Plasma-activated water assisted soy protein isolate-soluble soybean polysaccharide glycosylation complex, and preparation method and application thereof

Abstract

The application belongs to the technical fields of plant protein modification and food emulsion preparation, and discloses a plasma-activated water assisted soybean protein isolate-soluble soybean polysaccharide glycosylation compound, a preparation method and application thereof. A water phase medium is treated by a cold atmospheric pressure plasma jet to obtain plasma-activated water; then, the soybean protein isolate and the soluble soybean polysaccharide are dispersed in the plasma-activated water, stirred, hydrated, glycosylated at 80 DEG C, cooled in an ice water bath and freeze-dried to obtain a glycosylation compound. The compound is prepared into a water dispersion, mixed with soybean oil and homogenized to obtain an oil-in-water emulsion. The dispersion and interface performance of the compound can be regulated by adjusting the plasma treatment time, the sample treated for 40 s has a higher solubility and a lower polydispersity index, and the sample treated for 80 s has a higher glycosylation degree, foaming capacity and emulsifying activity. The obtained compound can be used as an emulsified food ingredient.

Description

Technical Field

[0001] This invention belongs to the field of plant protein modification and food emulsion preparation technology, specifically relating to a plasma-activated water-assisted soy protein isolate-soluble soy polysaccharide glycosylation complex, its preparation method, and its application. Background Technology

[0002] Soy protein isolate is characterized by its high protein content and relatively balanced amino acid composition, and is widely used in plant protein beverages, emulsified foods, and plant-based foods. Natural soy protein isolate typically has a relatively compact globular conformation, with some hydrophobic groups, thiol groups, and amino reactive sites embedded within the molecule, limiting its dispersibility in aqueous phases and its rapid adsorption capacity at the oil-water interface.

[0003] Glycosylation between proteins and polysaccharides allows the reducing end of the polysaccharide to covalently bind to the amino group of the protein, thereby regulating the protein's surface charge, hydrophilicity-hydrophobicity balance, and spatial conformation. In conventional wet thermal glycosylation, the accessibility of natural protein reaction sites is limited; extending the heating time to increase the reaction degree easily leads to protein aggregation and browning, making it difficult to simultaneously achieve optimal glycosylation degree, dispersion performance, and interfacial properties.

[0004] Cold atmospheric pressure plasma can generate a variety of active species in aqueous phases. When directly treating proteins or protein-polysaccharide mixtures with plasma, the active species may locally concentrate on the material surface, causing uneven treatment or localized over-oxidation. Therefore, there is a need for a complete method that first prepares plasma-activated water, then uses the plasma-activated water to promote appropriate protein conformational development and subsequent glycosylation, and further uses the resulting complex in emulsion preparation. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a plasma-activated water-assisted soy protein isolate-soluble soy polysaccharide glycosylation complex, its preparation method, and its application. This invention first treats an aqueous medium with a cold atmospheric pressure plasma jet, then uses the resulting plasma-activated water as the glycosylation reaction medium. This allows for the adjustment of the accessibility of the reaction sites of soy protein isolate, and the resulting glycosylation complex is used to prepare oil-in-water emulsions.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a plasma-activated water-assisted soy protein isolate-soluble soy polysaccharide glycosylation complex, comprising the following steps: First, the aqueous medium is subjected to cold atmospheric pressure plasma jet treatment to obtain plasma-activated water; Then, soy protein isolate and soluble soy polysaccharide are dispersed in the plasma-activated water to obtain a dispersion; the dispersion is then subjected to stirring, hydration, heating for saccharification, and cooling to obtain a reaction solution. Finally, the obtained reaction solution was freeze-dried to obtain the soybean protein isolate-soluble soybean polysaccharide glycosylated complex.

[0007] Preferably, the aqueous medium is a phosphate buffer solution with a concentration of 0.01 mol / L and a pH of 8.0, and the aqueous medium is subjected to cold atmospheric pressure plasma jet treatment, with a single treatment volume of 50 mL.

[0008] Preferably, the cold atmospheric pressure plasma jet has a working power of 300 W, uses air as the carrier gas, the jet nozzle is 4 cm away from the surface of the aqueous medium, and the processing time is 20 to 120 s.

[0009] Preferably, the mass ratio of soy protein isolate to soluble soy polysaccharide is 1:1, and the total mass concentration of soy protein isolate and soluble soy polysaccharide in the dispersion is 10 mg / mL.

