Preparation method of soybean protein isolate peptide covalent complex and covalent complex

Abstract

This invention discloses a method for preparing a covalent complex of soy protein isolate peptides and the covalent complex itself, belonging to the field of food manufacturing. The method includes the following steps: dispersing soy protein isolate in water and preheating it to induce thermal depolymerization, obtaining a pretreated protein solution; subjecting the pretreated protein solution to a glycosylation cross-linking reaction with chitosan oligosaccharide under the catalysis of transglutaminase, obtaining a chitosan oligosaccharide-soy protein isolate covalent complex; subjecting the obtained chitosan oligosaccharide-soy protein isolate covalent complex to ultrasound-assisted enzymatic hydrolysis using a complex protease for stepwise enzymatic hydrolysis, obtaining a chitosan oligosaccharide-soy protein isolate peptide covalent complex solution; and drying the obtained complex solution to obtain the chitosan oligosaccharide-soy protein isolate peptide covalent complex. This application employs a reverse route of glycosylation cross-linking followed by enzymatic hydrolysis, resulting in higher grafting density, more uniform molecular structure, and superior interfacial activity.

Description

Technical Field

[0001] This invention relates to the field of food manufacturing, and more specifically, to a method for preparing a covalent complex of soybean protein isolate peptides and the covalent complex itself. Background Technology

[0002] Soy protein isolate is a widely sourced plant protein with high nutritional value and good processing properties, making it widely used in the food industry. However, its natural structure is relatively compact, with molecules existing as 7S and 11S aggregates, which limits its solubility and functional properties under certain conditions. Enzymatic hydrolysis can yield lower molecular weight soy protein peptides, improving its solubility and imparting certain biological activities. However, enzymatic hydrolysis reduces the stability of the system, limiting its further application in emulsion systems and functional carriers.

[0003] Chitosan oligosaccharides are low-molecular-weight products obtained from the degradation of chitosan. They exhibit good water solubility, high biocompatibility, and both antibacterial and antioxidant activities. Currently, the binding of chitosan oligosaccharides with proteins or peptides is mostly achieved through physical complexation. However, the resulting non-covalent complexes have poor stability and are prone to dissociation under processing, storage, or pH changes. While traditional chemical grafting methods can achieve covalent bonding, they require the use of chemical cross-linking agents, posing residual risks and safety hazards, thus hindering their widespread application in the food industry.

[0004] Transglutaminase can mediate the covalent binding of proteins to chitosan oligosaccharides, but existing processes all follow a fixed pattern of 'enzymatic hydrolysis followed by glycosylation'. In this route, the free polypeptides produced by enzymatic hydrolysis are in a random coil state, and the grafting efficiency of transglutaminase is limited by the flexible conformation of the polypeptide chain; the glycosylation reaction occurs at random sites on the polypeptide chain, and the grafting density and molecular structure of the product are difficult to precisely control; at the same time, lysine residues on the polypeptide chain that can be recognized by transglutaminase are easily over-cleaved or undergo conformational changes during enzymatic hydrolysis, further reducing the glycosylation efficiency.

[0005] There is currently no effective solution to the above problems. Summary of the Invention

[0006] To address the problems in related technologies, this invention proposes a method for preparing a chitosan oligosaccharide-soybean protein isolate covalent complex and the covalent complex itself, thereby overcoming the aforementioned technical problems in existing related technologies.

[0007] The technical solution of this invention is implemented as follows:

[0008] According to one aspect of the present invention, a method for preparing a soybean protein isolate peptide covalent complex is provided, comprising the following steps:

[0009] S1. Disperse soy protein isolate in water and preheat it to induce thermal depolymerization, thus obtaining a pretreated protein solution.

[0010] S2. The pretreated protein solution obtained in step S1 is subjected to a glycosylation cross-linking reaction with chitosan oligosaccharide under the catalysis of transglutaminase to obtain a chitosan oligosaccharide-soybean protein isolate covalent complex.

[0011] S3. The chitosan oligosaccharide-soybean protein isolate covalent complex obtained in step S2 is subjected to ultrasonic-assisted enzymatic hydrolysis, and stepwise enzymatic hydrolysis is performed using a complex protease to obtain a chitosan oligosaccharide-soybean protein isolate peptide covalent complex solution.

[0012] S4. The complex solution obtained in step S3 is dried to obtain a chitosan oligosaccharide-soybean protein isolate covalent complex.

[0013] Optionally, the preheating temperature in step S1 is 70–95°C, and the time is 10–40 min.

[0014] Optionally, the mass concentration of the soy protein isolate solution in step S1 is 3% to 8%, and the pH value is 7.0 to 8.0.

