Method for regulating functional properties and oxidation resistance of potato protein

Abstract

The invention discloses a method for adjusting functional properties and oxidation resistance of potato protein, and relates to the technical field of food processing. The method comprises the following steps: extracting potato protein by adopting an alkali-solution and acid-isolation method, and modifying the potato protein by utilizing an ultrasonic technology; the ultrasonic frequency is set to be 20 kHz, the amplitude is set to be 40%, the ultrasonic time is 0-10 min, and the change rule of the potato protein structure and the functional property under different ultrasonic time is analyzed. After ultrasonic treatment, the particle size and polydispersity index of the potato protein are obviously reduced, the Zeta-potential is increased, but main strips are not changed; after 6 minutes of ultrasonic treatment, the particle size is 266.90 + / -2.26 nm, the PDI is 0.27 + / -0.03, and the Zeta-potential is-26.25 + / -0.93 mV. After 6 minutes of ultrasonic treatment, the solubility of potato protein is the highest, the foamability is improved by 1.18 times, the emulsifying activity is improved by 1.54 times, and the digestibility of simulated intestines is remarkably improved. Under the condition, the reducing power of the potato protein and the scavenging rates of DPPH, superoxide anions and hydroxyl radicals reach the maximum value.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, in particular to a method for regulating the functional properties and antioxidant properties of potato protein. Background Art

[0002] In recent years, in response to energy conservation and emission reduction, and the need for a broader food perspective, researchers have been searching for new proteins to supplement and replace animal proteins. Plant proteins, among them, have attracted considerable attention due to their low-carbon, sustainable, and environmentally friendly properties. Potato protein is considered a higher-quality plant protein compared to other plant proteins.

[0003] Research on potato protein in my country has primarily focused on protein recovery from starch processing wastewater, while its structure and properties remain understudied. Researchers have analyzed the properties of potato protein extracted using alcohol, salt, and alkali methods. They found that alcohol and alkali methods yielded higher extraction rates, while samples obtained using the alkali method exhibited superior foaming properties and comparable functional properties to those obtained using the alcohol method. Therefore, the alkali method is the most common extraction method. To further expand the application of potato protein, some researchers have modified it. By varying the pH of the potato protein, researchers found that at pH 10, the electrostatic repulsion of the potato protein was greater, resulting in a more ordered structure. However, in acidic environments, the potato protein formed soluble aggregates, resulting in decreased stability. Covalent cross-linking of potato protein with transglutaminase and peroxidase resulted in a gel with a more uniform three-dimensional network and larger pore size. Glycosylation improved the emulsifying properties of the potato protein. High-pressure treatment increased its surface hydrophobicity by 1.3 times, and its foam stability by 177%. It can be seen that physical modification technology can effectively improve the properties of potato protein.

[0004] Ultrasonic treatment is an emerging non-thermal processing technology that is widely used in physical modification of proteins due to its advantages of being green, convenient, and environmentally friendly. Ultrasonic waves destroy protein aggregates through cavitation, weaken the non-covalent interactions between protein molecules, change their structure, and thus improve their functional properties. The literature indicates that after ultrasonic treatment at 400W / 30min, the emulsifying activity of pepper seed protein increased from 42.64±1.52m 2 / g increased to 98.44±1.86m 2 / g. After 8 minutes of ultrasonic treatment, the solubility of wheat germ protein (pH 7.0) increased from 32.3±1.28% to 44.98±2.38%. After walnut protein was ultrasonically treated at 400W / 30min, its emulsification activity increased by 1.57 times and its emulsification stability increased by 2.67 times. Although ultrasonic treatment has been shown to be effective in modifying other plant proteins, research on the structure and properties of potato protein is still lacking. Summary of the Invention

[0005] In response to the above technical problems, the present invention provides a method for regulating the functional properties and antioxidant activity of potato protein.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for regulating the functional properties and antioxidant activity of potato protein comprises the following steps:

[0008] S1. Dissolve potato protein with a protein content of 68.44 ± 0.58% (N × 6.25) in 50-100 mL of deionized water to a protein concentration of 20-30 mg / mL;

[0009] S2. Adjust the pH of the potato protein to 6.5-7.5 using 1-2 mol / L NaOH to prepare a potato protein solution with a concentration of 15-25 mg / mL;

[0010] S3. Take 30-50 mL of potato protein solution and place it in a probe-type ultrasonic processor. Control the temperature of the water bath at 25-30° C., ultrasonic frequency at 20 kHz, amplitude at 30-40%, and ultrasonicate for 2-10 minutes.

[0011] Furthermore, the preparation of potato protein in step S1 comprises the following steps:

[0012] S1.1. Take fresh potato tubers, beat them into pulp, and filter them through 60-100 mesh gauze to collect the filtrate;

[0013] S1.2. Rinse the filter residue with 30-50 mL of distilled water and collect the filtrate.

[0014] S1.3. Combine the two filtrates and place them in a beaker. Adjust the pH of the filtrate to 9.0 with 1 mol / L NaOH. Then, add the filtrate to a magnetic stirring device at a stirring speed of 1500-2500 r / min. After magnetic stirring at 37°C for 30 minutes, place the filtrate in a centrifuge and centrifuge at 9000 r / min for 30 minutes to collect the precipitate.

[0015] S1.4. Transfer the supernatant after centrifugation to a beaker and adjust the pH to 4.5 with 1 mol / L hydrochloric acid to allow the protein to fully precipitate. Then, place the supernatant in a centrifuge and centrifuge at 9000 rpm for 30 minutes to collect the precipitate.