[0010] Preferably, the stirring is performed by magnetic stirring at room temperature for 0.5 h; the hydration temperature is 4°C for 12 h; the heating saccharification temperature is 80°C for 1 h; and the cooling is performed by ice-water bath cooling to 4°C. The freeze-drying conditions are -80°C under vacuum for 48 h.

[0011] The present invention also provides a soybean protein isolate-soluble soybean polysaccharide glycosylated complex obtained by the preparation method described in the above technical solution.

[0012] The present invention also provides the application of the soybean protein isolate-soluble soybean polysaccharide glycosylation complex described in the above technical solution in the preparation of oil-in-water emulsions.

[0013] Preferably, the method for preparing the oil-in-water emulsion includes the following steps: The soybean protein isolate-soluble soybean polysaccharide glycosylation complex was dispersed in water to obtain an aqueous dispersion of the complex. The aqueous dispersion of the complex was mixed with oil and then homogenized and emulsified to obtain the oil-in-water emulsion.

[0014] Preferably, the oil is soybean oil, and the volume ratio of the oil to the aqueous dispersion of the complex is 1:3.

[0015] Preferably, the concentration of the soybean protein isolate-soluble soybean polysaccharide glycosylation complex in the aqueous dispersion of the complex is 10 mg / mL.

[0016] Preferably, the homogenization emulsification speed is 10000 r / min and the time is 2 min.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention first prepares plasma-activated water, which is then used as the aqueous medium for the glycosylation reaction of soy protein isolate and soluble soy polysaccharides. This allows plasma-active species to form and distribute in the aqueous phase first, avoiding direct action of the plasma jet on the protein surface. Appropriate treatment can promote protein conformational unfolding and exposure of amino reaction sites, increase the early covalent binding between proteins and polysaccharides, and simultaneously inhibit the formation of colored late Maillard products.

[0018] This invention uses plasma treatment time in an aqueous medium as a functional adjustment parameter. The solubility of the complex obtained after 40 s treatment is 67.23%, which is 2.81 times that of natural soy protein isolate, and the polydispersity index is 0.451. The degree of glycosylation of the complex obtained after 80 s treatment is 28.90%, the foaming ability is 93.99%, the foam stability is 84.40%, and the emulsifying activity index is 32.28 m² / g. Therefore, the treatment time can be selected according to the product's requirements for aqueous dispersibility or rapid interface formation capability.

[0019] The glycosylated complex obtained by this invention can be dispersed in water and homogenized with soybean oil to form an oil-in-water emulsion, thus forming a complete technical process from plasma-activated water preparation, glycosylated complex preparation to emulsion preparation. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments are briefly described below. The following drawings are only some embodiments of the present invention; those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0021] Figure 1 This is a flowchart illustrating the preparation process of plasma-activated water, glycosylated complex, and oil-in-water emulsion according to the present invention. Figure 2 The results show the determination of degree of glycosylation (A) and degree of browning (B) under different plasma treatment times; Figure 3 The results are as follows: SDS-PAGE (A), protein secondary structure (B), disulfide bond conformation (C), aromatic residue microenvironment (D), and ultraviolet absorption (E). Figure 4 The results of measurements of intrinsic fluorescence (A), X-ray diffraction (B), surface free thiol groups (C), surface hydrophobicity (D), particle size, polydispersity index (E), and zeta potential (F) are shown. Figure 5This is a scanning electron microscope image of the sample; Figure 6 This is an atomic force microscope image of the sample; Figure 7 The apparent viscosity (E) and shear pressure (F) are the solubility (A), turbidity (B), foaming properties (C), emulsifying properties (D), and steady-state shear rheological properties of the emulsion. Figure 8 The results show the determination of the free radical scavenging abilities of DPPH and ABTS; Detailed Implementation

[0022] This invention provides a method for preparing a plasma-activated water-assisted soy protein isolate-soluble soy polysaccharide glycosylation complex, comprising the following steps: Plasma-activated water is obtained by treating an aqueous medium with a cold atmospheric pressure plasma jet. Soy protein isolate (SPI) and soluble soybean polysaccharide (SSPS) were dispersed in plasma-activated water, and then stirred, hydrated, heated for glycosylation, and cooled in sequence. The resulting reaction solution was freeze-dried to obtain the soy protein isolate-soluble soybean polysaccharide glycosylated complex.

[0023] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0024] This invention involves treating an aqueous medium with a cold atmospheric pressure plasma jet to obtain plasma-activated water.

