Chickpea protein isolate functionality improving method, chickpea protein isolate product and application
By performing heat treatment and pH adjustment in an acidic environment, chickpea protein isolate self-assembled to form amyloid fibers, solving the problem of insufficient foaming and emulsification, achieving functional improvement, and supporting industrial applications.
Patent Information
- Application Number
- CN202510595531.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-06-20
AI Technical Summary
The foaming and emulsifying properties of chickpea protein isolate are inferior to those of animal proteins, which restricts its use in industrial applications.
By heat-treating the aqueous solution of chickpea protein isolate in an acidic environment, adjusting the pH to 1.5-2.5, and performing centrifugation and heating treatment, the protein molecules are self-assembled to form amyloid fibers, thereby enhancing its functionality.
It effectively improves the foaming and emulsifying properties of chickpea protein isolate, making its functionality close to or even surpasses animal protein, and supports its widespread use in industrial applications.
Smart Images
Figure CN120167544A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of food protein processing, and particularly to a method for improving the functionality of chickpea protein isolate, a chickpea protein isolate product and its application. Background Art
[0002] In recent years, with the increasing demand for protein and the growing concern for healthy diets, people have started to consider using plant proteins to replace animal proteins. Chickpeas have a protein content of about 20% to 25%, and the composition of protein amino acids is balanced, rich in lysine lacking in cereal proteins, and essential amino acids account for 36% of the total amino acids, so they have attracted much attention. However, the foaming and emulsifying properties of chickpea protein isolate are inferior to those of animal proteins, which restricts the industrial application of chickpea protein isolate. Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention
[0003] The purpose of the present invention is to provide a method for improving the functionality of chickpea protein isolate, a chickpea protein isolate product and its application. The improvement method is green, environmentally friendly, low in economic cost and easy to operate, can effectively improve the functionality of chickpea protein isolate, and can provide strong support for the industrial application of chickpea protein isolate.
[0004] In a first aspect, a method for improving the functionality of chickpea protein isolate provided by the present invention includes: after the aqueous solution of chickpea protein isolate undergoes a hydration reaction, adjusting the pH to 1.5 - 2.5 and stirring for reaction, then centrifuging to obtain the supernatant, and reacting the supernatant at 75°C - 95°C and then ice-bathing; the functionality includes one of foaming property and emulsifying property.
[0005] Optionally, the concentration of chickpea protein isolate in the aqueous solution of chickpea protein isolate is 1% - 5%.
[0006] Optionally, the hydration reaction is carried out at 1°C - 8°C.
[0007] Optionally, the hydration reaction is carried out for 8h - 16h.
[0008] Optionally, hydrochloric acid is added to adjust the pH to 1.5 - 2.5.
[0009] Optionally, the chickpea protein isolate is stirred in ultrapure water at 20°C - 30°C for 2h - 5h to obtain the aqueous solution of chickpea protein isolate.
[0010] Optionally, after the stirring reaction, centrifugation is carried out at 2000rpm - 6000rpm.
[0011] Optionally, after the stirring reaction, centrifugation is carried out for 10min - 30min.
[0012] Optionally, the supernatant is reacted at 75°C - 95°C for 0.1 h - 12 h.
[0013] In a second aspect, the present invention also provides a chickpea protein isolate product prepared by using any one of the above optional functional improvement methods.
[0014] In a third aspect, the present invention also provides an application of a chickpea protein isolate product prepared by using any one of the above optional functional improvement methods.
[0015] Optionally, the application includes preparing an emulsion. Description of the Drawings
[0016] Figure 1 is a flowchart of a method for improving the functionality of chickpea protein isolate provided by the present invention;
[0017] Figure 2 is a graph showing the change in ThT fluorescence intensity of chickpea protein isolate products in Examples 1 to 9 and Comparative Example 1 of the present invention;
[0018] Figure 3 is a polyacrylamide gel electrophoresis change diagram of chickpea protein isolate products in Examples 1 to 9 and Comparative Example 1 of the present invention;
[0019] Figure 4 is a comparative diagram of the molecular flexibility change of chickpea protein isolate in Examples 1 to 9 and Comparative Example 1 of the present invention;
[0020] Figure 5 is a comparative diagram of the molecular hydrophobicity change of chickpea protein isolate in Examples 1 to 9 and Comparative Example 1 of the present invention;
[0021] Figure 6 is a diagram of the secondary structure content of chickpea protein isolate products in Examples 1 to 9 and Comparative Example 1 of the present invention;
[0022] Figure 7 is a transmission electron microscopy characterization diagram of chickpea protein isolate products in Examples 1, 5, 9 and Comparative Example 1 of the present invention;
[0023] Figure 8 is a comparative diagram of the emulsifying activity of chickpea protein isolate products in Examples 1 to 9 and Comparative Example 1 of the present invention;
[0024] Figure 9 is a comparative diagram of the foaming performance of chickpea protein isolate products in Examples 1 to 9 and Comparative Example 1 of the present invention;
[0025] Figure 10 is a comparative diagram of the absolute value of the potential of droplets in the protein emulsion corresponding to Example 9 and Blank Example 1 of the present invention;
[0026] Figure 11 This is a comparison chart of the average droplet size of the protein emulsions corresponding to Example 9 and Blank Example 1 of the present invention;
[0027] Figure 12 This is a comparison chart of the centrifugal stability of the protein emulsions corresponding to Example 9 and Blank Example 1 of the invention. Detailed implementation manners
[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention pertains.