[0016] S1.5. Combine the two precipitates, wash them with distilled water 2-3 times, and freeze-dry them to obtain potato protein with a mass content of 68.44±0.58% (N×6.25).

[0017] Furthermore, the ultrasonic process in step S3 adopts a pulse mode, ultrasonicating for 3-5 seconds and stopping for 3-5 seconds.

[0018] Compared with the prior art, the present invention has the following beneficial effects: potato protein is extracted using an alkali dissolution and acid precipitation method and physically modified by ultrasonic treatment. The ultrasonically treated potato protein has a smaller particle size, better dispersibility, and carries a large amount of negative charge, resulting in excellent solubility. Ultrasonic treatment improves the foaming and foam stability, emulsification, and emulsion stability of the potato protein, with the greatest improvement achieved when ultrasonic treatment is performed for 6 minutes. Furthermore, ultrasonic treatment for 6 minutes significantly improves the potato protein's simulated intestinal digestibility and antioxidant properties, including DPPH free radical scavenging rate, reducing power, and superoxide anion and hydroxyl radical scavenging rates. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The SDS-PAGE images of potato proteins under different ultrasonic times;

[0020] Figure 2 The particle size (A) and Zeta potential (B) of potato protein under different ultrasonic times;

[0021] Figure 3 UV-visible spectra (A) and FTIR spectra (B) of potato protein under different ultrasound times;

[0022] Figure 4 The solubility of potato protein under different ultrasound times;

[0023] Figure 5 The foaming properties (A) and foam stability (B) of potato protein under different ultrasonic times;

[0024] Figure 6 Emulsifying activity (A) and emulsifying stability (B) of potato protein under different ultrasound times;

[0025] Figure 7 The oil retention of potato protein under different ultrasound times;

[0026] Figure 8 The simulated intestinal digestibility of potato protein under different ultrasound times;

[0027] Figure 9 is the DPPH free radical scavenging ability of potato protein under different ultrasound times;

[0028] Figure 10 is the ABTS free radical scavenging ability of potato protein under different ultrasound times;

[0029] Figure 11is the ferrous iron chelation rate of potato protein under different ultrasonic times;

[0030] Figure 12 is the reducing power of potato protein under different ultrasound times;

[0031] Figure 13 is the hydroxyl radical scavenging ability of potato protein under different ultrasound times;

[0032] Figure 14 The superoxide anion radical scavenging ability of potato protein under different ultrasound times. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concept of the present invention.

[0034] Example 1

[0035] A method for regulating the functional properties and antioxidant activity of potato protein comprises the following steps:

[0036] S1. Potato protein with a protein content of 67.86% (N × 6.25) was dissolved in 50 mL of deionized water to a protein concentration of 21 mg / mL. N represents the nitrogen content measured in the sample, and 6.25 is a universal conversion factor for animal and plant proteins derived from the Kjeldahl method.

[0037] The preparation of potato protein includes the following steps:

[0038] S1.1. Take fresh potato tubers, beat them, and filter them through 60-mesh gauze to collect the filtrate;

[0039] S1.2. Rinse the filter residue with 30 mL of distilled water and collect the filtrate;

[0040] S1.3. Combine the two filtrates and place them in a beaker. Adjust the pH of the filtrate to 9.0 with 1 mol / L NaOH. Then, add the filtrate to a magnetic stirrer at 1500 rpm. After magnetic stirring at 37°C for 30 min, place the filtrate in a centrifuge and centrifuge at 9000 rpm for 30 min to collect the precipitate.

[0041] S1.4. Transfer the supernatant after centrifugation to a beaker and adjust the pH to 4.5 with 1 mol / L hydrochloric acid to allow the protein to fully precipitate. Then, place the supernatant in a centrifuge and centrifuge at 9000 rpm for 30 minutes to collect the precipitate.

[0042] S1.5. Combine the two precipitates, wash them twice with distilled water, and freeze-dry them to obtain potato protein with a mass content of 67.86% (N×6.25).

[0043] S2. Adjust the pH of the potato protein to 6.5 using 1 mol / L NaOH to prepare a potato protein solution with a concentration of 18 mg / mL;

[0044] S3. Take 30 mL of potato protein solution and place it in a probe-type ultrasonic processor. Control the water bath temperature at 25°C, ultrasonic frequency at 20 kHz, amplitude at 30%, and ultrasonicate for 10 min. Analyze the changes in the structure and functional properties of potato protein at different ultrasonic times. The ultrasonic process adopts pulse mode, ultrasonicating for 3 seconds and stopping for 3 seconds.

[0045] Example 2

[0046] A method for regulating the functional properties and antioxidant activity of potato protein comprises the following steps:

[0047] S1. After potato protein with a protein content of 68.44 (N×6.25) is dissolved in 70 mL of deionized water, the protein concentration is 25 mg / mL. The preparation of potato protein includes the following steps:

[0048] S1.1. Take fresh potato tubers, beat them into pulp, and filter them through 80-mesh gauze to collect the filtrate;

[0049] S1.2. Rinse the filter residue with 40 mL of distilled water and collect the filtrate;

[0050] S1.3. Combine the two filtrates and place them in a beaker. Adjust the pH of the filtrate to 9.0 with 1 mol / L NaOH. Then, add the filtrate to a magnetic stirrer at 2000 rpm. After magnetic stirring at 37°C for 30 min, place the filtrate in a centrifuge and centrifuge at 9000 rpm for 30 min to collect the precipitate.

