A casein hydrolysate-chlorogenic acid non-covalent complex emulsion, and a preparation method and application thereof

Abstract

A casein hydrolysate-chlorogenic acid non-covalent complex emulsion as well as a preparation method and application thereof belong to the technical field of food processing. In view of the fact that the prior art cannot simultaneously consider the alpha-glucosidase inhibitory activity, emulsification stability and antioxidant activity of the casein hydrolysate, the casein hydrolysate-chlorogenic acid complex is prepared through non-covalent interaction, and the complex is used as an emulsifier to construct a functional emulsion. The interaction mainly occurs through hydrogen bonds and hydrophobic interaction, resulting in the unfolding of protein molecular structure and the fluorescence quenching. Meanwhile, the functional emulsion has good emulsification activity and stability. The emulsion prepared by using the complex has the smallest particle size, the lowest turbidity and uniform droplet distribution. The functional emulsion obtained by the application has the antidiabetic effect and excellent emulsification performance, and has important application value in the development of functional foods.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a casein hydrolysate-chlorogenic acid non-covalent complex emulsion, its preparation method, and its application. Background Technology

[0002] Milk is rich in protein and is an important part of the daily diet, playing a significant role in human health. Casein is the main protein in milk, accounting for about 80% of the total milk protein, and includes αs1-casein, αs2-casein, β-casein, and κ-casein. Enzymatic hydrolysis of casein can produce various bioactive peptides with health-promoting effects, such as antioxidant peptides and anti-diabetic peptides. Studies have shown that casein hydrolysates can slow down starch digestion both in vitro and in vivo, and have α-glucosidase inhibitory effects, demonstrating potential anti-diabetic functions.

[0003] Although studies have shown that protein hydrolysis improves emulsification, the emulsification stability of the hydrolysate is generally reduced due to the strong hydrophilicity of the small peptides produced, which often form weak interfacial films. Furthermore, focusing on improving the α-glucosidase inhibitory activity of the hydrolysate may decrease its antioxidant activity. Therefore, it is necessary to combine this method with other modification approaches to simultaneously improve both the emulsification and antioxidant activity of the hydrolysate.

[0004] The modification of proteins by polyphenols has been extensively studied. Numerous studies have shown that the interaction between polyphenols and proteins or their hydrolysates affects their structural and functional properties, such as inducing protein structure unfolding, enhancing emulsifying activity, improving foaming properties, and strengthening antioxidant capacity. For example, the conjugation of porcine plasma protein hydrolysates with oxidized tannins or oxidized chlorogenic acid significantly improves both emulsifying stability and antioxidant activity. Furthermore, polyphenols are among the largest groups of antioxidants in the diet, and conjugating polyphenols with proteins can enhance their antioxidant properties. Studies have found that non-covalent complexes of clam protein hydrolysates and chlorogenic acid exhibit synergistic antioxidant effects. The interaction between proteins and polyphenols includes irreversible covalent interactions and reversible non-covalent interactions. Covalent interactions typically involve oxidation or formation in alkaline environments. Non-covalent interactions include hydrophobic interactions, van der Waals forces, hydrogen bonds, and electrostatic attraction. Non-covalent interactions avoid the use of chemical cross-linking agents and complex preparation processes, making them a simple, safe, and suitable method for food and pharmaceutical applications.

[0005] Chlorogenic acid is a dietary polyphenol widely found in plant-based foods such as coffee, green tea, and honeysuckle. The chlorogenic acid molecule contains five hydroxyl groups, exhibiting excellent free radical scavenging and protein-binding capabilities. Furthermore, chlorogenic acid has been shown to inhibit α-amylase and α-glucosidase, delaying glucose absorption and thus reducing the risk of type 2 diabetes. Currently, research on chlorogenic acid binding to proteins to enhance antioxidant and emulsifying properties has been extensively reported; however, research on the binding of chlorogenic acid to protein hydrolysates, especially the antidiabetic effects of these complexes, is rarely explored.

[0006] While casein hydrolysates possess α-glucosidase inhibitory activity, their emulsifying stability and antioxidant activity need improvement. Furthermore, research on the combination of chlorogenic acid with protein hydrolysates, particularly the anti-diabetic function of the complex, is insufficient. Therefore, those skilled in the art are eager to develop a novel functional food ingredient that simultaneously possesses anti-diabetic properties and good emulsifying performance. Summary of the Invention

[0007] To address the problem of the difficulty in simultaneously achieving α-glucosidase inhibitory activity, emulsification stability, and antioxidant activity in casein hydrolysates, thus hindering the production of functional food ingredients that combine anti-diabetic properties with good emulsification performance, this invention provides a casein hydrolysate-chlorogenic acid non-covalent complex emulsion, its preparation method, and its applications.