[0015] Optionally, in step S2, the mass ratio of chitosan oligosaccharide to soy protein isolate is 1:2 to 1:5, the amount of transglutaminase added is 5 to 20 U / g protein, the reaction temperature is 30 to 40°C, and the reaction time is 1 to 5 h.

[0016] Optionally, the complex protease in step S3 includes an endopeptidase and an exopeptidase.

[0017] Optionally, the endopeptidase is subtilisin, and the exopeptidase is flavor protease.

[0018] Optionally, the ultrasound-assisted enzymatic hydrolysis treatment in step S3 includes:

[0019] S31. Add Bacillus subtilis protease to the glycosylated crosslinked product obtained in step S2, and carry out the first enzymatic hydrolysis under ultrasonic-assisted conditions. The enzymatic hydrolysis temperature is 50-60℃ and the enzymatic hydrolysis time is 1-3h.

[0020] S32. After the first enzymatic hydrolysis is completed, flavor protease is added to the system, and the second enzymatic hydrolysis is carried out under ultrasonic-assisted conditions. The enzymatic hydrolysis temperature is 45-55℃ and the enzymatic hydrolysis time is 1-3h.

[0021] S33. After enzymatic hydrolysis, the enzyme is inactivated by heating, and the supernatant is collected by centrifugation to obtain a chitosan oligosaccharide-soybean protein isolate peptide covalent complex solution.

[0022] Optionally, the ultrasound power of the ultrasound-assisted ultrasound in steps S31 and S32 is 200-500W, and the ultrasound mode is intermittent ultrasound, with a 5-15s interval between ultrasound sessions.

[0023] Optionally, the enzyme inactivation temperature in step S33 is 85–95°C, and the enzyme inactivation time is 5–15 min.

[0024] According to another aspect of the present invention, a soybean protein isolate peptide covalent complex is provided, which is prepared by the above-described method for preparing a soybean protein isolate peptide covalent complex.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) Breaking through the traditional fixed process mode of first enzymatic hydrolysis and then glycosylation, a reverse route of first glycosylation cross-linking and then enzymatic hydrolysis is adopted. First, complete natural soy protein isolate and chitosan oligosaccharide are formed into a covalent complex under the catalysis of transglutaminase. A high molecular weight copolymer network is constructed with chitosan oligosaccharide as the backbone. Then, active peptides covalently grafted onto the chitosan oligosaccharide chain are released from this network by ultrasound-assisted enzymatic hydrolysis, forming a brush copolymer structure with chitosan oligosaccharide as the backbone and active peptides as side chains. This structure is a brush copolymer, which has a higher grafting density and a more uniform molecular structure than the random copolymer formed by random grafting of peptides and chitosan oligosaccharide in the traditional route, and also has better interfacial activity.

[0027] (2) A stepwise synergistic enzymatic hydrolysis strategy combining endopeptidases and exopeptidases is adopted. First, the peptide fragments are cleaved by Alcalase, and then exolytically modified by Flavorzyme, releasing specific active peptides covalently bound to the chitosan oligosaccharide backbone. This stepwise strategy can achieve precise control of peptide molecular weight and controllable release of active peptides.

[0028] (3) Introducing ultrasonic-assisted treatment during enzymatic hydrolysis, the shear force and microjets generated by ultrasonic cavitation effect can change the substrate conformation, increase the contact frequency between enzyme and substrate, promote product diffusion, significantly improve the enzymatic hydrolysis efficiency and the release rate of grafted peptides, while shortening the enzymatic hydrolysis time and reducing the amount of enzyme used.

[0029] (4) In view of the structural characteristics of soybean protein isolate in the form of 7S / 11S aggregates, a preheating treatment step is added before the reaction to induce thermal depolymerization of the natural aggregates, exposing more lysine residues and glutamine residues that can be recognized by transglutaminase, which significantly improves the glycosylation grafting efficiency. This pretreatment strategy is a targeted design of the present invention for the unique aggregate structure of soybean protein isolate. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a flowchart of a method for preparing a soybean protein peptide covalent complex according to an embodiment of the present invention;

[0032] Figure 2 These are SDS-PAGE electrophoresis spectra of different samples;

[0033] Figure 3 These are Fourier transform infrared spectra of different samples;

[0034] Figure 4 These are the ultraviolet absorption spectra of different samples;

[0035] Figure 5 This is a graph showing the ABTS free radical scavenging rate of different samples;

[0036] Figure 6 This is a graph showing the DPPH free radical scavenging rate of different samples; Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0038] Example 1

[0039] Please see Figure 1 This embodiment provides a method for preparing a covalent complex of soybean protein isolate peptides, specifically including the following steps:

[0040] S1. Preheating and depolymerization treatment: Weigh 5g of soy protein isolate and disperse it in deionized water. Adjust the pH of the system to 7.4 with 1mol / L sodium hydroxide solution. Heat the system to 85℃ under continuous stirring and keep it at that temperature for 30min to induce thermal depolymerization of the soy protein isolate. Then cool it to room temperature and add water to make the total volume 100mL to obtain a pretreated protein solution (mass concentration of 5%).