[0051] S1.4. Transfer the supernatant after centrifugation to a beaker and adjust the pH to 4.5 with 1 mol / L hydrochloric acid to allow the protein to fully precipitate. Then, place the supernatant in a centrifuge and centrifuge at 9000 rpm for 30 minutes to collect the precipitate.

[0052] S1.5. Combine the two precipitates, wash them twice with distilled water, and freeze-dry them to obtain potato protein with a mass content of 68.44 (N×6.25).

[0053] S2. Adjust the pH of the potato protein to 7 using 2 mol / L NaOH to prepare a potato protein solution with a concentration of 22 mg / mL;

[0054] S3. Take 40 mL of potato protein solution and place it in a probe-type ultrasonic processor. Control the temperature of the water bath at 27°C, ultrasonic frequency at 20 kHz, amplitude at 35%, and ultrasonicate for 10 min. Analyze the changes in the structure and functional properties of potato protein under different ultrasonic times. The ultrasonic process adopts pulse mode, ultrasonicating for 4 seconds and stopping for 4 seconds.

[0055] Example 3

[0056] A method for regulating the functional properties and antioxidant activity of potato protein comprises the following steps:

[0057] S1. Potato protein with a protein content of 69.01% (N×6.25) was dissolved in 100 mL of deionized water to a protein concentration of 30 mg / mL. The preparation of potato protein included the following steps:

[0058] S1.1. Take fresh potato tubers, beat them into pulp, and filter them through 100-mesh gauze to collect the filtrate;

[0059] S1.2. Rinse the filter residue with 50 mL of distilled water and collect the filtrate;

[0060] S1.3. Combine the two filtrates and place them in a beaker. Adjust the pH of the filtrate to 9.0 with 1 mol / L NaOH. Then, add the filtrate to a magnetic stirrer at 2500 rpm. After magnetic stirring at 37°C for 30 min, place the filtrate in a centrifuge and centrifuge at 9000 rpm for 30 min to collect the precipitate.

[0061] S1.4. Transfer the supernatant after centrifugation to a beaker and adjust the pH to 4.5 with 1 mol / L hydrochloric acid to allow the protein to fully precipitate. Then, place the supernatant in a centrifuge and centrifuge at 9000 rpm for 30 minutes to collect the precipitate.

[0062] S1.5. Combine the two precipitates, wash them three times with distilled water, and freeze-dry them to obtain potato protein with a mass content of 69.01% (N×6.25).

[0063] S2. Adjust the pH of the potato protein to 7.5 using 2 mol / L NaOH to prepare a potato protein solution with a concentration of 24 mg / mL;

[0064] S3. Take 50 mL of potato protein solution and place it in a probe-type ultrasonic processor. Control the temperature of the water bath at 30°C, set the ultrasonic frequency at 20 kHz, and the amplitude at 40% for 10 min. Analyze the changes in the structure and functional properties of potato protein under different ultrasonic times. The ultrasonic process adopts pulse mode, ultrasonicating for 5 seconds and stopping for 5 seconds.

[0065] 1. Structural characterization of potato protein

[0066] 1.1 SDS-PAGE analysis

[0067] The potato protein sample solution obtained by any example was diluted to 2 mg / mL, mixed evenly with the loading buffer at a volume ratio of 2:1, boiled in a water bath for 4 minutes, and centrifuged at 4000 rpm for 4 minutes; then 10 μL of the mixture was injected into the gel track and electrophoresed at a constant voltage of 130 V.

[0068] The results of SDS-PAGE analysis of potato protein are shown in Figure 1 . As can be seen from the figure, the potato protein extracted by the alkali dissolution and acid precipitation method is distributed in the range of 11-75kDa, among which the molecular weight regions with more bands are 11-25kDa and 35-50kDa. When the ultrasonic time is 2 minutes, the color of each band of potato protein is significantly deepened. This is because the physical effects generated by ultrasound, such as mechanical stirring, shearing, high-pressure impact, microjetting, cavitation, etc., promote the disaggregation of protein aggregates and increase solubility, so the color of the protein band becomes darker. As the ultrasonic time increases, the potato protein bands do not change significantly, and no new bands are generated. It can be seen that ultrasonic treatment increases the solubility of potato protein, but does not cause chemical cross-linking or degradation.

[0069] 1.2 Particle size and Zeta potential

[0070] The potato protein sample solution obtained in any example was diluted to 2 mg / mL and the particle size and potential were measured using a BT-Zeta100 particle size analyzer. The instrument was cleaned and zeroed with distilled water, using distilled water as the dispersant. The equilibration time was 120 s, the sample refractive index was 1.35, and the medium refractive index was 1.33.

[0071] The particle size, polydispersity index (PDI) and zeta potential of potato protein under different ultrasonic times are shown in Figure 2. Figure 2 As shown. Figure 2 A shows that the particle size of potato protein is 824.23±9.78nm and PDI is 0.44±0.01. After 2min of ultrasonic treatment, the particle size and PDI of potato protein were significantly reduced (p<0.05), which was consistent with SDS-PAGE. Figure 1The results of the analysis were consistent. When the ultrasonic time was extended between 2-6 minutes, the particle size of potato protein continued to decrease, but the PDI did not change significantly (p>0.05). This is because the cavitation effect generated by ultrasound weakened the aggregation of potato protein. The longer the treatment time, the greater the reduction in particle size. When the ultrasonic time was 8 minutes, the PDI of potato protein increased, but its particle size was not significantly different from that under 6 minutes of treatment (p>0.05). At 10 minutes of ultrasound, the particle size of potato protein reached its minimum value, but the PDI was higher than that under 2-6 minutes of treatment (p<0.05).