[0008] One objective of this invention is to provide a method for preparing a casein hydrolysate-chlorogenic acid non-covalent complex emulsion, the method comprising the following steps: S1. Preparation of casein hydrolysate: The casein solution was enzymatically hydrolyzed by adding neutral protease at 50°C and pH=7. After the enzymatic hydrolysis was completed, the solution was heated in a boiling water bath for 10 min to inactivate the enzyme. Then, it was immediately cooled to room temperature with running tap water, centrifuged, the supernatant was collected and freeze-dried to obtain casein hydrolysate. S2. Preparation of casein hydrolysate-chlorogenic acid non-covalent complex: Chlorogenic acid was dissolved in a 0.01 mol / L phosphate buffer solution with pH=7.0 and magnetically stirred to prepare a chlorogenic acid stock solution. The casein hydrolysate obtained in S1 was mixed with the chlorogenic acid stock solution to obtain a casein hydrolysate-chlorogenic acid non-covalent complex. The non-covalent complex was placed in a shaker and incubated in the dark to obtain a casein hydrolysate-chlorogenic acid non-covalent complex solution. S3. Preparation of casein hydrolysate-chlorogenic acid non-covalent complex emulsion: Add 0.2% xanthan gum solution to the casein hydrolysate-chlorogenic acid non-covalent complex solution obtained in S2 and mix. Homogenize with 5% (v / v) medium-chain triglycerides at room temperature and at 12,000 rpm for 2 min. Then, sonicate in an ice bath to prepare the casein hydrolysate-chlorogenic acid non-covalent complex emulsion.

[0009] In a preferred embodiment of the present invention, the casein solution in S1 is 5%, w / w; the enzymatic hydrolysis time is 2 h; the ratio of neutral protease to casein is 1% (w / w); and the centrifugation conditions are: 10000×g centrifugation for 15 min.

[0010] In a preferred embodiment of the present invention, the α-glucosidase inhibitory activity of the casein hydrolysate prepared in S1 is 76.14% ± 0.018%, IC50. 50 =7.19 mg / mL.

[0011] In a preferred embodiment of the present invention, the mass fraction of the chlorogenic acid mother liquor in S2 is 10 mg / mL.

[0012] In a preferred embodiment of the present invention, the final concentration of chlorogenic acid in the casein hydrolysate-chlorogenic acid non-covalent complex in S2 is 100 μmol / g.

[0013] In a preferred embodiment of the present invention, the conditions for dark incubation in S2 are: dark incubation for 2 h at 25°C and 150 r / min on a shaker.

[0014] In a preferred embodiment of the present invention, the mixing volume ratio of the casein hydrolysate-chlorogenic acid non-covalent complex solution to the xanthan gum solution in S3 is 2:1. In a preferred embodiment of the present invention, the conditions for ultrasonic treatment in S3 are: treatment time of 3 min, pulse mode of 2 s on and 2 s off, and power output of 300 watts.

[0015] A second objective of this invention is to provide a casein hydrolysate-chlorogenic acid non-covalent complex emulsion, which is prepared using the method described above.

[0016] A third objective of this invention is to provide the application of the above-mentioned casein hydrolysate-chlorogenic acid non-covalent complex emulsion in the preparation of food with anti-diabetic function and emulsifying properties, wherein the food is for non-therapeutic purposes.

[0017] Compared with existing technologies, the beneficial effects of this invention are as follows: Addressing the shortcomings of existing technologies in simultaneously achieving α-glucosidase inhibitory activity, emulsifying stability, and antioxidant activity in casein hydrolysates, this invention prepares a casein hydrolysate-chlorogenic acid complex through non-covalent interactions and uses it as an emulsifier to construct functional emulsions. Specifically, through the non-covalent interaction between casein hydrolysate (CH) and chlorogenic acid (CA), its structure, emulsifying properties, and anti-diabetic potential were explored. Mixing with polyphenols significantly enhanced the anti-diabetic potential of CH, such as α-glucosidase inhibitory activity and ABTS and DPPH free radical scavenging capabilities, thanks to the addition of chlorogenic acid and phenolic hydroxyl groups. Furthermore, due to structural unfolding, reduced particle size, and exposure of hydrophobic residues in CH, the complexation with chlorogenic acid significantly enhanced the emulsifying activity of CH. It was also found that among complexes with different concentrations of added chlorogenic acid, CH-CA100 showed the greatest overall improvement in α-glucosidase inhibitory activity, antioxidant activity, and emulsifying properties compared to CH. In summary, compared with the CH control group, the complex provided by this invention showed reduced particle size, significantly increased emulsifying properties, α-glucosidase inhibitory activity, and antioxidant activity.