[0029] See Figure 1 , the present invention provides a method for enhancing the functionality of chickpea protein isolate, including:
[0030] S1. Hydrating the aqueous solution of chickpea protein isolate;
[0031] S2. Adjusting the pH to 1.5 - 2.5, stirring the reaction, and then centrifuging to obtain the supernatant;
[0032] S3. Reacting the supernatant at 75°C - 95°C and then performing an ice bath.
[0033] In fact, by subjecting the hydrated solution of chickpea protein isolate to heat treatment in an acidic environment, it can promote the self-assembly of chickpea protein isolate molecules to form amyloid fibers with a nanoscale diameter, a micron-scale length, and a high aspect ratio, thereby effectively improving the foaming property and emulsifying property of the protein isolate product.
[0034] Specifically, by regulating the acidic environment and performing centrifugation and heat treatment, the formation of chickpea protein isolate amyloid fibers can be precisely regulated, thereby enhancing the functionality.
[0035] In some embodiments, the concentration of chickpea protein isolate in the aqueous solution of chickpea protein isolate used in step S1 is 1% - 5%. In fact, the chickpea protein isolate product can be added to ultrapure water in advance and stirred at 20°C - 30°C for 2 h - 5 h to prepare the aqueous solution of chickpea protein isolate.
[0036] In some embodiments, when performing step S1, the aqueous solution of chickpea protein isolate can be hydrated at 1°C - 8°C for 8h - 16h. In addition, when performing step S2, hydrochloric acid is added to adjust the pH to 1.5 - 2.5, followed by stirring reaction, and then centrifuged at 2000rpm - 6000rpm for 10min - 30min to separate the supernatant.
[0037] In some embodiments, when performing step S3, the supernatant is reacted at 75°C - 95°C for 0.1h - 12h. In fact, with the increase of the hydrothermal treatment time, it can promote the formation of amyloid fiber structure by protein molecules.
[0038] Example 1
[0039] This Example 1 provides a method for enhancing the functionality of chickpea protein isolate, including the following steps:
[0040] S1. Dispersing chickpea protein isolate in ultrapure water, stirring at room temperature for 2h to prepare a 2% aqueous solution of chickpea protein isolate, and hydrating the aqueous solution of chickpea protein isolate at 4°C for 12h;
[0041] S2. After the hydration reaction, adding hydrochloric acid to adjust the pH of the system to 2.0, followed by stirring reaction, then loading into a centrifuge tube and centrifuging at a speed of 4000rpm for 15min to collect the supernatant;
[0042] S3. Heating the supernatant in a water bath at 85°C for 0.25h, then ice-bathing and freeze-drying to obtain the enhanced chickpea protein isolate product.
[0043] Examples 2 to 9
[0044] Examples 2 to 9 respectively provide a method for enhancing the functionality of chickpea protein isolate. The difference from Example 1 is that the duration of water bath heating in step S3 is different, as shown in Table 1 below.
[0045] Blank Example 1
[0046] This Blank Example provides a method for preparing an aqueous solution of hydrated chickpea protein isolate, including: dispersing chickpea protein isolate in ultrapure water, stirring at room temperature for 2h to prepare a 1% aqueous solution of chickpea protein isolate, and hydrating the aqueous solution of chickpea protein isolate at 4°C for 12h.
[0047] Comparative Example 1
[0048] This Comparative Example 1 provides a method for enhancing the functionality of chickpea protein isolate. The difference from Example 1 is that the duration of water bath heating in step S3 is different, as shown in Table 1 below.
[0049] Table 1 Water bath heating time in Examples 1 to 9 and Comparative Example 1
[0050] Heating duration / h Heating duration / h Example 1 0.25 Example 6 6 Example 2 0.5 Example 7 8 Example 3 1 Example 8 10 Example 4 2 Example 9 12 Example 5 4 Comparative Example 1 0
[0051] Product Testing
[0052] The thioflavin T powder was stirred and dissolved in a sodium phosphate buffer solution (10 mmol / L phosphate, pH=7.4, 150 mmol / L sodium chloride) to obtain a ThT mother solution with a concentration of 0.8 mg / mL, and then the ThT mother solution was diluted 50 times with the sodium phosphate buffer solution, and then filtered through a 0.2 μm aqueous phase filter membrane to obtain a ThT working solution; the chickpea protein isolate solutions obtained in Examples 1 to 9 and Comparative Example 1 were stirred and dissolved in ultrapure water to prepare a protein sample solution with a concentration of 2%, 1 μL of the protein sample solution was taken and fully mixed with 1 mL of the ThT working solution, and the fluorescence excitation wavelength was set to 440 nm and the emission wavelength was set to 482 nm, and the relationship curve between the fluorescence intensity and the water bath heating time was plotted as shown in FIG. Figure 2 shown.