[0041] S2. Glycosylation cross-linking reaction: Chitosan oligosaccharide (mass ratio of chitosan oligosaccharide to soy protein isolate is 1:3) is added to the pretreated protein solution obtained in step S1, and transglutaminase (enzyme activity 147 U / g, added amount is 10 U / g protein) is added. The reaction is carried out at 37℃ for 3 h to allow the soy protein isolate and chitosan oligosaccharide to undergo a covalent cross-linking reaction, and a chitosan oligosaccharide-soy protein isolate covalent complex solution is obtained.

[0042] S3. Ultrasonic-assisted stepwise enzymatic hydrolysis:

[0043] S31. Add subtilisin (Alcalase, added at 1% of the substrate protein mass) to the glycosylated cross-linked product obtained in step S2, and perform the first enzymatic hydrolysis under ultrasonic-assisted conditions. The ultrasonic power is 300W, and the intermittent ultrasonic mode is used (10s interval between ultrasonic cycles). The enzymatic hydrolysis temperature is 55℃ and the enzymatic hydrolysis time is 2h.

[0044] S32. After the first enzymatic hydrolysis is completed, flavor enzyme (0.5% of the substrate protein mass) is added to the system. The second enzymatic hydrolysis is carried out under ultrasonic-assisted conditions. The ultrasonic power is 300W, and the intermittent ultrasonic mode is used (10s interval between ultrasonic cycles). The enzymatic hydrolysis temperature is 50℃ and the enzymatic hydrolysis time is 2h.

[0045] S33. After enzymatic hydrolysis, heat to 90℃ for 10 min to inactivate the enzyme, cool to room temperature, centrifuge the hydrolysate (10000 r / min, 15 min), collect the supernatant, and obtain the chitosan oligosaccharide-soybean protein isolate peptide covalent complex solution.

[0046] S4. Drying treatment: The complex solution obtained in step S3 is dialyzed for 48 hours to remove small molecules, and then freeze-dried under vacuum to obtain chitosan oligosaccharide-soybean protein isolate peptide covalent complex.

[0047] Example 2

[0048] Based on Example 1, the preheating temperature in step S1 is set to 70°C and the preheating time is 30 min, while the other conditions remain unchanged.

[0049] Example 3

[0050] Based on Example 1, the preheating temperature in step S1 is set to 95°C and the preheating time is 30 min, while the other conditions remain unchanged.

[0051] Example 4

[0052] Based on Example 1, the mass ratio of chitosan oligosaccharide to soy protein isolate in step S2 was changed to 1:1, while the other conditions remained unchanged.

[0053] Example 5

[0054] Based on Example 1, the mass ratio of chitosan oligosaccharide to soy protein isolate in step S2 was changed to 1:5, while the other conditions remained unchanged.

[0055] Example 6

[0056] Based on Example 1, the ultrasonic power in step S3 is set to 200W, while the other conditions remain unchanged.

[0057] Example 7

[0058] Based on Example 1, the ultrasonic power in step S3 is set to 400W, while the other conditions remain unchanged.

[0059] Example 8

[0060] Based on Example 1, in step S3, only Bacillus subtilis protease is used for single-enzyme hydrolysis (without adding flavor protease), and the other conditions remain unchanged.

[0061] Example 9

[0062] Based on Example 1, in step S3, only flavor protease is used for single-enzyme hydrolysis (without adding Bacillus subtilis protease), and the other conditions remain unchanged.

[0063] Compare with Example 1

[0064] This comparative example provides a traditional method for preparing chitosan oligosaccharide-soybean protein isolate peptide covalent complexes via a pre-enzymatic hydrolysis followed by glycosylation route, specifically including the following steps:

[0065] T1. Weigh 5g of soy protein isolate and disperse it in deionized water, adjust the pH to 7.4, and add water to 100mL to obtain a soy protein isolate solution.

[0066] T2. Add Bacillus subtilis protease (1%) to the above solution and hydrolyze at 55°C for 2 hours. Then add flavor protease (0.5%) and hydrolyze at 50°C for 2 hours. After hydrolysis, inactivate the enzyme at 90°C for 10 minutes. Centrifuge and collect the supernatant to obtain soy protein isolate hydrolysate.