[0018] This invention optimizes the enzymatic hydrolysis process, identifying a 2-hour neutral protease hydrolysis as the optimal step. The resulting casein hydrolysate exhibits the highest α-glucosidase inhibitory activity, laying the foundation for the antidiabetic activity of the composite product. Furthermore, the optimal final concentration of chlorogenic acid was determined to be 100 μmol / g CH, at which point the emulsifying properties, antidiabetic activity, and antioxidant activity of the complex are synergistically maximized, overcoming the limitation of single modification in achieving multiple functions. Moreover, the emulsion performance is significantly improved; under the conditions defined in this invention, the emulsion produced has the smallest particle size, increased potential, and under an optical microscope, the droplet size is uniform and the dispersibility is optimal, with significantly improved interfacial stability.

[0019] The present invention provides a method for preparing a casein hydrolysate-chlorogenic acid non-covalent complex and emulsion, offering insights into the development of functional food ingredients with excellent bioactivity and emulsifying properties, demonstrating the great potential of the complex as a functional food ingredient with anti-diabetic functions. The results of this study demonstrate the influence of the non-covalent interaction between CH and chlorogenic acid on the bioactivity and emulsifying effect of CH, and showcase a novel method for developing casein peptides with high α-glucosidase inhibitory activity, antioxidant activity, and emulsifying properties. Future research may utilize cell and animal models to confirm the α-glucosidase inhibitory and antioxidant activities of the CH-CA complex, and explore the preparation of stable emulsions to deliver other bioactive substances. Furthermore, further research is needed on the interaction mechanism between CH and CA, which is crucial for elucidating the bioactivity of the complex. Attached Figure Description

[0020] Figure 1The graph shows the antidiabetic potential of the complex in Example 1; A is the Fourier transform infrared spectrum, B is the ultraviolet absorption spectrum, and C is the intrinsic fluorescence spectrum. Figure 2 The images shown are scanning electron microscope images of the complex in Example 1; A is CH-CA0, B is CH-CA25, C is CH-CA50, D is CH-CA100, E is CH-CA200, and F is CH-CA250. Figure 3 The graphs show the physical property test results of the complex in Example 1; A is the particle size distribution graph, B is the zeta potential distribution graph, C is the surface hydrophobicity distribution graph, and D is the free thiol content distribution graph. Figure 4 Figure A shows the antidiabetic potential of the complex in Example 1; Figure B shows the statistical chart of ABTS free radical scavenging ability and Figure B shows the statistical chart of DPPH free radical scavenging ability. Figure 5 Figure A shows the emulsification performance results of the complex in Example 1; Figure B is a statistical graph of emulsification characteristics and a statistical graph of interfacial tension. Figure 6 is The emulsion performance results of the complex emulsion in Example 2 are shown in the figure; A is the particle size statistics chart, and B is the Zeta potential energy statistics chart. Figure 7 The following graphs show the rheological properties of the complex emulsion in Example 2; A is the apparent viscosity graph, and B is the rheological property graph. Figure 8 The images shown are optical microscope images of the complex emulsion in Example 2; A is CH-CA0, B is CH-CA25, C is CH-CA50, D is CH-CA100, E is CH-CA200, and F is CH-CA250. Detailed Implementation

[0021] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0023] The casein powder used in the following examples was purchased from Heilongjiang Feihe Dairy Co., Ltd. (casein content 85.6g / 100g); Neutrase 0.8L (0.8 AU / g) was purchased from Novozyme Biotechnology Co., Ltd. (Tianjin, China); chlorogenic acid (≥98%), α-glucosidase (100U), and pNPG (p-nitrophenyl-α-D-glucopyranoside) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China).

[0024] Example 1: Preparation of a casein hydrolysate-chlorogenic acid non-covalent complex S1. Preparation of casein hydrolysate: A casein solution (5%, w / w) was enzymatically hydrolyzed for 2 h at 50 °C and pH=7 with the addition of neutral protease (neutral protease to casein ratio of 1%, w / w). After hydrolysis, the solution was heated in a boiling water bath for 10 min to inactivate the enzyme, and then immediately cooled to room temperature with running tap water. The solution was centrifuged at 10000×g for 15 min, the supernatant was collected and freeze-dried to obtain casein hydrolysate. S2. Preparation of casein hydrolysate-chlorogenic acid non-covalent complex: Chlorogenic acid was dissolved in a 0.01 mol / L phosphate buffer solution with pH=7.0 and magnetically stirred to prepare a chlorogenic acid solution with a mass fraction of 10 mg / mL. The casein hydrolysate obtained in S1 was mixed with the chlorogenic acid solution to obtain casein hydrolysate-chlorogenic acid non-covalent complexes with final chlorogenic acid concentrations of 25, 50, 100, 200, and 250 μmol / g, respectively. The non-covalent complexes were incubated in the dark at 25℃ and 150 r / min for 2 h to obtain casein hydrolysate-chlorogenic acid non-covalent complex solutions with different final chlorogenic acid concentrations (abbreviated as CH-CA0, CH-CA25, CH-CA50, CH-CA100, CH-CA200, and CH-CA250, respectively). The solutions were then freeze-dried at -80℃, and the freeze-dried casein hydrolysate-chlorogenic acid non-covalent complex powder was sealed and stored at -20℃ for later use.

