate, the unfolding of the molecular structure increases the flexibility of protein molecules. With the increase of time, the bubble diameter gradually expands and shows an obvious shape change. All bubbles rapidly enlarge within a short time. At the same time, the morphology of the composite also decreases with the increase of catechin concentration. Compared with the natural SPI solution (0%) without catechin addition, the SPI protein molecules are connected by catechin, forming a stable network by reducing the size of the initial bubbles and delaying the increase of bubbles.

[0084] 4. Determination test of emulsifying property and emulsifying activity

[0085] The SPI prepared in Examples 1-3 and Comparative Examples 1-5 was respectively formulated into a sample solution with a protein concentration of 10 mg / mL. Then, 9 mL of the sample solution was added with 3 mL of soybean oil (i.e., the volume ratio of the two was 3:1), and after being whipped with a high-speed homogenizer at 10,000 r / min for 1 min, an emulsion was obtained for standby measurement. 100 μL was respectively extracted from the bottom of each prepared emulsion, and 10 mL of a 0.1% SDS solution was added. After mixing evenly, the absorbance value was measured at 500 nm, using the SDS solution as a blank, and the measurement was carried out again after 10 min. The emulsifying property and emulsifying stability were calculated respectively according to the following formulas:

[0086]

[0087] In the formula, C is the sample concentration (g / mL); Ф is the proportion of the oil phase in the emulsion, 0.25; dilution is the dilution factor; A0 is the absorbance value of the initial emulsion; A 10 is the absorbance value after 10 min.

[0088] Figure 4 are the measurement results of the influence of the catechin addition amount on the emulsifying property and emulsifying stability of soy protein isolate.

[0089] From Figure 4 it can be seen that with the increase of the catechin addition amount, the emulsifying property and emulsifying stability of the composite system show a trend of first increasing and then decreasing, reaching the maximum value when the catechin addition amount is 1%. Compared with the control group, the emulsifying property and emulsifying stability are increased by 15.1% and 82% respectively. The addition of an appropriate amount of catechin can change the protein-protein interaction, reduce the free energy, thereby reducing the interfacial tension at the oil-water interface. Due to the interaction between catechin and SPI, the emulsifying performance of SPI is improved, so that the flexibility and solubility of the modified protein are increased, and further the adsorption ability of the protein on the oil-water surface is increased.

[0090] 5. Gel property measurement test

[0091] The SPI prepared in Examples 1-3 and Comparative Examples 1-5 was respectively formulated into a solution with a protein mass concentration of 12%, stirred at 20 °C for 30 min, then heated in a 90 °C water bath for 30 min, cooled and placed in a 4 °C refrigerator for 12 h, and finally the solution was placed at room temperature for 30 min. The required parameters were measured by a texture analyzer. The P / 0.5 probe was selected and the measurement was carried out in a puncture mode, and the apex of the test peak was recorded, which was the hardness (N) of the measured gel.

[0092] Figure 5 are the measurement results of the influence of the catechin addition amount on the gel property of soy protein isolate. From Figure 5It can be seen that the addition amounts of catechins with different contents all significantly increased the gel properties of SPI. When the addition amount of catechins was 0.25%, the gel properties of SPI showed the maximum value, and the gel strength increased by 37.9% compared with the control group. Then, when the addition amount of catechins continued to increase, the gel properties of SPI decreased instead. This may be because at first, with the addition of catechins, active hydroxyl groups that can bind to protein amino acid residues were provided for the solution, promoting the formation of the protein network structure in the solution. As the addition amount of catechins continued to increase, covalent cross-linking occurred between the sulfhydryl groups of SPI and catechins. The hydroxyl groups of catechins were easily oxidized to ortho-quinones, and the sulfhydryl groups of proteins and quinones formed sulfhydryl-quinone covalent products through an addition reaction, preventing the generation of disulfide bonds during the heat-induced gelation process and resulting in a decrease in gel strength.

[0093] 6. Observation of Gel Microstructure (Atomic Force Microscope)

[0094] The atomic force microscope (AFM) was used to observe the microstructure of the protein gel. The SPI solutions prepared in Examples 1-3 and Comparative Examples 1-5 were diluted to a concentration of 20 μg / mL. 4 μL of the solution was aspirated and applied on a glass slide, and a gel was formed by heating in a water bath at 90 °C, and then it was placed on the stage and observed in the tapping mode.