[0025] In this invention, the aqueous phase medium is preferably a phosphate buffer solution, the concentration of which is preferably 0.01 mol / L and the pH preferably 8.0; the single treatment volume of the aqueous phase medium is preferably 50 mL. The phosphate buffer solution is only used as the starting aqueous phase medium for preparing plasma-activated water, and the reaction medium obtained after plasma treatment is uniformly referred to as plasma-activated water.

[0026] In this invention, the working power of the cold atmospheric pressure plasma jet is preferably 300 W, and the carrier gas is preferably air; the distance between the jet nozzle and the surface of the aqueous medium is preferably 4 cm, and the processing time is preferably 20–120 s, more preferably 40–80 s. A processing time of 40 s is beneficial for obtaining a complex with high solubility and relatively uniform particle distribution; a processing time of 80 s is beneficial for obtaining a complex with high glycosylation degree, foaming ability, and emulsifying activity.

[0027] After obtaining plasma-activated water, the present invention disperses soy protein isolate and soluble soy polysaccharide in the plasma-activated water to obtain a dispersion; and then performs stirring, hydration, heating for saccharification and cooling in sequence.

[0028] In this invention, the preferred mass ratio of soy protein isolate to soluble soy polysaccharide is 1:1; the preferred total mass concentration of soy protein isolate and soluble soy polysaccharide in the dispersion is 10 mg / mL; and the mixture obtained by mixing soy protein isolate and soluble soy polysaccharide at a mass ratio of 1:1 is abbreviated as PS.

[0029] In this invention, the stirring is preferably magnetic stirring, the stirring temperature is preferably room temperature, and the stirring time is preferably 0.5 h; the hydration temperature is preferably 4°C, and the hydration time is preferably 12 h. Through stirring and low-temperature hydration, soy protein isolate and soluble soy polysaccharides can be fully dispersed and hydrated in plasma-activated water.

[0030] In this invention, the heating glycosylation is preferably carried out under magnetic stirring conditions, the heating temperature is preferably 80°C, and the heating time is preferably 1 h; after heating, it is preferably placed in an ice-water bath for cooling immediately to terminate the glycosylation reaction and limit subsequent browning.

[0031] After cooling, the reaction solution obtained in this invention is freeze-dried to obtain a soybean protein isolate-soluble soybean polysaccharide glycosylated complex.

[0032] The present invention also provides a soybean protein isolate-soluble soybean polysaccharide glycosylated complex obtained by the preparation method described in the above technical solution.

[0033] The present invention also provides the application of the soybean protein isolate-soluble soybean polysaccharide glycosylation complex described in the above technical solution in the preparation of oil-in-water emulsions.

[0034] In this invention, the method for preparing the oil-in-water emulsion preferably includes the following steps: The soybean protein isolate-soluble soybean polysaccharide glycosylation complex was dispersed in water to obtain an aqueous dispersion of the complex. The aqueous dispersion of the complex was mixed with oil and then homogenized and emulsified to obtain the oil-in-water emulsion.

[0035] In this invention, the concentration of the glycosylated complex in the aqueous dispersion of the complex is preferably 10 mg / mL.

[0036] In this invention, the oil is preferably vegetable oil, more preferably soybean oil; the volume ratio of the oil to the aqueous dispersion of the complex is preferably 1:3.

[0037] In this invention, the preferred rotation speed for homogenizing emulsification is 10,000 r / min, and the preferred time is 2 min. During homogenization, the glycosylated complex is adsorbed at the oil-water interface and forms an interfacial layer, thereby obtaining an oil-in-water emulsion.

[0038] To further illustrate the present invention, the following detailed description of the soybean protein isolate-soluble soybean polysaccharide glycosylation complex, its preparation method, and its application, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1:

[0040] A cold atmospheric pressure plasma jet apparatus (PG-1000 ZD, Nanjing Suman Plasma Technology Co., Ltd.) was used, with the operating power set to 300 W and air as the carrier gas. 50 mL of 0.01 mol / L, pH 8.0 phosphate buffer solution was taken, and the jet nozzle was positioned 4 cm above the liquid surface. Treatment was performed for 0, 20, 40, 60, 80, 100, and 120 seconds. No plasma treatment was performed in the 0-second group; plasma-activated water was obtained for the corresponding treatment time in each of the other groups.