[0025] Effect Experiment: 1. Structural properties (1) Fourier transform infrared spectrum The casein hydrolysate-chlorogenic acid non-covalent complex prepared in Example 1 was subjected to Fourier transform infrared spectroscopy (FTIR). The sample to be tested was mixed with potassium bromide at a weight ratio of 1:100, ground evenly, and then compressed into tablets. The tablets were scanned at room temperature in the wavenumber range of 4000-400 cm⁻¹. -1 The inner diameter is 0.01 cm. -1 The wavenumber was scanned 32 times, with a resolution of 4 cm. -1 .

[0026] Fourier transform infrared spectroscopy (FTIR) was used to reflect the interaction between casein hydrolysate (CH) and chlorogenic acid (CA). In the protein spectrum, the amide A band (located at 3500-3300 cm⁻¹) was observed. -1 This corresponds to the stretching vibrations of NH and -OH. The amide I band (approximately 1650 cm⁻¹) corresponds to this. -1 The amide II band (approximately 1540 cm⁻¹) represents the C=O stretching vibration, which directly reflects the secondary structure of proteins. -1 It is associated with NH bending vibration and CN tensile vibration.

[0027] The results are as follows Figure 1 As shown in section A, the binding of CH and CA significantly altered the functional group interactions and secondary conformation of the peptide chain. A red shift occurred in the amide A band, with the peak shifting from 3279.60 cm⁻¹ to 3303.91, 3304.22, 3304.43, 3312.52, and 3304.19 cm⁻¹, respectively. -1 Furthermore, the peak width changed; the reason for this is likely due to the OH stretching vibration between CH and CA, resulting in hydrogen bond interactions. Additionally, the peak value of amide II changed from 1652.52 cm⁻¹. -1 Moved to 1659.03 cm -1 This is related to the stretching vibration of the CN- bond. The shifts in the amide I and amide II bands indicate that CN and NH bonds may be involved in the reaction.

[0028] (2) Ultraviolet absorption spectrum Ultraviolet-visible absorption spectroscopy is a useful technique for identifying complexes between proteins and small molecules; structural changes in proteins can also be detected by analyzing changes in the absorption peaks of different amino acid residues.

[0029] The casein hydrolysate-chlorogenic acid non-covalent complex prepared in Example 1 was diluted with phosphate buffer to 0.5 mg / mL (10 mM, pH 7.0), and the wavelength was scanned at 200-400 nm using a UV spectrophotometer.

[0030] The results are as follows Figure 1 As shown in section B, CH exhibits two absorption peaks at approximately 280 nm and 220 nm. The peak at 280 nm is attributed to a π-π transition peptide bond occurring in the C=O groups of tyrosine and tryptophan residues, while the peak at 220 nm is attributed to the polypeptide backbone structure. Clearly, both peaks increase with increasing CA concentration, indicating an effective and specific biochemical interaction between CH and the aromatic residues of CA, leading to the formation of a new complex and changes in the absorption spectrum. Furthermore, the addition of CA to the CFP solution resulted in a new peak at approximately 324 nm, and its intensity increased with increasing CA concentration. This is likely due to the oxidation of tyrosine to dityrosine.

[0031] (3) Endogenous fluorescence spectrum Tyrosine and tryptophan produce strong intrinsic fluorescence at an excitation wavelength of 280 nm, so changes in their emission spectra can help explore the nature of protein-polyphenol interactions.

[0032] The casein hydrolysate-chlorogenic acid non-covalent complex prepared in Example 1 was diluted with phosphate buffer to 0.5 mg / mL (10 mM, pH 7.0) and excited at 280 nm. The fluorescence emission spectra from 300 nm to 500 nm (slit width: 5 nm) were recorded using an F-7100 fluorescence spectrophotometer (Hitachi, Japan).

[0033] The results are as follows Figure 1 Part C shows the fluorescence intensity curves of the synergistic effect of CH with different concentrations of CA. The Tyr and Trp residues of the six samples are at 308 cm⁻¹. -1 349 cm -1 There are distinct characteristic peaks on both sides. Visually, the fluorescence intensity decreases with the addition of CA, and the decrease in fluorescence intensity intensifies with increasing CA uptake, indicating that CA may bind to CH, inhibiting the intrinsic fluorescence of CH and causing fluorescence quenching. Furthermore, when the CA concentration increases to 250 μmol / g protein, the maximum peak wavelength of CH significantly redshifts from 349 nm to 365 nm. This phenomenon may be due to the formation of a CH-CA complex, which is non-fluorescent and alters the microenvironment of tyrosine and tryptophan residues in CFP, making them more hydrophilic.