[0072] Depend on Figure 2 As shown in Figure B, the zeta potential of potato protein is -29.69 ± 0.09 mV, indicating that potato protein contains a large number of negatively charged amino acids, consistent with literature results. After 2 minutes of ultrasonic treatment, the zeta potential of potato protein increased, and the negative charge decreased. When ultrasonicated for 4 and 6 minutes, the zeta potential of potato protein decreased, but then increased again. A comprehensive consideration of the trends in particle size, PDI, and zeta potential revealed that potato protein after 6 minutes of ultrasonic treatment exhibited smaller particle size, better dispersibility, and lower zeta potential.

[0073] 1.3 UV-visible spectrum

[0074] The potato protein sample solution obtained by any example was diluted to 1 mg / mL, and the UV-visible absorption spectrum of the sample was measured using a UV-visible spectrophotometer with distilled water as a reference. The UV spectrum of the potato protein after ultrasonic treatment is as follows: Figure 3 As shown in Figure A. As can be seen from the figure, potato protein has an absorption peak at 280nm, which is a typical absorption peak of protein. As the ultrasonic treatment time increases, the absorption peak intensity decreases, but the maximum absorption wavelength does not change significantly, indicating that ultrasonic treatment changes the microenvironment of hydrophobic amino acid residues and promotes the dissolution of protein. The change trend is similar to Figure 2 The particle size change trends shown are basically consistent.

[0075] 1.4 FTIR spectroscopy

[0076] Drop a small amount of sample solution onto the ATR element and collect infrared spectra with a scanning range of 400-4000 cm -1 , the instrument resolution is 2cm -1 .

[0077] Figure 3 B is the FTIR spectrum of potato protein, where the -1 The peak is protein amide I band, 1537 cm -1The peak at 1397 cm is the amide II band. -1 Amide III band, 3272 cm -1 The amide A band is located at the bottom, representing the OH and NH stretching vibrations of the intermolecular hydrogen bonds. After 2 minutes of ultrasonic treatment, the transmittance of the potato protein amide band I, II and A bands increased, indicating that the 2-minute ultrasonic treatment significantly weakened the aggregation of the protein, increased the solubility of the protein, and enhanced its interaction with water. Figure 2 The particle size analysis results are consistent with those shown. As for amide bands I and II, the transmittance is highest when the ultrasonic time is 2 minutes, followed by the sample with ultrasonic time of 6 minutes; however, ultrasonic treatment has no effect on their position, that is, ultrasonic treatment only weakens the aggregation of proteins and does not change the potato protein skeleton, which is consistent with SDS-PAGE. Figure 1 and UV spectrum Figure 3 The results of A analysis were consistent with the trend of the effect of ultrasonic treatment on the structure of rapeseed and flax protein isolates.

[0078] 2 Analysis of functional properties of potato protein

[0079] 2.1 Solubility

[0080] Take 10 mL of the treated sample in a centrifuge tube, centrifuge at 4000 r / min for 30 min, discard the supernatant, dry it in an oven at 65 ° C to constant weight, weigh the precipitate, and calculate the solubility as shown in formula (1):

[0081]

[0082] Where m0 is the mass of protein in 10 mL of solution, g; m1 is the mass of precipitate, g.

[0083] The UV spectrum of potato protein after ultrasonic treatment is as follows Figure 3 As shown in Figure A. As can be seen from the figure, potato protein has an absorption peak at 280nm, which is a typical absorption peak of protein. As the ultrasonic treatment time increases, the absorption peak intensity decreases, but the maximum absorption wavelength does not change significantly, indicating that ultrasonic treatment changes the microenvironment of hydrophobic amino acid residues and promotes the dissolution of protein. The change trend is similar to Figure 2 The particle size change trends shown are basically consistent.

[0084] Figure 3 B is the FTIR spectrum of potato protein, where the -1 The peak is protein amide I band, 1537 cm -1 The peak at 1397 cm is the amide II band. -1 Amide III band, 3272 cm -1The amide A band is located at the bottom, representing the OH and NH stretching vibrations of the intermolecular hydrogen bonds. After 2 minutes of ultrasonic treatment, the transmittance of the potato protein amide band I, II and A bands increased, indicating that the 2-minute ultrasonic treatment significantly weakened the aggregation of the protein, increased the solubility of the protein, and enhanced its interaction with water. Figure 2 The particle size analysis results are consistent with those shown. As for amide bands I and II, the transmittance is highest when the ultrasonic time is 2 minutes, followed by the sample with ultrasonic time of 6 minutes; however, ultrasonic treatment has no effect on their position, that is, ultrasonic treatment only weakens the aggregation of proteins and does not change the potato protein skeleton, which is consistent with SDS-PAGE. Figure 1 ) and UV spectrum Figure 3 The results of A analysis were consistent with the trend of the effect of ultrasonic treatment on the structure of rapeseed and flax protein isolates.