[0067] T3. Add chitosan oligosaccharide (mass ratio of chitosan oligosaccharide to hydrolysate is 1:3) and transglutaminase (10U / g) to the obtained hydrolysate, and react at 37℃ for 3h.

[0068] T4. After the reaction was completed, the mixture was dialyzed for 48 hours and then freeze-dried to obtain a chitosan oligosaccharide-soybean protein isolate covalent complex.

[0069] Compare with Example 2

[0070] The preparation method of this comparative example differs from that of Example 1 in that chitosan oligosaccharide is not added in step S2, while the other conditions are the same as those in Example 1.

[0071] Compare with Example 3

[0072] The preparation method of this comparative example differs from that of Example 1 in that no preheating treatment is performed in step S1 (i.e., the soy protein isolate is directly dissolved in water and proceeds to step S2 without heating and heat preservation). The other conditions are the same as those in Example 1.

[0073] Compare with Example 4

[0074] The preparation method of this comparative example differs from that of Example 1 in that ultrasound assistance is not performed in step S3 (i.e., no ultrasound field is applied during enzymatic hydrolysis), while the other conditions are the same as those in Example 1.

[0075] Experimental Example 1

[0076] The products obtained in Example 1 and Comparative Examples 1-4 were analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 2 As shown.

[0077] Electrophoresis results showed that pretreated soy protein isolate (lane SPI) exhibited typical 7S (α′, α, β subunits, approximately 42-72 kDa) and 11S (acidic subunit approximately 37 kDa, basic subunit approximately 20 kDa) bands, consistent with the characteristic molecular weight composition of soy protein isolate. After single-enzyme hydrolysis with flavor protease (lane SPI-COS-F), the 7S and 11S subunit bands were significantly weakened, while multiple newly formed low molecular weight bands appeared in the 15-35 kDa region, indicating that the flavor protease effectively hydrolyzed and cleaved the soy protein isolate. After single-enzyme hydrolysis with subtilisin (lane SPI-COS-A), the weakening of the 7S and 11S subunit bands and the generation of low molecular weight bands were also observed, but the band distribution differed from that of SPI-COS-F, indicating that different proteases had different hydrolysis sites, resulting in polypeptide fragments with different molecular weight distributions.

[0078] After cross-linking with transglutaminase without the addition of chitosan oligosaccharide (lane SPI-CL), a distinct diffuse band appeared in the high molecular weight region (>100kDa), while the 7S and 11S subunit bands were further weakened, and some low molecular weight bands disappeared. This indicates that transglutaminase successfully catalyzed the cross-linking reaction between polypeptide chains, forming high molecular weight cross-linked aggregates. This is due to the formation of heteropeptide bonds between soybean protein isolate molecules catalyzed by transglutaminase, and is unrelated to the covalent grafting of chitosan oligosaccharide.

[0079] Samples prepared using the traditional route of enzymatic hydrolysis followed by glycosylation (lane P-SPI-COS) showed further enhancement and upward migration of the diffuse bands in the high molecular weight region, indicating that chitosan oligosaccharides were successfully covalently grafted onto soybean protein isolate peptides via transglutaminase catalysis, forming a higher molecular weight covalent complex. Compared to the cross-linked products, the products from the traditional route exhibited more diffuse bands in the high molecular weight region, consistent with the typical electrophoretic characteristics of polysaccharide-protein covalent complexes.

[0080] Example 1

[0081] The electrophoretic bands of the final product (SPI-COS-P) exhibit significantly different characteristics: the intensity of the diffuse bands in the high molecular weight region is significantly lower than that of the product from the traditional route, while a series of clear and sharp low molecular weight bands appear in the 10-35 kDa region. This indicates that, through ultrasound-assisted stepwise enzymatic hydrolysis, the active peptides covalently grafted onto the chitosan oligosaccharide backbone were successfully released, forming a composite structure with chitosan oligosaccharide as the backbone and active peptides as side chains. This band distribution characteristic is distinct from both the diffuse low molecular weight bands of the single-enzyme hydrolysis products (SPI-COS-F, SPI-COS-A) and the high molecular weight diffuse bands of the product from the traditional route (P-SPI-COS), reflecting the unique molecular structural characteristics of the glycosylation-follow-enzymatic hydrolysis route of this invention.

[0082] Furthermore, the low molecular weight region bands of lane SPI-COS-P showed better sharpness and concentration than those of SPI-COS-F and SPI-COS-A, indicating that the peptide molecular weight distribution in the complex prepared by the method of the present invention is more concentrated and the molecular structure is more regular.