[0034] (4) Scanning electron microscope The degree of structural relaxation of the complex is also related to its emulsifying properties, because a loose structure allows the protein to maintain sufficient electrostatic repulsion, stabilize the system and regulate the stability of the oil-water interface layer, thereby obtaining good emulsifying properties.

[0035] The casein hydrolysate-chlorogenic acid non-covalent complexes prepared in Example 1 were placed on a processing stage, coated and sprayed with gold, and observed using an S-480 scanning electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 5 kV and a magnification of 5000×.

[0036] like Figure 2 As shown, the CH-CA0 sample mostly exhibits a sheet-like structure. When combined with different concentrations of CA, the sheet-like structure of CH breaks down, and the complex displays a fragmented state. Molecular aggregation forms more layered block structures, which appear incomplete and coarse compared to the original protein structure. It is evident that the addition of CA alters the dense structure of the protein, causing it to unfold and become looser. The CH-CA100 sample, in particular, has a smaller and more dispersed particle size, consistent with the particle size results.

[0037] 2. Physical properties (1) Particle size and zeta potential determination Particle size primarily affects emulsifying activity and the initial size or distribution of emulsion droplets. The particle size and zeta potential of the casein hydrolysate-chlorogenic acid non-covalent complex prepared in Example 1 were determined using a Mastersizer 2000 (Malvern Instruments Co., Ltd., Worcestershire, UK), with refractive indices of 1.33 and 1.46 for the dispersion medium and emulsion particles, respectively.

[0038] like Figure 3 As shown in Part A, the average particle size of the non-covalently bound CH-CA complex was lowest when the chlorogenic acid content was 100 μmol / g protein. The average particle size of CH-CA0 was 228.93 ± 5.55, while that of CH-CA100 was 220.97 ± 1.65. This may be related to the molecular unfolding caused by the interaction between CH and CA, as well as the exposure of charged groups and amino acid residues inside the protein.

[0039] Furthermore, the reduction in nanocomposite size may also be due to the formation of bridging between protein molecules, resulting in a stable distance between protein micelles; the average particle size of the complexes formed when combined with other concentrations of chlorogenic acid was significantly increased (P<0.05). This may be because the phenolic hydroxyl groups in CA crosslink with CH and induce aggregation, leading to an increase in the particle size of the composites.

[0040] like Figure 3As shown in Part B, the average surface charge of CH is -27.35 ± 1.32 mV, and the addition of different concentrations of CA did not cause significant changes (p > 0.05). This is because CA mainly binds to CH through hydrogen bonds and hydrophobic interactions, without significantly altering the net charge density of the protein surface. Furthermore, most of the hydroxyl groups on the CA molecule are unionized or only slightly ionized, making the surface charge negligible when the pH of the system remains constant at 7.

[0041] (2) Surface hydrophobicity and free thiol groups The casein hydrolysate-chlorogenic acid non-covalent complexes prepared in Example 1 were diluted in 0.01 M sodium phosphate buffer (pH 7.0) to a concentration range of 0.05-0.25 mg / mL. 4 mL of the sample diluent (0.05, 0.1, 0.15, 0.20, and 0.25 mg / mL, respectively) was thoroughly mixed with 20 μL of ANS (8 mmol / L) (1 mL, 5 μL). After being placed in the dark for 10 minutes, the relative fluorescence intensity of each solution was measured using a fluorescence spectrophotometer. The excitation wavelength was set to 390 nm, and the emission wavelength was set to 470 nm. The relationship between fluorescence intensity and protein concentration was fitted using linear regression, and the slope of the curve was used to define surface hydrophobicity. Each sample was analyzed in triplicate.

[0042] The casein hydrolysate-chlorogenic acid non-covalent complex prepared in Example 1 was dissolved at a concentration of 1 mg / mL in Tris-glycine buffer (pH 8.0) containing 8 M urea. 5 mL of the test solution was mixed with 20 μL of Ellman's reagent and incubated at room temperature in the dark for 15 minutes. The absorbance at 412 nm was measured using a SpectraMax i3x multi-functional microplate reader (MolecularDevices Co., Ltd., CA, USA). The formula for calculating the free SH content is as follows: .

[0043] In the formula: 73.53 is the conversion factor; A 412 1 is the absorbance of CH-CA at a wavelength of 412 nm; D is the dilution factor, which is 1 in this experiment; C is the concentration of the CH-CA solution, which is 1 mg / mL in this experiment.