[0041] Soy protein isolate and soluble soybean polysaccharide were added to plasma-activated water treated with different methods at a 1:1 mass ratio to form dispersions with a total mass concentration of 10 mg / mL. Each dispersion was magnetically stirred at room temperature for 0.5 h, then hydrated at 4 °C for 12 h. Subsequently, a glycosylation reaction was carried out by magnetic stirring at 80 °C for 1 h. After the reaction, the mixture was immediately cooled to 4 °C in an ice-water bath and freeze-dried (under vacuum at -80 °C for 48 h) to obtain glycosylated complexes (corresponding to different treatment times, which will be abbreviated as 0 s samples, 20 s samples, 40 s samples, 60 s samples, 80 s samples, 100 s samples, and 120 s samples, respectively).

[0042] 2) Determination of sample structure and physicochemical properties 1. Determination of glycosylation degree: Dilute the sample to 0.5 mg / mL, mix with OPA reagent at a volume ratio of 1:20, react at 35℃ for 2 min, and measure the absorbance at 340 nm. The glycosylation degree is calculated according to formula (1): (1) In equation (1), A1 is the absorbance of SPI before glycosylation, and A2 is the absorbance of the glycosylated sample.

[0043] Figure 2 A represents the results of glycosylation degree determination. The glycosylation degree of the 0 s sample was 12.34%. With the extension of plasma treatment time, the glycosylation degree increased and reached 28.90% in the 80 s sample, which was 2.34 times that of the 0 s sample. The glycosylation degree of the 100 s and 120 s samples decreased to 26.27% and 24.67%, respectively. This indicates that moderate plasma-activated water treatment is beneficial to the covalent bonding of carbonyl and amino groups, while excessive treatment time limits the exposure of reactive sites.

[0044] 2. Determination of browning degree: Adjust the sample solution to 5 mg / mL, take 4 mL of sample solution and mix it with 2 mL of 200 g / L SDS solution and 2 mL of 0.1 mol / L borax buffer. Replace the sample solution with an equal volume of phosphate buffer as a blank, and measure the absorbance at 420 nm.

[0045] Figure 2 B represents the results of the browning degree measurement. The browning absorbance at 420 nm decreased continuously from 0.759 for the 0 s sample to 0.479 for the 120 s sample, indicating that under the treatment conditions of this invention, plasma-activated water promotes the early glycosylation reaction without increasing the accumulation of colored late Maillard products.

[0046] 3. SDS-PAGE assay: A 5% stacking gel and a 10% separating gel were used. 10 μL of 1 mg / mL sample solution was mixed with the loading buffer at a volume ratio of 1:5 and heated at 90℃ for 5 min. 10 μL of sample was added to each well, and electrophoresis was performed at a constant voltage of 120 V. After electrophoresis, Coomassie Brilliant Blue R-250 was used for staining, destaining, and photographing.

[0047] Figure 3 A represents the results of SDS-PAGE analysis. The glycosylated sample bands broadened, became less clear, and migrated towards higher molecular weight regions, indicating that soluble soybean polysaccharides and soybean protein isolate formed a covalent complex with a broad molecular weight distribution, and that none of the treatments caused significant protein backbone breakage.

[0048] 4. Raman spectroscopy analysis: The sample was evenly spread on a glass slide, and a 532 nm laser with an excitation power of 10 mW was used to collect spectra in the range of 400–2000 cm⁻¹. The acquisition time was 10 s, and 10 scans were performed cumulatively. The intensity of the phenylalanine peak at 1003 cm⁻¹ was normalized, and the secondary structure, disulfide bond conformation, and aromatic residue microenvironment of the protein were analyzed.

[0049] Figure 3 B represents the results of protein secondary structure determination. In the SPI, the α-helix and β-sheet contents were 29% and 34%, respectively, decreasing to 19% and 27% in the 0 s sample; in the 60 s sample, the α-helix content decreased to 12%, while the β-turn content increased to 35%, whereas in the 120 s sample, the α-helix content rebounded to 30%, and the random coil content decreased to 17%. This indicates that moderate treatment promotes structural unfolding, while excessively long treatment times cause local rearrangement within the aggregates.

[0050] Figure 3C represents the results of disulfide bond conformation determination. The proportion of the ggg conformation decreased from 49.63% in the SPI to 34.67% in the 0 s sample, and further to 10.50% in the 80 s sample; the proportion of the ggt conformation increased from 29.91% in the SPI to 38.82% in the 0 s sample, and further to 73.40% in the 80 s sample, indicating that moderate treatment promotes protein unfolding and disulfide bond rotation and rearrangement.