[0095] The results showed that when catechins were not added, SPI had disordered aggregation and uneven distribution; when catechins were added, the gel microstructure of the aggregates transformed from irregular and uneven to regular, uniform, and dense. The average surface roughness (Ra) of SPI at different addition amounts of catechins was 5.153 nm, 1.090 nm, 1.134 nm, 1.158 nm, 2.723 nm, 3.392 nm, 3.706 nm, and 4.387 nm respectively. The decrease and then increase of Ra indicated that with the increase of the addition amount of catechins, the aggregation degree of SPI showed a trend of first decreasing and then increasing, and when the addition amount of catechins was 0.25%, the Ra value was the smallest, and the gel structure was dense, uniform, and stable.

[0096] 7. Determination of Soluble / Insoluble Aggregate Content

[0097] The SPI prepared in Examples 1-3 and Comparative Examples 1-5 was respectively formulated into a solution with a protein concentration of 1%, and then centrifuged (8000 r / min, 30 min, 4 °C). The precipitate obtained after centrifugation was the insoluble aggregate of SPI, and its protein content was determined. The supernatant after centrifugation was first filtered through a 0.45 μm microfiltration membrane to remove insoluble large protein particles, and then dialyzed with a 100 kDa dialysis bag. The part retained was the soluble aggregate with a molecular weight less than 100 kDa, and its content was determined.

[0098] Figure 7Content determination results of insoluble aggregates, soluble aggregates with molecular weight less than 100 kDa, and aggregates with molecular weight less than 100 kDa in SPI prepared in Examples 1-3 and Comparative Examples 1-5.

[0099] It can be seen from Figure 7 that as the addition amount of catechin increases, the content of insoluble aggregates shows a trend of first decreasing and then slightly increasing. The insoluble aggregates are the precipitated part after centrifugation of the solution. The less this part is, the more the content of soluble aggregates, which further indicates that the complex of catechin and SPI has higher solubility. After the solution is dialyzed, the content of soluble aggregates with molecular weight > 100 kDa shows a trend of first increasing and then decreasing with the increase of catechin addition amount, which is consistent with the trend of solubility. When the addition amount of catechin is 1%, the content of soluble aggregates is the highest (P > 0.05). It can also be known from the solubility index that the solubility is the highest at this time, indicating that when the addition amount of catechin is 1%, it can effectively intervene in the thermal aggregation problem of commercial SPI caused by heat processing, thereby improving the problem of decreased solubility of commercial SPI caused by spray drying treatment, and further improving some functional properties of SPI.

[0100] 8. Thermal stability determination

[0101] The thermal stability of the protein was determined using a differential scanning calorimeter (DSC). 5.5 mg of the sample was placed in an aluminum crucible, the drying nitrogen gas flow rate was 20 mL / min, the heating rate was 10 °C / min, and the temperature range was 30 - 180 °C. The results were analyzed using Pyris software, and the maximum denaturation temperature (T max ) and the enthalpy value (ΔH) were recorded.

[0102] Heat treatment can promote denaturation, thereby affecting the structure and functional properties of SPI. Thermal denaturation involves the transition of proteins from the folded to the unfolded state, which can be measured using a differential scanning calorimeter (DSC). The DSC experimental results are shown in Table 1. It can be seen from Table 1 that when the sample temperature is increased from 30 °C to 180 °C, as the addition amount of catechin gradually increases, the denaturation temperature of the complex peak gradually increases, and the enthalpy value increases. Among them, when the addition amount of catechin is 1%, the denaturation temperature of the complex at this time is 81.90 ± 0.63 a °C, which is 19.12% higher than that of SPI, indicating that the heat resistance of SPI is improved after the addition of catechin, and further indicating that catechin can effectively intervene in the thermal aggregation behavior of SPI.

[0103] 9. Determination of the content of protein secondary structure

[0104] The circular dichroism spectrum of the samples was scanned using a multi-functional spectrometer to analyze the changes in the secondary structure of SPI prepared in Examples 1-3 and Comparative Examples 1-5. The sample solution was prepared using a 10 mM phosphate buffer solution at pH 7.0, with the SPI concentration in the solution being 0.1 mg / mL. The scanning wavelength was set at 190-260 nm and the optical path was 1 mm at room temperature. The resulting data should note to deduct the circular dichroism spectrum corresponding to the buffer solution, and the data was processed using CDPro software.

[0105] From the measurement results of the circular dichroism spectrum, it can be seen that as the addition amount of catechin increases, the secondary structure of the protein gradually changes from a disordered extended state to an ordered structure. When the addition amount of catechin is 1%, the content of α-helix structure is the highest, and the composite system is the most stable. Correspondingly, the peak temperature of C-SPI is the highest (P < 0.05). When the addition amount of catechin exceeds 1%, based on the measurement results of the binding affinity between bound catechin and soy protein isolate, it can be known that the ability of SPI to encapsulate catechin is limited. Continuing to add catechin, the active hydroxyl groups contained therein destroy the stable state of the complex, resulting in a slight decrease in the thermal stability of the sample.