[0085] 2.2 Foamability

[0086] Dilute the potato protein sample solution obtained in any of the examples to 10 mg / mL, take 20 mL (V0) and place it in a 50 mL beaker. Homogenize at 10,000 rpm for 1 minute. The total volume of the solution and foam after homogenization is recorded as V1. Let the homogenized liquid stand for 0-30 minutes, and record the total volume of the solution and foam at 10, 20, and 30 minutes (V2). The foamability and foam stability are calculated as shown in formulas (2) and (3):

[0087]

[0088] After ultrasonic treatment for different time, the foaming property of potato protein Figure 5 .Depend on Figure 5 A It can be seen that after ultrasonic treatment, the foaming property of potato protein first increases and then decreases, reaching a maximum value at 6 minutes of ultrasonic treatment, which is significantly different from the non-ultrasonic sample (p<0.05), but not significantly different from the sample after ultrasonic treatment for 2-8 minutes (p>0.05). The cavitation effect of ultrasound makes the protein structure loose, the aggregates dissociate, and the solubility increases, making it easier for the protein to adsorb to the water and air interface, thereby improving the foaming property of the protein (Zou et al., 2020). Therefore, compared with the untreated sample, the foaming property of potato protein after 6 minutes of ultrasonic treatment increased by 1.18 times. However, when the ultrasonic time is too long, such as Figure 2 As shown, the PDI of potato protein increases, indicating that partial aggregation may occur between protein molecules, leading to a decrease in foaming performance. Foaming performance, on the other hand, is related to the hydrophilic-hydrophobic balance of the protein. When sonication is prolonged, the hydrophilic groups of potato protein are overexposed, and the hydrophilicity continues to increase, which in turn hinders the ability to reduce the surface tension of water, resulting in a decrease in foaming performance.

[0089] In terms of foam stability, ultrasonic treatment for 6 and 8 minutes significantly improved the foam stability of potato protein at 30 minutes, but had little effect on its foam stability at 10 and 20 minutes. Ultrasonic treatment increases the solubility of potato protein, making it easier to form an interfacial film. Furthermore, this improved solubility strengthens the interactions between the interfacial protein and neighboring molecules, resulting in little change in foam stability overall.

[0090] 2.3 Emulsification

[0091] The potato protein sample solution obtained in any of the examples was diluted to 2 mg / mL, 24 mL was mixed with 8 mL of soybean oil, and homogenized at 10,000 rpm for 1 minute to form a uniform emulsion. At 0, 10, and 30 minutes, 50 μL of the emulsion was taken from the bottom of the beaker and mixed with 5 mL of a 1 g / L SDS solution. The absorbance at 500 nm was measured, with the 1 g / L SDS solution used as a reference. Emulsifying activity and emulsifying stability were calculated according to formulas (4) and (5):

[0092]

[0093] Where A0 is the absorbance at 0 min; A 10 A is the absorbance of the emulsion when it is left standing for 10 min; 30 is the absorbance of the emulsion after standing for 30 min; D is the dilution factor, 100; ρ is the mass concentration of protein in the solution before emulsion formation, g / L; θ is the volume fraction of the oil phase in the emulsion, 0.25; φ is the diameter of the cuvette, 0.01 m.

[0094] The ability of proteins to form and stabilize emulsions is crucial in food processing. Emulsification is used to characterize the ability of proteins to adsorb at the oil-water interface and stabilize the interface. The emulsifying activity and emulsion stability of potato proteins are shown in Figure 6 As shown in the figure, the emulsifying activity of potato protein was greatest after 6 minutes of ultrasonic treatment, 1.54 times that of the untreated sample and significantly higher than the emulsifying activity of samples treated under other ultrasonic conditions (p < 0.05). Furthermore, after 6 minutes of ultrasonic treatment, the emulsifying stability of potato protein was significantly higher than that of other samples (p < 0.05); compared to the untreated sample, its emulsifying stability increased by 22.64% at 10 minutes and by 18.09% at 30 minutes. In summary, 6 minutes of ultrasonic treatment significantly improved the emulsifying properties of potato protein.

[0095] After 6 min of ultrasonic treatment, the particle size and PDI of potato protein were significantly reduced. Figure 2 As shown, and the solubility is the greatest, as Figure 4As shown in Figure 3 , potato protein exhibits a good hydrophilic-lipophilic balance, allowing it to easily adsorb at the oil-water interface, thereby exhibiting excellent emulsifying activity. Furthermore, its good solubility improves the stability of the interfacial film. Therefore, after 6 minutes of ultrasound treatment, potato protein exhibits excellent emulsifying activity and emulsification stability, a trend similar to the emulsifying effect of ultrasound treatment on Zanthoxylum bungeanum seed protein.

[0096] 2.4 Oil retention

[0097] Accurately weigh a certain mass of potato protein sample (m1) and place it in a centrifuge tube (m2). Add soybean oil at a protein to oil ratio of 1:5 (w:v) so that the protein sample is completely submerged in the oil. After gently stirring, let it stand at room temperature for 30 minutes. Then, centrifuge it at 5000 rpm for 15 minutes to discard the upper unabsorbed oil phase. Use filter paper to absorb the excess soybean oil from the bottom and inner walls of the centrifuge tube. Reweigh the total mass of the centrifuge tube and sample (m3). Oil absorption is calculated using Equation (6).

[0098] Oil retention (g / g) = (m3-m1-m2) / m1 (6)

[0099] Potato protein oil holding capacity Figure 7 As shown in the figure, the oil retention of potato protein is 1.19 ± 0.003 g / g protein, which is similar to the oil retention of potato protein extracted by alkaline dissolution method reported in existing literature. However, the oil retention of potato protein decreases after ultrasonic treatment, and the decrease is significant with the extension of ultrasonication time (p < 0.05).