[0051] Figure 3 D represents the measurement results of the aromatic residue microenvironment. Tyrosine I 850 / I 830 The concentration of tryptophan I increased from 0.70 for SPI and 0.49 for PS to 1.00 for the 0 s sample, and reached 1.34 for the 80 s sample; normalized tryptophan I 760 The decrease from 0.37 in the 0 s sample to 0.28 in the 80 s sample indicates that moderate treatment improves the solvent accessibility of aromatic residues. The partial rebound or decline after 100 s and 120 s reflects the re-embedding of residues during the aggregation process.

[0052] 5. Ultraviolet absorption spectroscopy analysis: Dilute the sample with deionized water to 1 mg / mL and collect the ultraviolet absorption spectrum in the range of 240–350 nm.

[0053] Figure 3 E represents the ultraviolet absorption spectrum. The absorption intensity of the glycosylated sample in the 250–290 nm region was generally higher than that of SPI and PS, with the highest absorption intensity observed in the 80 s sample. This indicates that moderate treatment increased the exposure of aromatic residues such as tyrosine and tryptophan and altered the early Maillard products and their surrounding polar environment.

[0054] 6. Intrinsic fluorescence analysis: The sample was diluted with deionized water to 1 mg / mL, the excitation wavelength was 280 nm, the emission scanning range was 310–410 nm, and the excitation and emission slit widths were both 5 nm.

[0055] Figure 4 A represents the intrinsic fluorescence spectrum. The maximum emission wavelengths of both SPI and PS are approximately 345 nm. The 0 s sample redshifts to 351 nm, and the 60 s sample further redshifts to 353 nm. The fluorescence intensity of the 40–80 s samples is relatively low, indicating that the aromatic residues are exposed to a polar environment and undergo glycosylation quenching. The maximum emission wavelengths of the 100 s and 120 s samples shift back to 347–348 nm, indicating that after prolonged treatment, some aromatic residues re-enter the hydrophobic region during aggregation.

[0056] 7. X-ray diffraction analysis: Cu Kα radiation was used, with an operating voltage of 40 kV and an operating current of 40 mA. The scanning range was 2θ = 5 to 90°, and the scanning rate was 2° / min.

[0057] Figure 4 B is the X-ray diffraction pattern. All samples showed broad protein diffraction peaks at approximately 9° and 19–21° at 2θ. Physical mixing, glycosylation, and plasma-activated water treatment altered the peak width and relative intensity, but no new strong and sharp diffraction peaks appeared, indicating that the treatment changed the local ordered stacking without forming a new overall crystal phase.

[0058] 8. Determination of surface free thiol content: Disperse the sample in Tris-Gly buffer to 1 mg / mL, centrifuge at 8000 r / min for 20 min, take the supernatant and mix with 4 mg / mL DTNB reagent, react in the dark for 1 h, and measure the absorbance at 412 nm. The surface free thiol content is calculated according to formula (2): (2) In equation (2), ΔA 412 The absorbance at 412 nm after deducting the blank is given. D is the dilution factor, C is the protein concentration in the sample solution in mg / mL, and 73.53 is the conversion factor.

[0059] Figure 4 C represents the results of the determination of surface free thiol content. The surface free thiol content of SPI, PS, and 0 s samples were 5.23, 7.86, and 13.96 μmol / g, respectively; the content increased to 16.41 μmol / g in the 80 s sample, and decreased to 11.60 and 9.37 μmol / g in the 100 s and 120 s samples, respectively. This indicates that moderate treatment promotes thiol exposure and disulfide bond exchange, while excessive treatment time leads to thiol oxidation or re-embedding.

[0060] 9. Determination of surface hydrophobicity: Disperse the sample in phosphate buffer at pH 8.0 to a concentration of 5 mg / mL, add 200 μL of bromophenol blue solution (1 mg / mL), mix, and centrifuge at 8000 r / min for 20 min; take the supernatant, dilute it 10 times with phosphate buffer, and measure the absorbance at 595 nm. The bromophenol blue binding amount of the sample is calculated according to formula (3): (3) In equation (3), A blank A represents the absorbance of the blank supernatant at 595 nm. sample The absorbance of the sample supernatant at 595 nm is given.

[0061] Figure 4D represents the results of surface hydrophobicity determination. The bromophenol blue binding amount decreased from 51.26 μg in SPI to 38.63 μg in PS and 31.80 μg in the 0 s sample, and then increased with increasing treatment time, reaching 68.27 μg in the 80 s sample, and decreasing to 62.83 and 56.07 μg in the 100 s and 120 s samples, respectively. This indicates that moderate treatment exposes nonpolar residues, while excessive treatment promotes the aggregation of hydrophobic regions or their re-masking by polysaccharide segments.