[0053] Thioflavin T fluorescence can be used to characterize the formation of protein β-sheet structure layers, and the increase in its fluorescence intensity can indicate the formation of amyloid fiber structure. Figure 2 It can be seen that as the heating time of chickpea protein isolate in water bath increases, the fluorescence intensity increases and the content of the characteristic structure of protein amyloid fibers increases, which indicates that the fibrillation process of chickpea protein isolate is completed.
[0054] The protein subunit composition and protein molecular weight change of the chickpea protein isolate products obtained in Examples 1 to 9 and Comparative Example 1 were determined respectively, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, using 5% concentrated gel and 15% separation gel) was used. 10 μL of protein sample solution (concentration 20 mg / mL) was added to each sample well and electrophoresed at a constant voltage. After electrophoresis for 40 minutes in Servicebio's Tris-Glycine SDS-PAGE SWE high-resolution fast electrophoresis buffer at a voltage of 200 V, Coomassie Brilliant Blue G-250 staining was used to observe the protein bands as shown in FIG. Figure 3 shown.
[0055] from Figure 3 It can be seen that the acidic subunit and basic subunit of the 11S part of the protein are degraded with the increase of water bath heating treatment time, and the band of the 7S part also disappears with the increase of treatment time. This shows that the chickpea protein isolate is structurally hydrolyzed into small molecular polypeptides during the treatment process, which is a hallmark structural change of protein in the fibrosis process.
[0056] The flexibility and hydrophobicity of the chickpea protein isolate molecules obtained in Examples 1 to 9 and Comparative Example 1 were measured separately. Trypsin was dissolved in Tris-HCl buffer (0.05 mol, pH = 8.0) to prepare a trypsin solution with a concentration of 1 mg / mL. Then, 250 μL of the trypsin solution was mixed with 4 mL of the protein sample solution (1 mg / mL) and enzymatically hydrolyzed at 37 °C for 10 min. 4 mL of trichloroacetic acid solution (5 mg / mL) was added to terminate the enzymatic hydrolysis. After centrifugation at 4000 rpm for 15 min, the supernatant was collected, and the absorbance at 280 nm was measured using a UV-visible spectrophotometer. The results are as Figure 4 shown; the hydrophobic strength was characterized by ABS-NH4 (1-anilinonaphthalene-8-sulfonic acid ammonium salt), with an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The results are as Figure 5 shown. It can be seen from Figure 4 and Figure 5 that with the increase in the heating treatment duration, the exposure of the hydrophobic sites of the protein molecules is promoted, and hydrophobic interaction is the main driving force for the formation of amyloid fibrils.
[0057] The secondary structures of the chickpea protein isolate products obtained in Examples 1 to 9 and Comparative Example 1 were measured separately using Fourier transform infrared spectroscopy (FTIR). The resolution was set to 4 cm -1 , the scanning frequency was 32 Hz, and the scanning wave number was 400 to 4000. The obtained data were subjected to baseline calibration, deconvolution, and smoothing to obtain the changes in the protein secondary structure content as shown in Figure 6 . It can be seen from Figure 6 that during the amyloid fibrillization process of protein molecules, the secondary structure of the protein changes, and consistent with Figure 2 is the increase in the β-sheet content.
[0058] The chickpea protein isolate products in Examples 1, 5, 9, and Comparative Example 1 were separately diluted to 0.2 mg / mL with ultrapure water, coated on the carbon support film of the copper grid, and left standing for 15 min. After removing the excess moisture with filter paper, negative staining was performed with phosphotungstic acid (3%, W / V). After 3 min, the phosphotungstic acid was removed with filter paper, and observation was carried out using a transmission electron microscope at 100 kV as shown in Figure 7 . It can be seen from Figure 7 that with the increase in the heating duration, the microscopic structure and morphology of the protein changed, and amyloid fibrils were significantly formed, changing from a granular morphology to a worm-like fiber morphology.