[0083] Experiment Example 2

[0084] Fourier transform infrared (FTIR) spectroscopy was performed on the products obtained in Example 1 and Comparative Examples 1-4. After freeze-drying, the samples were thoroughly ground and mixed with KBr in a specific ratio, compressed into tablets, and then scanned using a Fourier transform infrared spectrometer with a scanning range of 4000-400 cm⁻¹. -1 The resolution is set to 4cm. -1 The number of scans was 32, and the results are as follows: Figure 3 As shown in Table 1.

[0085] Table 1. Characteristic peak positions of Fourier transform infrared spectra of different samples

[0086] Fourier transform infrared spectroscopy results showed that pretreated soy protein isolate (H-SPI) was at 3405.74 cm⁻¹. -1The amide A band exhibits a characteristic absorption peak at 3396.52 cm⁻¹, which mainly corresponds to the stretching vibration of the NH bond and reflects the strength of hydrogen bonding between protein molecules. The amide A band of natural soy protein isolate (untreated SPI) is typically at 3396.52 cm⁻¹. -1 Nearby, after preheating treatment, the blue color shifted to 3405.74 cm. -1 This indicates that preheating weakens the hydrogen bonding between protein molecules and causes the structure to tend to expand, which is consistent with the expectation that preheating depolymerization will destroy the tight aggregates of proteins.

[0087] After single-enzyme hydrolysis with flavor protease (SPI-COS-F), the peak position of amide A was 3405.42 cm⁻¹. -1 The results were largely consistent with those of H-SPI; after hydrolysis by Bacillus subtilis protease (SPI-COS-A), the peak position of amide A shifted to 3305.32 cm⁻¹. -1 The results indicate that different proteases have significantly different effects on the protein hydrogen bond network, which may be related to the different hydrolysis sites and action modes of the two proteases. The effect of subtilisin on the protein hydrogen bond network is less than that of flavor protease.

[0088] After cross-linking with transglutaminase without the addition of chitosan oligosaccharide (SPI-CL), the peak position of amide A band showed a significant red shift to 3304.51 cm⁻¹. -1 The red shift of the amide A band indicates that the cross-linking reaction formed new intermolecular isopeptide bonds, which enhanced intermolecular hydrogen bonding and made the protein molecular structure more compact and ordered.

[0089] The amide A peak of the product from the traditional enzymatic hydrolysis followed by glycosylation route (P-SPI-COS) is located at 3302.94 cm⁻¹. -1 Compared to the crosslinking control (SPI-CL), there was a slight blue shift, indicating that the covalent grafting of chitosan oligosaccharides altered the intermolecular interactions to some extent, but retained the basic structure of the crosslinked network. The amide A peak of the final product (SPI-COS-P) in Example 1 was located at 3304.51 cm⁻¹. -1 Consistent with the cross-linking control (SPI-CL), this indicates that although ultrasound-assisted enzymatic hydrolysis releases grafted peptides, it does not disrupt the covalent connection between the chitosan oligosaccharide backbone and the peptides, thus preserving the basic structure of the complex.

[0090] In the amide I band (1600-1700 cm) -1 The amide I band in the H-SPI spectrum primarily corresponds to the stretching vibration of the C=O bond and is highly sensitive to changes in protein secondary structure. The peak position of the amide I band is 1653.55 cm⁻¹. -1 After enzymatic hydrolysis, SPI-COS-A (1653.61 cm⁻¹) was obtained. -1) and SPI-COS-F (1652.08 cm) -1 The amide I band peak position of SPI-CL showed little change compared to H-SPI, indicating that enzymatic digestion had limited impact on the secondary structure of the protein. After cross-linking treatment, SPI-CL (1653.60 cm⁻¹) showed a smaller change. -1 The amide I band peak position of the product (P-SPI-COS) is basically consistent with that of H-SPI, indicating that the cross-linking reaction has little effect on the protein secondary structure. The traditional route product (P-SPI-COS, 1653.60 cm⁻¹) -1 The peak position trend of the product is basically consistent with that of the crosslinked product. It is noteworthy that the amide I band peak of the final product (SPI-COS-P) in Example 1 is at 1653.55 cm⁻¹. -1 The results are completely consistent with H-SPI, indicating that the secondary structure of the complex prepared by the method of the present invention is restored to an ordered state similar to that of the pretreated protein after undergoing multiple enzymatic reactions, which confirms that the formation of the "brush-like" copolymer structure is beneficial to the stability of the protein secondary structure.

[0091] Furthermore, the glycosylated sample (P-SPI-COS) exhibited a characteristic absorption peak of glycosidic COC in the 1050-1150 cm⁻¹ region, which was not found in the H-SPI, SPI-COS-F, SPI-COS-A and cross-linked samples, directly confirming the successful covalent grafting of chitosan oligosaccharides.