[0062] 10. Determination of particle size and polydispersity index: Disperse the sample in phosphate buffer at pH 8.0 to 1 mg / mL, centrifuge at 10000 r / min for 20 min, take the supernatant and equilibrate at 25℃ for 2 min before determining the hydrodynamic particle size and polydispersity index.

[0063] Figure 4 E represents the results of particle size and polydispersity index determination. The average particle size and polydispersity index of SPI were 398.70 nm and 0.806, respectively, while those of PS decreased to 209.27 nm and 0.552, respectively. The average particle size of the glycosylated samples increased from 247.30 nm in the 0 s sample to 352.93 nm in the 80 s sample and 379.30 nm in the 120 s sample. The polydispersity index of the 40 s sample was the lowest at 0.451, indicating that the 40 s sample formed a relatively homogeneous hydrated complex, while the particle heterogeneity increased with further extension of the treatment time.

[0064] 11. Measurement of zeta potential: Disperse the sample in phosphate buffer at pH 8.0 to 1 mg / mL, centrifuge at 10000 r / min for 20 min, take the supernatant and equilibrate at 25℃ for 2 min before measuring the zeta potential.

[0065] Figure 4 F represents the measurement results of the zeta potential. The zeta potential gradually shifted negatively from -7.84 mV for SPI, -9.32 mV for PS, and -10.78 mV for the 0 s sample, reaching -15.53 mV for the 80 s sample; the zeta potential shifted back to approximately -13.1 mV for the 100 s and 120 s samples. This indicates that moderate treatment increased the negative charge on the surface of the complex and electrostatic repulsion between particles, while excessive treatment time resulted in charge shielding or embedding of charged groups.

[0066] 12. Scanning electron microscopy observation: The freeze-dried sample was fixed on conductive adhesive and sputtered with gold. The surface morphology of the sample was observed at 200x and 2000x magnification under an accelerating voltage of 20 kV.

[0067] Figure 5The images are scanning electron microscope images of the samples. SPI mainly presents as a relatively smooth, collapsed spherical structure; after glycosylation, the samples gradually form cracks, open lamellae, and porous regions. The 120 s sample shows larger and more densely bound particle domains, indicating that moderate processing is beneficial for structure development, while excessive processing time promotes heterogeneous aggregation.

[0068] 13. Atomic force microscopy observation: Dilute the sample appropriately, add 5 μL to a fresh mica sheet and air dry. Under environmental conditions, use closed-loop AC mode for scanning, with a scanning range of 2 μm × 2 μm, a pixel size of 256 × 256, and a scanning frequency of 1.796 Hz.

[0069] Figure 6 The images are atomic force microscopy (AFM) images of the samples. The 80 s sample shows more obvious nanoscale protrusions, while the peak shapes of the 100 s and 120 s samples tend to be heterogeneous, reflecting the transformation of the complex from a homogeneous hydrated state to an interfacial active aggregate and a heterogeneous aggregate state.

[0070] 14. Determination of solubility: Disperse 20 mg of sample in 10 mL of phosphate buffer (pH 8.0), shake in a 25°C water bath for 1 h, then centrifuge at 12000 r / min for 15 min. Determine the protein concentration in the supernatant using the Bradford method. Solubility is calculated according to formula (4): (4) In equation (4), C s C0 is the protein concentration in the supernatant, and C0 is the initial protein concentration in the dispersion.

[0071] Figure 7 A represents the solubility measurement results. The solubility of SPI, PS, and 0 s samples were 23.90%, 25.48%, and 49.09%, respectively. The solubility of the 20 s and 40 s samples increased to 52.33% and 67.23%, respectively. Thereafter, the solubility decreased, with the 120 s sample dropping to 13.13%. This indicates that the 40 s treatment formed a complex with good hydration and dispersion uniformity, while excessively long treatment times promoted particle aggregation and reduced solubility.

[0072] 15. Turbidity determination: Disperse the sample with deionized water at a mass ratio of 1:100, equilibrate at 25℃, and use deionized water as a blank to measure the absorbance at 500 nm. The turbidity coefficient is calculated according to formula (5): (5) In equation (5), T is the turbidity coefficient, and A 500 The absorbance at 500 nm after subtracting the blank is L, which is the optical path length of the cuvette.