[0059] The chickpea protein isolate products prepared in Examples 1 to 9 and Comparative Example 1 were respectively configured into 2% protein sample solutions using ultrapure water. 3 mL of corn oil and 1 mL of the protein sample solution were mixed at a rotation speed of 10,000 rpm for 2 min to prepare an emulsion. 10 μL of the emulsion was diluted 400 times with a SDS solution with a concentration of 1 mg / mL, and then the absorbance of the diluted emulsion was measured at 500 nm using an ultraviolet-visible spectrophotometer. The SDS solution was used as a blank control, and the emulsion activity index (EAI) was calculated according to the following formula. The results are shown as Figure 8 shown below.
[0060]
[0061] where C is the concentration of the protein sample solution (g / mL), is the volume fraction of corn oil in the emulsion, and A 500 is the absorbance of the diluted emulsion at 500 nm.
[0062] The chickpea protein isolate products in Examples 1, 5, 9, and Comparative Example 1 were respectively diluted to 0.2 mg / mL with ultrapure water. Then, 10 mL of the protein sample solution was added into a 50 mL beaker and homogenized at a rotation speed of 10,000 rpm for 60 s. The foam capacity (FC) was calculated according to the following formula. The results are shown as Figure 9 shown below.
[0063]
[0064] where V0 is the volume of the protein sample solution before homogenization, and V 60 is the volume of the sample solution after 60 s of homogenization.
[0065] From Figure 8 and Figure 9 it can be seen that with the increase of the heating time and the formation of amyloid fibrils, the emulsifying activity of the protein increased from about 23 m 2 / g at 0 h to about 55 m 2 / g at 12 h; its foaming ability increased from about 53% at 0 h to more than 130% after 6 h of fibrillation, and the foaming ability was greater than 130% thereafter.
[0066] The chickpea protein isolate product prepared in Example 9 was formulated into a 1% protein solution using ultrapure water. The protein solution corresponding to Example 9 (CPIF) and the hydration solution of Blank Example 1 (CPI) were respectively mixed with corn oil at a volume ratio of 9:1, and after stirring for 2 min at 10,000 rpm using a high-speed shear emulsifier, a protein emulsion was prepared by circulating at 40 Mpa for 3 min using a high-pressure homogenizer. The droplet potential in the protein emulsions corresponding to Example 9 and Blank Example 1 was measured using a Nano ZSP nanoparticle size and zeta potential analyzer (Malvern Instruments, UK), and the results are as shown in Figure 10 ; The droplet size in the protein emulsions corresponding to Example 9 and Blank Example 1 was measured using a Mastersizer 3000 laser diffraction particle size analyzer, and the results are as shown in Figure 11 ; An accelerated centrifugation test was performed on the protein emulsions corresponding to Example 9 and Comparative Example 1 at 3000 rpm for 3600 s using a LUMiFuge stability analyzer (LUM GmbH, Germany), and their stability is as shown in Figure 12 .
[0067] It can be seen from Figure 10 that the electrostatic repulsion of the droplets in the protein emulsion corresponding to Example 9 is stronger, which is more conducive to preventing droplet aggregation and flocculation compared to Blank Example 1, thus effectively improving the dispersion stability of the emulsion droplets; it can be seen from Figure 11 that the droplet size in the protein emulsion corresponding to Example 9 is smaller, indicating that the present invention can improve the emulsifying performance through acidic heat treatment, and it can be seen from Figure 12 that the chickpea protein isolate can form amyloid fibrils after acidic heat treatment, so that the centrifugal stability index is significantly higher than that of Blank Example 1.
[0068] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes fall within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and can be implemented or realized in various ways.
Claims
1. A method for improving the functionality of chickpea protein isolate, characterized in that: include: After the chickpea protein isolate aqueous solution is hydrated, the pH is adjusted to 1.5-2.5 and stirred for reaction, and then the supernatant is centrifuged and reacted at 75°C-95°C, and then ice-bathed; the functionality includes one of foaming and emulsifying properties.
2. The method for improving functionality according to claim 1, characterized in that: The concentration of chickpea protein isolate in the chickpea protein isolate aqueous solution is 1%-5%; and / or, the hydration reaction is carried out at 1° C.-8° C.; and / or, the hydration reaction is carried out for 8h-16h.
3. The method for improving functionality according to claim 1, characterized in that: Adding hydrochloric acid to adjust the pH to 1.5-2.5; and / or, stirring the chickpea protein isolate in ultrapure water at 20° C.-30° C. for 2 h-5 h to obtain a chickpea protein isolate aqueous solution.
4. The method for improving functionality according to claim 1, characterized in that: The reaction is stirred and then centrifuged at 2000 rpm-6000 rpm; and / or, the reaction is stirred and then centrifuged for 10 min-30 min; and / or, the supernatant is reacted at 75° C.-95° C. for 0.1 h-12 h.
5. A chickpea protein isolate product obtained by the functionality enhancement method according to any one of claims 1 to 4.
6. An application of a chickpea protein isolate product obtained by the functional enhancement method according to any one of claims 1 to 4, characterized in that: The use comprises the preparation of emulsions.