[0100] The oil-holding capacity of proteins is related to various factors, such as surface area, hydrophobicity, surface charge, and surface morphology. Ultrasonic treatment reduces the particle size of potato protein and its surface hydrophobicity, thereby reducing its oil-holding capacity. It has been reported that ultrasonic treatment for 2-4 minutes has little effect on the oil-holding capacity of casein micelles, but ultrasonic treatment for 6 minutes significantly increases the oil-holding capacity of casein micelles. This suggests that ultrasonic treatment has different effects on the oil-holding capacity of different proteins.

[0101] 2.5 In vitro simulated intestinal digestion

[0102] A 50 mL potato protein sample was mixed with 10 mg of trypsin and digested at 37°C. At designated time points, an equal volume of 20% (w / v) trichloroacetic acid was added to the digestion solution. The mixture was centrifuged at 10,000 × g for 20 minutes, and the absorbance of the supernatant at 280 nm was measured to represent the soluble nitrogen content generated by the digestion.

[0103] The simulated intestinal digestibility of potato protein under different ultrasonic treatment times is as follows: Figure 8As shown in the figure, during the simulated intestinal digestion process, the soluble nitrogen content in the digestive juice increased with the extension of digestion time, indicating that potato protein has good digestibility.

[0104] Ultrasonic treatment significantly improved the simulated intestinal fluid digestibility of potato protein. When the sonication duration was 2-6 minutes, the simulated intestinal fluid digestibility of potato protein increased significantly with increasing sonication time (p < 0.05). However, when the sonication duration was 8-10 minutes, the simulated intestinal fluid digestibility did not change significantly with increasing sonication time (p > 0.05). Short-term sonication increases protein solubility, thereby enhancing its digestibility. However, long-term sonication changes the surface hydrophobicity of the protein, stabilizing intermolecular interactions and thus preventing significant changes in its digestibility.

[0105] 2.6DPPH free radical scavenging ability

[0106] 3 mL of the 8 mg / mL sample solution was added to 3 mL of a 0.1 mmol / L DPPH ethanol solution, shaken evenly, and allowed to stand in the dark at room temperature for 30 minutes. The mixture was then centrifuged at 4000 rpm for 10 minutes. The absorbance of the supernatant was measured at 517 nm, and the solution was zeroed using a mixture of equal volumes of water and ethanol. The DPPH radical scavenging activity was calculated as shown in Equation (7):

[0107]

[0108] Where A1 is the absorbance of the supernatant; A2 is the absorbance of the solution with an equal volume of ethanol solution replacing DPPH; A0 is the absorbance of the solution with an equal volume of water replacing the sample.

[0109] DPPH radical scavenging activity of potato protein after ultrasonic treatment Figure 9 As shown in the figure, potato protein exhibits a certain DPPH radical scavenging capacity, with the scavenging rate of the unsonicated sample reaching 44.93 ± 0.63%. Ultrasonic treatment promoted the dissolution of potato protein, enhancing its DPPH radical scavenging capacity. The DPPH radical scavenging capacity of potato protein reached its highest level at 55.91 ± 0.76% after 6 minutes of ultrasonic treatment. Subsequently, the DPPH radical scavenging rate of potato protein decreased with increasing ultrasonication time, likely due to partial protein cross-linking and increased particle size caused by prolonged ultrasonication, which reduced its DPPH radical scavenging capacity.

[0110] 2.7 ABTS free radical scavenging ability

[0111] Prepare ABTS working solution and place in the dark for 12-16 hours. During the measurement, take an appropriate amount of working solution and dilute it with pH 7.4 phosphate buffer so that the absorbance at a wavelength of 734 nm is 0.70±0.02. Take 0.5mL of 8mg / mL sample solution, add 3.5mL of ABTS diluent, react at room temperature for 10 minutes, and measure the absorbance at 734nm. The ABTS free radical scavenging ability is calculated as shown in formula (8):

[0112]

[0113] Where A0 is the absorbance of the working solution after dilution with pH 7.4 phosphate buffer; A is the absorbance of the sample.

[0114] The ABTS free radical scavenging rate of potato protein is shown in Figure 10 As can be seen from the figure, the ABTS free radical scavenging rate of potato protein is 58.24±0.76%. When the ultrasonic treatment lasts for 2 minutes, the ABTS free radical scavenging rate of potato protein does not change significantly (p>0.05). When the ultrasonic treatment time is further extended, the ABTS free radical scavenging rate of potato protein increases significantly with the extension of ultrasonic treatment time (p<0.05), reaching a maximum at 10 minutes of ultrasonic treatment. ABTS is a water-soluble free radical. Ultrasonic treatment improves the solubility of potato protein, thereby increasing its ABTS free radical scavenging rate.

[0115] 2.8 Ferrous chelation force

[0116] Separately, 0.2 mL of the 8 mg / mL sample solution was mixed with 3.65 mL of deionized water and 0.05 mL of a 2 mmol / L FeCl2 solution. The mixture was allowed to stand at room temperature for 30 seconds. 0.1 mL of 5 mmol / L ferrozine was then added and mixed thoroughly. The mixture was allowed to stand at room temperature for 10 minutes, then centrifuged at 4000 g / min for 5 minutes. The absorbance was measured at 562 nm. The chelating force was calculated as follows:

[0117]

[0118] Where A is the absorbance of the supernatant; A0 is the absorbance of the control group after replacing the sample with water.