[0044] like Figure 3As shown in section C, the surface hydrophobicity of CH increased from 994.07±7.85 to 1333.53±7.10 after binding to CA of 100 μmol / g protein. Furthermore, the surface hydrophobicity of CH-CA25 and CH-CA200 also showed a certain increase. This phenomenon may be attributed to the many hydrophilic amino acids involved in the non-covalent binding of CH to CA, leading to the exposure of previously hydrophobic sites within CH. The surface hydrophobicity of CH decreased significantly after binding to CA of 50 and 250 μmol / g protein. This may be because when polyphenols interact non-covalently with proteins, a large number of phenolic hydroxyl groups attached to the aromatic ring of the polyphenol are introduced into the protein molecule surface, thereby reducing the protein surface hydrophobicity. Alternatively, it may be due to the aggregation of proteins caused by polyphenols, leading to a decrease in hydrophobicity.

[0045] SH content is a key indicator of changes in the tertiary and quaternary structure of proteins. The content of free sulfhydryl groups increases significantly after CH binds to different concentrations of chlorogenic acid, such as... Figure 3 As shown in section D, the cleavage of SS bonds and the exposure of free SH groups in proteins lead to an increase in free SH content, indicating that the protein structure is unfolded. Therefore, the increased free sulfhydryl content in the CH-CA polyphenol complex suggests that the addition of CA affects protein structure, exposing more sulfhydryl groups and opening the structure. A similar increase in free sulfhydryl groups was also observed in soybean meal hydrolysate-proanthocyanidin conjugates. Compared to the soybean meal hydrolysate control group, increasing the concentration of proanthocyanidins significantly increased the free SH content of the complex. Furthermore, the change in free sulfhydryl content was related to the type of polyphenol bound to the protein.

[0046] 3. Anti-diabetic potential The in vitro hypoglycemic activity of the casein hydrolysate-chlorogenic acid non-covalent complex sample prepared in Example 1 was evaluated using an α-glucosidase inhibitory activity assay. Furthermore, the antioxidant capacity of the sample was closely related to its anti-diabetic potential; the complex was measured at a concentration of 10 mg / mL.

[0047] (1) α-glucosidase inhibition Sample solutions were prepared using phosphate-buffered saline (PBS, 0.2 M, pH=7) for p-nitrophenyl-α-D-glucopyranoside (PNPG, 2.5 mM), α-glucosidase solution (0.2 U / mL), and the casein hydrolysate-chlorogenic acid non-covalent complex sample prepared in Example 1. 50 μL of α-glucosidase solution was premixed with 50 μL of the sample solution, incubated at 37°C for 5 min, and then 50 μL of PNPG solution was added to initiate the reaction. After reacting at 37°C for 15 min, 100 μL of sodium carbonate solution (0.2 M) was added to terminate the reaction. The absorbance was measured at 405 nm. The α-glucosidase inhibitory activity was calculated as follows: .

[0048] In the formula: Asample includes the absorbance values ​​of the sample and enzyme solution; Asample blank uses PBS instead of the absorbance value of the enzyme solution; Ablank control uses PBS instead of the absorbance value of the sample solution; Ablank uses PBS instead of the absorbance values ​​of the sample solution and enzyme solution.

[0049] like Figure 4 As shown in Part A, the inhibitory effects of the CH-CA0 control group (without chlorogenic acid) and the CH-CA non-covalent complexes containing different concentrations of chlorogenic acid on α-glucosidase were compared. The results showed that the CH-CA complex significantly enhanced the inhibitory effect of CH on α-glucosidase (P<0.05). The inhibitory effect of CH on α-glucosidase was 63.65% ± 1.96%. After binding with chlorogenic acid, the inhibitory activity of CH-CA100 and CH-CA200 on α-glucosidase reached a maximum of 80%. This indicates that the complex formed by the non-covalent binding of chlorogenic acid and myofibrillar protein has a synergistic effect on the inhibition of α-glucosidase.

[0050] (2) ABTS and DPPH free radical scavenging rates The 2,2′-abazapine (3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging activity of the SMH-polyphenol complex was determined in the casein hydrolysate-chlorogenic acid non-covalent complex samples prepared in Example 1. ABTS+• working solutions were prepared by mixing ABTS reagent (7 mmol / L) and potassium persulfate (2.45 mmol / L) at a 1:1 volume ratio and allowing the solution to stand at room temperature in the dark for 12-16 hours. Before use, the working solution was diluted with PBS to achieve an absorbance of 0.70 ± 0.02 at 734 nm. 180 μL of ABTS+• and 20 μL of CH-CA solution were added to each well of a 96-well plate, respectively. For the blank control, 20 μL of PBS was used instead of the sample. The absorbance measurement was recorded as A0. The ABTS free radical scavenging activity was calculated using the following equation: .