[0073] Figure 7B represents the turbidity measurement results. The turbidity decreased from 0.541 for SPI and 0.418 for PS to 0.285 for the 0 s sample, reaching a minimum value of 0.163 for the 80 s sample; the turbidity rebounded to 0.211 and 0.207 for the 100 s and 120 s samples, respectively, indicating that moderate treatment reduced strong scattering aggregates, while excessive treatment time increased the non-uniformity of particle distribution.

[0074] 16. Determination of foaming performance: Homogenize the 2 mg / mL sample dispersion at 10000 r / min for 2 min, immediately transfer it to a graduated cylinder, and record the liquid volume V0 before homogenization, the total volume V1 immediately after homogenization, and the total volume V2 after standing for 15 min. The foaming capacity FC and foam stability FS are calculated according to equations (6) and (7), respectively: (6) (7) Figure 7 C represents the results of foaming performance measurement. The foaming capacity increased from 13.62% of SPI, 16.17% of PS, and 36.27% of the 0 s sample to 93.99% of the 80 s sample, and the foam stability increased from 68.30% of SPI to 84.40% of the 80 s sample. The foaming capacity of the 100 s and 120 s samples decreased to 65.95% and 45.46%, respectively, indicating that the 80 s sample has better gas-water interface adsorption and film stability, while the heterogeneous aggregates formed by over-treatment reduce the interfacial migration capacity.

[0075] Example 2: 1) Emulsion preparation Samples at SPI, PS, 0 s, 20 s, 40 s, 60 s, 80 s, 100 s, and 120 s were prepared into 10 mg / mL sample dispersions with water. 15 mL of the sample dispersion was mixed with 5 mL of soybean oil and homogenized at 10000 r / min for 2 min to obtain an oil-in-water emulsion.

[0076] 2) Determination of emulsion-related indicators 1. Determination of Emulsifying Activity Index and Emulsifying Stability Index: Immediately after emulsion preparation and after standing for 10 min, 100 μL of emulsion was taken from the bottom of the container, diluted with 5 mL of sodium dodecyl sulfate solution with a mass concentration of 1 g / L, and vortexed for 1 min. The absorbance was measured at 500 nm. The Emulsifying Activity Index (EAI) and Emulsifying Stability Index (ESI) were calculated according to formulas (8) and (9), respectively: (8) (9) In equations (8) and (9), A0 and A 10The absorbance values ​​were measured at 0 min and 10 min after emulsion preparation, respectively. N is the dilution factor, C is the protein concentration in the sample dispersion in g / mL, and φ is the oil phase volume fraction, taken as 0.25.

[0077] Figure 7 D represents the results of emulsifying performance determination. The emulsifying activity index increased from 15.11 m² / g for SPI, 21.13 m² / g for PS, and 24.71 m² / g for the 0 s sample to 32.28 m² / g for the 80 s sample. The emulsifying stability indices for SPI and PS were 33.73 min and 38.58 min, respectively, while the emulsifying stability index for the glycosylated complex ranged from 23.59 to 25.26 min. Therefore, the current results indicate that the 80 s sample has higher initial emulsifying activity, but this does not necessarily mean that its 10 min emulsifying stability index has been improved.

[0078] 2. Determination of steady-state shear rheological properties of emulsion: A MARS 60 rheometer was used with a parallel plate clamp with a diameter of 40 mm and a plate spacing of 1 mm. The measurement temperature was 25℃. The shear rate was increased from 0.1 s⁻¹ to 100 s⁻¹, and the apparent viscosity and shear stress of the emulsion were recorded.

[0079] Figure 7 E and Figure 7 F represents the results of the steady-state shear rheological properties of the emulsions. The apparent viscosity of all emulsions decreased with increasing shear rate, exhibiting shear-thinning characteristics. The apparent viscosity of the emulsion prepared by the glycosylated complex was lower than that of the SPI emulsion, indicating that it has lower flow resistance under the existing formulation and measurement conditions.

[0080] Example 3: 1. Determination of ABTS free radical scavenging ability: In the ABTS test, 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution were mixed in equal volumes and placed at 4℃ in the dark for 12 h. The absorbance at 734 nm was diluted with anhydrous ethanol to 0.70±0.02. 1 mL of sample solution was mixed with 3 mL of ABTS working solution and reacted in the dark for 6 min before being measured at 734 nm. The ABTS free radical scavenging rate was calculated according to formula (10): (10) In formula (10), A0 is the absorbance of the control when distilled water is used to replace the sample, and A1 is the absorbance after the sample is added.