[0119] The ABTS free radical scavenging rate of potato protein is shown in Figure 10As can be seen from the figure, the ABTS free radical scavenging rate of potato protein is 58.24±0.76%. When the ultrasonic treatment lasts for 2 minutes, the ABTS free radical scavenging rate of potato protein does not change significantly (p>0.05). When the ultrasonic treatment time is further extended, the ABTS free radical scavenging rate of potato protein increases significantly with the extension of ultrasonic treatment time (p<0.05), reaching a maximum at 10 minutes of ultrasonic treatment. ABTS is a water-soluble free radical. Ultrasonic treatment improves the solubility of potato protein, thereby increasing its ABTS free radical scavenging rate.

[0120] 2.9 Reduction Power

[0121] Separately, mix 2.5 mL of the 8 mg / mL sample solution with 2.5 mL of 0.2 mol / L pH 6.6 phosphate buffer and 2.5 mL of 1% potassium ferricyanide solution. Incubate in a 50°C water bath for 20 minutes. After cooling to room temperature, add 2.5 mL of 10% trichloroacetic acid (TCA) solution. Centrifuge the mixture at 4000 rpm for 10 minutes. Add 2.5 mL of the supernatant to 2.5 mL of ultrapure water and 500 μL of 0.1% FeCl₃ solution. Mix thoroughly, let stand for 10 minutes, and measure the absorbance of the mixture at 700 nm, which is the reducing power.

[0122] Depend on Figure 12 It can be seen that the reducing power of potato protein is the highest after 6 min of ultrasonic treatment. Ultrasonic treatment weakens the aggregation of potato protein and reduces its particle size. Figure 2 As shown in A, its solubility is increased, such as Figure 4 As shown, the reducing power is enhanced. When the ultrasonication time is too long, it causes partial cross-linking between protein molecules, and its reducing power decreases. Compared with the sample without ultrasonication, the reducing power of potato protein increased by 39.86% after 6 minutes of ultrasonication.

[0123] 2.10 Hydroxyl radical scavenging ability

[0124] To a test tube, add 2 mL of 6 mmol / L ferrous sulfate solution, 2 mL of 6 mmol / L salicylic acid solution, and 1 mL of 8 mg / mL sample solution, mix well, and then add 2 mL of 6 mmol / L hydrogen peroxide solution. Let the mixture stand at room temperature for 30 minutes, and measure the absorbance at 510 nm. The hydroxyl radical scavenging activity is calculated as shown in formula (10):

[0125]

[0126] Where A1 is the absorbance of the sample solution; A2 is the absorbance of an equal volume of deionized water solution instead of salicylic acid solution; A0 is the absorbance of an equal volume of deionized water solution instead of the sample.

[0127] The hydroxyl radical scavenging ability of potato protein is shown in Figure 13 As shown in the figure, the hydroxyl radical scavenging rate of potato protein is low, but it increases significantly after ultrasonic treatment (p < 0.05). After 6 minutes of ultrasonic treatment, the hydroxyl radical scavenging rate of potato protein is 35.60 ± 0.53%, 3.34 times that of the untreated sample. This is because the particle size of potato protein is minimized under these conditions, its solubility is improved, and the amino acid residues with antioxidant activity are fully exposed, thereby improving its antioxidant properties.

[0128] 2.11 Superoxide Anion Scavenging Ability

[0129] Add 3 mL of 50 mmol / L Tris-HCl buffer (pH 8.2) to the test tube, followed by 0.2 mL of the 8 mg / mL sample solution. The mixture is then reacted in a 25°C water bath for 10 minutes. At the same temperature, 0.2 mL of a 30 mmol / L pyrogallol solution is added and allowed to react for 4 minutes. The reaction is quenched with 0.5 mL of concentrated hydrochloric acid, and the absorbance is measured at 325 nm. The superoxide anion scavenging rate is calculated as shown in formula (11):

[0130]

[0131] Where A1 is the absorbance of the sample solution; A2 is the absorbance of the solution with an equal volume of deionized water instead of pyrogallol; A0 is the absorbance of the solution with an equal volume of deionized water instead of the sample.

[0132] The hydroxyl radical scavenging ability of potato protein is shown in Figure 13 As shown in the figure, the hydroxyl radical scavenging rate of potato protein is low, but it increases significantly after ultrasonic treatment (p < 0.05). After 6 minutes of ultrasonic treatment, the hydroxyl radical scavenging rate of potato protein is 35.60 ± 0.53%, 3.34 times that of the untreated sample. This is because the particle size of potato protein is minimized under these conditions, its solubility is improved, and the amino acid residues with antioxidant activity are fully exposed, thereby improving its antioxidant properties.

[0133] Ultrasonic treatment weakened potato protein aggregation, significantly reduced its particle size, and weakened its UV absorption at 280 nm, leading to a darker electrophoretic band. However, it did not alter its primary functional groups or generate new protein bands. In other words, ultrasonic treatment only physically modified potato proteins. However, after ultrasonic treatment, potato protein aggregates disaggregated, exposing some positively charged amino acid residues or other positively charged molecules, increasing their zeta potential.