[0051] DPPH-ethanol solution was prepared at a concentration of 0.2 mmol / L. Subsequently, 600 μL of DPPH-ethanol solution was added to 400 μL of the casein hydrolysate-chlorogenic acid non-covalent complex sample solution prepared in Example 1. After thorough mixing, the mixture was reacted at room temperature in the dark for 30 minutes, and the absorbance was measured at a wavelength of 517 nm. In the blank control, 400 μL of 80% methanol was used instead of the sample, and the absorbance measurement was recorded as A0. DPPH free radical scavenging was calculated using the following formula:

[0052] Oxidative stress is closely associated with diabetes and may lead to the development and progression of diabetes-related complications. Therefore, antioxidant activity is crucial for the anti-diabetic potential of samples. This study evaluated the antioxidant properties of the CH-CA0 control group and the CH-CA complex using ABTS / DPPH radical scavenging analysis.

[0053] like Figure 4 As shown in section BC, compared with pure CH, the ABTS and DPPH radical scavenging abilities of CH after non-covalent binding with CA were significantly improved (P<0.05), especially the DPPH radical scavenging ability. The ABTS radical scavenging ability increased by 9.29%, and the DPPH radical scavenging ability increased by 85.92%. This may be due to the formation of additional hydroxyl groups in the CH-CA complex, thereby inhibiting free radical chain reactions and oxidation processes. In summary, the non-covalent binding of CA with CH significantly increases the antioxidant activity of CH. Therefore, the interaction between CH and CA provides a promising approach for developing novel antioxidants and functional foods with anti-diabetic potential.

[0054] 3. Emulsifying properties (1) Emulsifying properties and emulsion stability 3 mL of soybean oil was mixed with 9 mL of the casein hydrolysate-chlorogenic acid non-covalent complex sample (1 mg / mL) prepared in Example 1. The mixture was homogenized at 12000 rpm for 2 minutes. 50 μL of the emulsion was added from the bottom of the container to 5 mL of SDS (concentration 1 mg / mL). The absorbance of the mixture at 500 nm was measured using a UV spectrophotometer at 0 and 10 minutes. The following formulas were used to calculate EAI and ESI: ; .

[0055] In the formula: A0 and an A 10 The values ​​represent the absorbance obtained at 0 minutes and 10 minutes, where D represents the dilution factor, C represents the sample concentration, L represents the oil phase volume fraction, and θ represents the optical path length (1 cm).

[0056] like Figure 5As shown in Section A, the EAI (Extracellular Activated Iodide) shows an overall upward trend. This may be due to the influence of hydrogen and covalent bonds during non-covalent binding, leading to a rearrangement of the CH protein structure, thereby enhancing the affinity at the oil-water interface. When CH binds to CA, the EAI of the CH-CA100 and CH-CA250 groups is significantly higher than that of CH-CA0, but the CH-CA250 group has the lowest emulsifying stability. This may be because the continuous increase in polyphenol concentration leads to protein aggregation, thereby reducing the emulsifying stability of CH-CA; therefore, the improvement in emulsifying activity may stem from the reduction in CH-CA100 particle size and the increase in surface hydrophobicity. Similar reports indicate that binding with polyphenols can significantly improve the emulsifying properties of soybean protein hydrolysates, even with an increase in interfacial tension relative to the hydrolysates.

[0057] (2) Interfacial tension The interfacial tension (γ-0) of CH and the casein hydrolysate-chlorogenic acid non-covalent complex sample prepared in Example 1 was measured using a contact angle meter. In short, a syringe (aqueous phase) was immersed below the surface of the test cuvette (soybean oil), 4 μL of the sample was manually injected, and the droplet morphology was monitored for 1200 seconds using a charge-coupled device (CCD) camera. The interfacial tension was calculated from the droplet shape using the Yang-Laplace equation.

[0058] like Figure 5 As shown in Part B, compared to CH, the interfacial tension of CH-CA50, CH-CA100, and CH-CA250 increased. This may be because when CH interacts with CA, the hydrophobic side chains of the amino acids stack, and the hydrophobic domains of CH cannot fully penetrate into the oil-water interface, thus affecting the protein's affinity and altering its conformation at the interface. The interfacial tension of the CH-CA100 group was slightly higher than that of the control group, but the EAI was significantly increased. This may be because, although the complex formed by chlorogenic acid and CH did not significantly reduce the interfacial tension when adsorbed at the oil-water interface, it improved the dispersibility of the emulsion by optimizing the spreading and arrangement of interfacial molecules, ultimately exhibiting higher emulsifying activity.