[0092] Experimental Example 3

[0093] The products obtained in Example 1 and Control Examples 1-4 were subjected to ultraviolet spectral analysis. The samples were completely dissolved in deionized water and diluted to a concentration of 1 mg / mL. The absorbance values ​​of the samples were measured using an ultraviolet spectrophotometer in the wavelength range of 200-450 nm. The results are as follows. Figure 4 As shown.

[0094] UV spectroscopy results showed that pretreated soy protein isolate (HSPI) exhibited a characteristic absorption peak near 280 nm, which was attributed to the π→π* transitions of aromatic amino acids (tryptophan and tyrosine). The UV absorption peak of untreated natural soy protein isolate (SPI) is typically located near 280 nm. After preheating, the UV absorption intensity of HSPI was enhanced compared to untreated SPI, indicating that preheating stretched the protein structure, exposing some of the aromatic amino acid residues embedded within the molecule to the solvent.

[0095] After hydrolysis with flavor protease alone (SPI-COS-F), the UV absorption intensity was further enhanced compared to HSPI, indicating that enzymatic hydrolysis further expanded the protein structure, exposing more aromatic amino acid residues and leading to enhanced UV absorption. After hydrolysis with subtilisin alone (SPI-COS-A), the UV absorption intensity was also significantly enhanced compared to HSPI, but the degree of enhancement differed slightly from SPI-COS-F, indicating that differences in the hydrolysis sites of different proteases resulted in varying degrees of exposure of aromatic amino acids in their products.

[0096] The UV absorption intensity of the product after cross-linking with transglutaminase without the addition of chitosan oligosaccharide (SPI-CL) was lower than that of all hydrolysates, indicating that the cross-linking reaction caused protein molecules to aggregate and some aromatic amino acid residues to be re-embedded inside the molecule, resulting in reduced UV absorption. The UV absorption intensity of the product from the traditional enzymatic hydrolysis followed by glycosylation route (P-SPI-COS) was further reduced, and the absorption peak position showed a slight red shift, indicating that the covalent grafting of chitosan oligosaccharide further altered the microenvironment of aromatic amino acid residues and regulated the tertiary structure of the protein.

[0097] Compared to the samples mentioned above, the UV absorption intensity of the final product of Example 1 (SPI-COS-P) is between that of the cross-linked product (SPI-CL) and the product of the conventional route (P-SPI-COS), and the peak shape is sharper. This indicates that after ultrasonic-assisted stepwise enzymatic hydrolysis, the active peptides covalently grafted onto the chitosan oligosaccharide backbone are released, and the molecular structure changes from a highly cross-linked, dense state to a brush-like structure with chitosan oligosaccharide as the backbone and active peptides as side chains. The microenvironment of aromatic amino acid residues also changes accordingly. Notably, the UV absorption peak position of SPI-COS-P is basically consistent with that of the product of the conventional route (P-SPI-COS), indicating that the covalent connection between chitosan oligosaccharide and peptides is maintained during enzymatic hydrolysis.

[0098] Experiment Example 4

[0099] The products obtained in Example 1 and Control Examples 1-4 were subjected to ABTS and DPPH free radical scavenging activities assays, and the results are as follows: Figure 5-6 As shown.

[0100] The method for determining ABTS free radical scavenging activity is as follows: An equal volume of ABTS aqueous solution (7 mmol / L) and potassium persulfate aqueous solution (4 mmol / L) is mixed to prepare an ABTS⁺ solution, which is then stored at room temperature in the dark for 12-16 hours. The ABTS⁺ solution is diluted with deionized water to adjust the OD value at 734 nm to 0.70 ± 0.02. 90 μL of sample (1 mg / mL) is mixed with 3.9 mL of ABTS⁺, and the mixture is allowed to stand at room temperature in the dark for 5 minutes. The absorbance is measured at 734 nm. Deionized water is used as a blank control instead of the sample. The ABTS free radical scavenging rate is calculated using the following formula:

[0101] ABTS radical scavenging rate (%) = (A0 - A1) / A0 × 100%

[0102] Where A0 is the absorbance of the blank group and A1 is the absorbance of the sample.

[0103] The method for determining the DPPH free radical scavenging activity is as follows: DPPH was weighed and dissolved in anhydrous ethanol to prepare a 0.2 mmol / L DPPH solution, which was then stored in the dark for later use. 100 μL of the DPPH solution was added to 100 μL of the complex solution, mixed thoroughly, and reacted at room temperature in the dark for 30 min. The absorbance of the complex was measured at 517 nm. Simultaneously, anhydrous ethanol (100 μL) was used as a blank control instead of the DPPH solution, and deionized water (100 μL) was mixed with the DPPH solution as a control control; the absorbance of the mixture was measured. The DPPH free radical scavenging rate was calculated using the following formula:

[0104] DPPH radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0105] Where A0 is the absorbance of the control group (deionized water + DPPH solution), A1 is the absorbance of the sample group (sample + DPPH solution), and A2 is the absorbance of the blank group (sample + anhydrous ethanol).