[0081] Figure 8The results show the determination of ABTS free radical scavenging ability. The ABTS free radical scavenging rate increased from 51.91% for SPI and 56.72% for PS to 73.22% for the 0 s sample. The glycosylated samples maintained a high level at different treatment times, with the 60 s sample reaching 75.52% and the 120 s sample reaching 74.86%, indicating that the hydrophilic antioxidant groups formed by glycosylation remained effective over a wide treatment range.

[0082] 2. Determination of DPPH free radical scavenging ability: In the DPPH test, 1 mL of sample solution was mixed with 1 mL of 0.1 mmol / L DPPH ethanol solution, and reacted at 25℃ in the dark for 30 min. The absorbance was measured at 517 nm. The DPPH free radical scavenging rate was calculated according to formula (11): (11) In formula (11), A2 is the absorbance of the control composed of distilled water and DPPH solution, A3 is the absorbance of the sample after reacting with DPPH solution, and A4 is the absorbance of the blank sample composed of the sample and anhydrous ethanol.

[0083] Figure 8 The results show the determination of DPPH radical scavenging ability. The DPPH radical scavenging rate increased from 36.31% for SPI and 37.03% for PS to 69.10% for the 0 s sample, 72.61% for the 80 s sample, and decreased to about 60% for the 100 s and 120 s samples. This indicates that glycosylation significantly improves the DPPH radical scavenging ability. Moderate plasma-activated water treatment can further improve the activity, while excessive treatment time will reduce the scavenging ability due to oxidation or re-embedding of hydrogen-donating groups.

[0084] Although the above embodiments have described the present invention in detail, these embodiments are only some, not all, implementations of the present invention. Equivalent substitutions or modifications made by those skilled in the art without departing from the inventive concept are all within the scope of protection of the present invention.

Claims

1. A method for preparing a plasma-activated water-assisted soy protein isolate-soluble soy polysaccharide glycosylation complex, characterized in that, Includes the following steps: First, the aqueous medium is subjected to cold atmospheric pressure plasma jet treatment to obtain plasma-activated water; Then, soy protein isolate and soluble soy polysaccharide are dispersed in the plasma-activated water to obtain a dispersion; the dispersion is then subjected to stirring, hydration, heating for saccharification, and cooling to obtain a reaction solution. Finally, the obtained reaction solution was freeze-dried to obtain the soybean protein isolate-soluble soybean polysaccharide glycosylated complex.

2. The preparation method according to claim 1, characterized in that, The aqueous medium is a phosphate buffer solution with a concentration of 0.01 mol / L and a pH of 8.

0. The aqueous medium is subjected to cold atmospheric pressure plasma jet treatment, with a single treatment volume of 50 mL.

3. The preparation method according to claim 1 or 2, characterized in that, The cold atmospheric pressure plasma jet has a working power of 300 W, uses air as the carrier gas, the jet nozzle is 4 cm away from the surface of the aqueous medium, and the processing time is 20 to 120 seconds.

4. The preparation method according to claim 1, characterized in that, The mass ratio of soy protein isolate to soluble soy polysaccharide is 1:1, and the total mass concentration of soy protein isolate and soluble soy polysaccharide in the dispersion is 10 mg / mL. The stirring is performed by magnetic stirring at room temperature for 0.5 h, the hydration temperature is 4 °C and the time is 12 h, the heating saccharification temperature is 80 °C and the time is 1 h, the cooling is performed by ice-water bath cooling to 4 °C, and the freeze-drying conditions are -80 °C under vacuum for 48 h.

5. The soybean protein isolate-soluble soybean polysaccharide glycosylated complex obtained by the preparation method according to any one of claims 1 to 4.

6. The application of the soybean protein isolate-soluble soybean polysaccharide glycosylation complex according to claim 5 in the preparation of oil-in-water emulsions.

7. The application according to claim 6, characterized in that, The method for preparing the water-in-oil emulsion includes the following steps: The soy protein isolate-soluble soy polysaccharide glycosylation complex was dispersed in water to obtain an aqueous dispersion of the complex; the aqueous dispersion of the complex was then mixed with oil and homogenized and emulsified to obtain the oil-in-water emulsion.

8. The application according to claim 7, characterized in that, The volume ratio of the oil to the aqueous dispersion of the complex is 1:3, and the oil is soybean oil.

9. The application according to claim 7, characterized in that, The concentration of the soybean protein isolate-soluble soybean polysaccharide glycosylation complex in the aqueous dispersion of the complex was 10 mg / mL.

10. The application according to claim 7, characterized in that, The homogenization emulsification was performed at a speed of 10,000 r / min for 2 min.

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