[0106] 10. Influence of different inlet air temperatures for spray drying on the solubility of soy protein isolate

[0107] Figure 8 This is the influence of the inlet air temperature for spray drying on the solubility of polyphenol / soy protein. According to the line graph, the solubility of soy protein isolate continuously decreases as the inlet air temperature increases. The solubility is the highest at 140 °C and then starts to decrease, and is the lowest at 180 °C. This is because during the spray drying process, due to the relatively high temperature, the peptide chains of soy protein isolate are stretched, and many groups are easily attracted and combined with each other, forming a large number of heat-induced aggregates, resulting in a decrease in the solubility of soy protein isolate.

[0108] Table 1 shows the influence of different addition amounts of catechin on the thermal stability of soy protein isolate

[0109] Table 1

[0110]

[0111] Table 2 shows the influence of different addition amounts of catechin on the secondary structure of soy protein isolate.

[0112] Table 2

[0113]

[0114] Table 3 shows the comparison results between the technical solution provided by the present invention and the traditional process.

[0115] Table 3

[0116]

[0117]

[0118] The industrial advantages of the present invention are reflected in the production line compatibility. A single production line can produce different functional SPI by adjusting the polyphenol ratio. Energy conservation and consumption reduction: The energy consumption is reduced by 30% with an inlet air temperature of 140°C compared to the traditional process (200°C).

[0119] Innovation statement: Through the synergistic regulation of polyphenol-protein complexation and drying parameters, the present invention reduces the content of insoluble aggregates by 57.5% and simultaneously realizes the directional improvement of functional properties. The whole process of the process is free of chemical pollution and is applicable to the industrial production of functional plant protein products.

[0120] The embodiments described above are only used to describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An optimization method for the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation, characterized in that It includes the following steps: (1) Crush the low-temperature defatted soybean meal and sieve it to a 200-mesh sieve pore, dissolve it in distilled water, adjust the pH of the solution to 7.8, stir evenly for 2 h and then centrifuge. Adjust the pH of the supernatant to 4.5, centrifuge again, dissolve the precipitate with distilled water, stir evenly again and then centrifuge. Dissolve the precipitate with distilled water and adjust the pH of the obtained solution to 7.0 to obtain a soy protein isolate solution; (2) Adjust the SPI solution to a mass concentration of 10 - 50 mg / mL and a pH of 7.0 - 7.5; (3) Add polyphenol compounds to the SPI solution, and the addition amount is 0.25% - 1.75% of the mass of SPI; (4) Load the mixed solution into a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da and dialyze at 2 - 6 °C for 12 - 72 hours; (5) Spray-dry the dialyzed solution, control the inlet air temperature at 140 - 180 °C, the outlet air temperature at 65 - 75 °C, and the feed liquid flow rate at 3.0 - 4.0 mL / min.

2. The optimization method of the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation according to claim 1, wherein, The polyphenol compound is a catechin substance, selected from at least one of epigallocatechin gallate, epigallocatechin, and epicatechin.

3. The optimization method of the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation according to claim 1, characterized in that The molecular weight cut-off of the dialysis bag is 8000 - 14000 Da, the dialysis temperature is 2 - 6 °C, and the dialysis time is 24 - 72 h.

4. The optimization method of the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation according to claim 1, wherein The inlet air temperature of the spray drying is 135 - 145 °C, the outlet air temperature is 68 - 72 °C, and the feed liquid flow rate is 3.5 ± 0.3 mL / min.

5. The optimization method of the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation according to any one of claims 1-4, characterized in that, The corresponding relationship between the polyphenol addition amount and functional regulation is as follows: when the addition amount is 1.0% of the SPI mass, the emulsion activity index of the obtained product is ≥ 85 m 2 / g.

6. The optimization method of the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation according to any one of claims 1-4, characterized in that, The corresponding relationship between the polyphenol addition amount and functional regulation is: when the addition amount is 0.75% of the mass of SPI, the solubility ≥ 80%.

7. The optimization method of the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation according to any one of claims 1-4, characterized in that, The corresponding relationship between the polyphenol addition amount and functional regulation is: when the addition amount is 1.0% of the mass of SPI, the foaming property ≥ 75%.

8. The method for optimizing the high-temperature spray drying process of soy protein isolate based on polyphenol composite regulation according to any one of claims 1-4, characterized in that The corresponding relationship between the polyphenol addition amount and functional regulation is: when the addition amount is 0.25% of the mass of SPI, the gel hardness ≥ 1.60 N.

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