[0106] ABTS free radical scavenging rate determination results ( Figure 5 The results showed that the ABTS radical scavenging rate of pretreated soy protein isolate (H-SPI) was low, approximately 6%. After hydrolysis with a single enzyme of Bacillus subtilis protease (SPI-COS-A), the scavenging rate increased to approximately 32%; after hydrolysis with a single enzyme of flavor protease (SPI-COS-F), the scavenging rate increased to approximately 22%, indicating that the active peptides released by enzymatic hydrolysis all possess certain antioxidant activity, and the hydrolysates from different proteases exhibit varying activities. After cross-linking with transglutaminase without the addition of chitosan oligosaccharides (SPI-CL), the scavenging rate was approximately 20%, lower than that of the hydrolysate, indicating that the cross-linking reaction encapsulated some antioxidant active groups. The scavenging rate of the product from the traditional enzymatic hydrolysis followed by glycosylation route (P-SPI-COS) was approximately 38%, indicating that the covalent grafting of chitosan oligosaccharides enhanced the antioxidant activity of the complex to some extent. The ABTS radical scavenging rate of the final product of Example 1 of this invention (i.e., SPI-COS-P) reached approximately 65%, significantly higher than all control samples.

[0107] DPPH free radical scavenging rate determination results ( Figure 6The results showed a similar trend. The DPPH scavenging rate of pretreated soy protein isolate (H-SPI) was approximately 16.5%, that of Bacillus subtilis protease monoenzyme hydrolysate (SPI-COS-A) increased to approximately 28.5%, that of flavor protease monoenzyme hydrolysate (SPI-COS-F) increased to approximately 31.5%, that of cross-linked control (SPI-CL) was approximately 29.5%, that of conventional route product (P-SPI-COS) was approximately 36.0%, and that of the final product of this invention (SPI-COS-P) reached approximately 45.0%, which was also significantly better than all control samples.

[0108] The above results indicate that the chitosan oligosaccharide-soybean protein isolate peptide covalent complex (SPI-COS-P) prepared in Example 1 of this invention exhibits significantly superior antioxidant activity compared to preheated protein, single-enzyme hydrolysate, cross-linked product, and product from the traditional route. This is attributed in two ways: firstly, the antioxidant activity inherent in chitosan oligosaccharide is covalently grafted onto the peptides, resulting in a synergistic effect; secondly, the brush-like copolymer structure formed by the "preheating depolymerization—glycosylation cross-linking—ultrasound-assisted stepwise enzymatic hydrolysis" route of this invention, in which the active peptides are covalently linked to the chitosan oligosaccharide backbone, maintains good molecular flexibility and spatial accessibility while avoiding the embedding and inactivation of active groups, thereby maximizing antioxidant activity.

[0109] Experimental Example 5

[0110] The DPP-IV (dipeptidyl peptidase-IV) inhibitory activity of the products obtained in Example 1 and Control Examples 1-4 was determined. Samples were dissolved in deionized water to prepare solutions with different concentration gradients. 25 μL of sample was mixed with 25 μL of DPP-IV enzyme solution (final concentration 0.01 U / mL), and 50 μL of substrate Gly-Pro-pNA (final concentration 0.2 mmol / L) was added. The mixture was reacted at 37 °C for 60 min, and the absorbance was measured at 405 nm. The DPP-IV inhibition rate was calculated using the following formula:

[0111] DPP-IV inhibition rate (%) = [1 - (A1 - A2) / A0] × 100%

[0112] Where A0 is the absorbance value of the control group (deionized water instead of the sample), A1 is the absorbance value of the sample group, and A2 is the absorbance value of the blank group (deionized water instead of the enzyme solution).

[0113] The measurement results show that the DPP-IV inhibition rate (IC50) of the final product of Example 1 of this invention is [missing information]. 50 =0.28mg / mL) was significantly better than control 1 (IC50). 50=0.51 mg / mL), indicating that the chitosan oligosaccharide-soybean protein isolate peptide covalent complex prepared by the method of the present invention has good hypoglycemic activity potential. This may be attributed to the fact that in the brush copolymer structure formed by the method of the present invention, the active peptide segments are covalently linked to the chitosan oligosaccharide backbone, effectively mimicking the spatial configuration of the natural substrate DPP-IV, thereby enhancing the enzyme inhibitory activity.