[0134] Potato protein also has higher solubility than some other plant proteins, such as quinoa, walnut, and rapeseed proteins. Ultrasound-induced cavitation weakens protein aggregation, reduces particle size, and increases potato protein solubility, reaching its maximum at 6 minutes of sonication. However, prolonged sonication may cause some protein or other molecules in the system to aggregate, leading to a decrease in solubility; this is consistent with the effect of ultrasonic treatment on the solubility of walnut protein. Due to the increased solubility, the foaming and foam stability of the potato protein treated with ultrasound were significantly improved at 6 minutes.

[0135] After 6 min of ultrasonic treatment, the particle size and PDI of potato protein were significantly reduced. Figure 1 As shown, and the solubility is the greatest, as Figure 4 As shown in Figure 3 , potato protein exhibits a good hydrophilic-lipophilic balance and readily adsorbs at the oil-water interface, thus exhibiting excellent emulsifying activity. Furthermore, its good solubility improves the stability of the interfacial film. Therefore, after 6 minutes of ultrasonic treatment, potato protein exhibits excellent emulsifying activity and emulsification stability, a trend similar to the effect of ultrasonic treatment on the emulsifying properties of Zanthoxylum bungeanum seed protein. However, ultrasonic treatment is not conducive to improving the oil-binding capacity of potato protein.

[0136] Potato protein exhibits excellent digestibility in simulated intestinal fluid. Ultrasound-induced cavitation promotes the disaggregation of potato protein, reducing particle size and increasing solubility. This exposes more enzymatic hydrolysis sites and increases the accessibility of digestive enzymes. Therefore, sonication for 2-6 minutes significantly improves potato protein digestibility in simulated intestinal fluid. However, when sonication duration exceeds 8 minutes, digestibility no longer significantly changes with prolonged sonication.

[0137] Potato protein possesses a certain antioxidant capacity. This capacity is related not only to its electron-donating and hydrogen-donating abilities, or to the presence of amino acid residues with chelating properties, but also to the presence of small amounts of phenolic acids and flavonoids. Ultrasonic treatment not only promotes the dissolution of potato protein but also exposes active amino acid residues, thereby improving its DPPH free radical scavenging rate, ABTS free radical scavenging rate, reducing power, and superoxide anion and hydroxyl radical scavenging rates. When the sonication time was 6 minutes, the DPPH free radical scavenging rate, reducing power, superoxide anion scavenging rate, and hydroxyl radical scavenging rate of potato protein were all maximized.

[0138] Potato protein was extracted using an alkali-acid precipitation method and physically modified by ultrasonic treatment. When the ultrasonic treatment time was 6 minutes, the potato protein had a smaller particle size, better dispersibility, and carried a large amount of negative charge, which showed excellent solubility. After ultrasonic treatment, the foaming and foam stability, emulsification and emulsion stability of the potato protein were all improved, and the improvement effect was the best when the ultrasonic treatment time was 6 minutes. In addition, the simulated intestinal digestibility and antioxidant properties of the potato protein were significantly improved after 6 minutes of ultrasonic treatment, including DPPH free radical scavenging rate, reducing power, superoxide anion and hydroxyl radical scavenging rate. Therefore, ultrasonic treatment for 6 minutes can be used to improve the properties of potato protein and expand the application range of potato protein.

[0139] It should be understood that the above-described specific embodiments of the present invention are merely illustrative or illustrative of the principles of the present invention and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents thereof.

Claims

1. A method for regulating the functional properties and antioxidant activity of potato protein, characterized in that: The following steps are involved: S1. Dissolve potato protein with a protein content of 68.44 ± 0.58% (N × 6.25) in 50-100 mL of deionized water to a protein concentration of 20-30 mg / mL; S2. Adjust the pH of the potato protein to 6.5-7.5 using 1-2 mol / L NaOH to prepare a potato protein solution with a concentration of 15-25 mg / mL; S3. Take 30-50 mL of potato protein solution and place it in a probe-type ultrasonic processor. Control the temperature of the water bath at 25-30° C., ultrasonic frequency at 20 kHz, amplitude at 30-40%, and ultrasonicate for 2-10 minutes.

2. The method for regulating the functional properties and antioxidant activity of potato protein according to claim 1, wherein: The preparation of potato protein in step S1 comprises the following steps: S1.

1. Take fresh potato tubers, beat them into pulp, and filter them through 60-100 mesh gauze to collect the filtrate; S1.

2. Rinse the filter residue with 30-50 mL of distilled water and collect the filtrate. S1.

3. Combine the two filtrates and place them in a beaker. Adjust the pH of the filtrate to 9.0 with 1 mol / L NaOH. Then, add the filtrate to a magnetic stirring device at a stirring speed of 1500-2500 r / min. After magnetic stirring at 37°C for 30 minutes, place the filtrate in a centrifuge and centrifuge at 9000 r / min for 30 minutes to collect the precipitate. S1.

4. Transfer the supernatant after centrifugation to a beaker and adjust the pH to 4.5 with 1 mol / L hydrochloric acid to allow the protein to fully precipitate. Then, place the supernatant in a centrifuge and centrifuge at 9000 rpm for 30 minutes to collect the precipitate. S1.

5. Combine the two precipitates, wash them with distilled water 2-3 times, and freeze-dry them to obtain potato protein with a mass content of 68.44±0.58% (N×6.25).

3. The method for regulating the functional properties and antioxidant activity of potato protein according to claim 1, characterized in that: The ultrasonic process in step S3 adopts a pulse mode, ultrasonicating for 3-5 seconds and stopping for 3-5 seconds.

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