[0114] Test results show that the chitosan oligosaccharide-soybean protein isolate peptide covalent complexes prepared in Examples 1-9 of this invention have more regular molecular structures, better spectroscopic properties, and stronger antioxidant and DPP-IV inhibitory activities compared to control examples 1-4. Among them, Example 1 (preheating depolymerization + glycosylation crosslinking + ultrasound-assisted stepwise enzymatic hydrolysis) exhibits the best overall performance.

[0115] In summary, by utilizing the technical solution of this invention, and breaking through the traditional fixed process mode of first enzymatic hydrolysis and then glycosylation, a reverse synergistic route of preheating depolymerization, glycosylation crosslinking, and ultrasound-assisted stepwise enzymatic hydrolysis is adopted. Using chitosan oligosaccharide as the backbone, a brush-like copolymer structure with chitosan oligosaccharide as the main body and active peptides as side chains is constructed, solving the technical problems of low grafting efficiency, uncontrollable molecular structure, and short-lasting functional activity in existing composite systems. The resulting complex has broad application prospects in plant-based functional foods, emulsion systems, and nutrient delivery.

[0116] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a soybean protein isolate peptide covalent complex, characterized in that, Includes the following steps: S1. Disperse soy protein isolate in water and preheat it to induce thermal depolymerization, thus obtaining a pretreated protein solution. S2. The pretreated protein solution obtained in step S1 is subjected to a glycosylation cross-linking reaction with chitosan oligosaccharide under the catalysis of transglutaminase to obtain a chitosan oligosaccharide-soybean protein isolate covalent complex. S3. The chitosan oligosaccharide-soybean protein isolate covalent complex obtained in step S2 is subjected to ultrasonic-assisted enzymatic hydrolysis, and stepwise enzymatic hydrolysis is performed using a complex protease to obtain a chitosan oligosaccharide-soybean protein isolate peptide covalent complex solution. S4. The complex solution obtained in step S3 is dried to obtain a chitosan oligosaccharide-soybean protein isolate covalent complex.

2. The method for preparing the soybean protein isolate peptide covalent complex according to claim 1, characterized in that, The preheating temperature in step S1 is 70–95°C, and the time is 10–40 min; the mass concentration of the soy protein isolate solution is 3%–8%, and the pH value is 7.0–8.

0.

3. The method for preparing the soybean protein isolate peptide covalent complex according to claim 1, characterized in that, In step S2, the mass ratio of chitosan oligosaccharide to soy protein isolate is 1:2 to 1:5, the amount of transglutaminase added is 5 to 20 U / g protein, the reaction temperature is 30 to 40°C, and the reaction time is 1 to 5 h.

4. The method for preparing the soybean protein isolate peptide covalent complex according to claim 1, characterized in that, The complex protease described in step S3 includes endopeptidase and exopeptidase.

5. The method for preparing the soybean protein isolate peptide covalent complex according to claim 4, characterized in that, The endopeptidase is Bacillus subtilis protease, and the exopeptidase is flavor protease.

6. The method for preparing the soybean protein isolate peptide covalent complex according to claim 5, characterized in that, The ultrasound-assisted enzymatic hydrolysis treatment in step S3 includes: S31. Add Bacillus subtilis protease to the glycosylated crosslinked product obtained in step S2, and carry out the first enzymatic hydrolysis under ultrasonic-assisted conditions. The enzymatic hydrolysis temperature is 50-60℃ and the enzymatic hydrolysis time is 1-3h. S32. After the first enzymatic hydrolysis is completed, flavor protease is added to the system, and the second enzymatic hydrolysis is carried out under ultrasonic-assisted conditions. The enzymatic hydrolysis temperature is 45-55℃ and the enzymatic hydrolysis time is 1-3h. S33. After enzymatic hydrolysis, the enzyme is inactivated by heating, and the supernatant is collected by centrifugation to obtain a chitosan oligosaccharide-soybean protein isolate peptide covalent complex solution.

7. The method for preparing the soybean protein isolate peptide covalent complex according to claim 6, characterized in that, The ultrasound power of the ultrasound-assisted ultrasound in steps S31 and S32 is 200-500W, and the ultrasound mode is intermittent ultrasound, with a 5-15s interval between ultrasound sessions.

8. The method for preparing the soybean protein isolate peptide covalent complex according to claim 6, characterized in that, The enzyme inactivation temperature in step S33 is 85-95℃, and the enzyme inactivation time is 5-15 min.

9. The method for preparing the soybean protein isolate peptide covalent complex according to claim 1, characterized in that, The drying process described in step S4 is vacuum freeze drying.

10. A soybean protein isolate peptide covalent complex, characterized in that... It is prepared by the method described in any one of claims 1-9.

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