[0059] Example 2: Preparation of casein hydrolysate-chlorogenic acid non-covalent complex emulsion 0.2% xanthan gum solution was added to the casein hydrolysate-chlorogenic acid non-covalent complex solution obtained in Example 1 at a volume ratio of 2:1. The mixture was homogenized at room temperature using 5% (v / v) medium-chain triglycerides and homogenized for 2 min at 12,000 rpm using a homogenizer (IKA, Stauffen, Germany). Subsequently, the mixture was ultrasonically treated in an ice bath using a Scientz ultrasonic processor (Ningbo, China) (ultrasonic treatment conditions: treatment time 3 min, pulse mode 2 s on, 2 s off, power output 300 W) to prepare casein hydrolysate-chlorogenic acid non-covalent complex emulsions (abbreviated as CH-CA0, CH-CA25, CH-CA50, CH-CA100, CH-CA200, CH-CA250, respectively).

[0060] Results data: 1. Emulsion properties (1) Particle size and Zeta potential The median particle size (D) of the casein hydrolysate-chlorogenic acid non-covalent complex emulsion prepared in Example 2 50 The zeta potential of the fresh emulsion was determined using a laser particle size analyzer (SYNC, USA) and a nanoparticle size analyzer (Malvern Instruments Ltd., UK).

[0061] With increasing chlorogenic acid concentration (25-100 μmol / g protein) in the emulsion, the overall particle size of the emulsion showed a decreasing trend. This may be because the addition of chlorogenic acid induces further unfolding of the CH structure, which exposes additional hydrophobic domains. Polyphenols alter protein conformation through hydrogen bonding, hydrophobicity, and π-π and ionic interactions, thereby changing the interfacial properties of protein particles. Figure 6 As shown in Part A, the CH-CA100-stabilized emulsion has the smallest particle size, consistent with the results for the nanocomposite particle size. With increasing chlorogenic acid content, the particle size of the composite-stabilized emulsion gradually increases. This may be due to the aggregation of the composite caused by the increased polyphenol content. The molecular weight and hydroxyl content of polyphenols may affect their ability to form non-covalent bonds with proteins, thus influencing emulsion droplet aggregation during emulsification and storage. Meanwhile, with increasing CA concentration, the absolute potential of the composite-stabilized emulsion shows a concentration-dependent increasing trend (e.g., ...). Figure 6 (As shown in Part B). A high net charge on the emulsion surface can increase the thickness of the emulsion interfacial film and the electrostatic repulsion, thereby improving the emulsion stability.

[0062] (2) Rheological properties The rheological properties of the casein hydrolysate-chlorogenic acid non-covalent complex emulsion prepared in Example 2 were determined using a rheometer. In viscosity testing mode, the gap was set to 0.5 mm, the testing temperature to 25°C, and the range was 0.1-100 s. -1 The apparent viscosity of the emulsion was determined at a shear rate of 1%. Frequency scanning was performed at 1% strain, with a frequency range of 0.1–10 Hz.

[0063] like Figure 7 As shown in Part A, with increasing shear rate, the apparent viscosity of CH-CA emulsions at different CA concentrations gradually decreases, eventually reaching equilibrium, exhibiting a "shear-thinning" characteristic. When the shear rate is sufficient to overcome Brownian motion, the emulsion droplets become more ordered and have low flow resistance, thus exhibiting low viscosity. Compared to the CH-CA0 control group, the composite provided by this invention has a relatively low apparent viscosity. Among them, the CH-CA100 composite-stabilized emulsion has the lowest apparent viscosity.

[0064] like Figure 7 As shown in Part B, the elastic modulus (G′) and loss modulus (G″) of CH-CA emulsions at different CA concentrations vary with frequency. The G′ value of all emulsions is consistently greater than the G″ value within the test frequency range, indicating the formation of a weak gel-like network structure, primarily exhibiting elastic behavior and good stability. Compared to CH-CA0-stabilized emulsions, the protein-polyphenol complex solution-stabilized emulsions show increased G′ and G″ values, indicating enhanced interdroplet interactions. This may also be due to the non-covalent interaction between CH and CA, thereby improving the interfacial and emulsifying properties of CH.

[0065] (3) Optical microscope The microstructure of the casein hydrolysate-chlorogenic acid non-covalent complex emulsion prepared in Example 2 was observed using an optical microscope system (Olympus BX53, Japan). The fresh emulsion was diluted 20 times and observed under an optical microscope at 40× magnification. The microstructure images were obtained using imaging software.

[0066] like Figure 8 As shown, in the microstructure of CH-based emulsions, the oil droplets are relatively large; in contrast, the oil droplets in emulsions based on low-concentration complexes are smaller, enhancing the physical stability of the emulsions. Among them, the CH-CA100-based emulsion exhibits the best microstructure morphology, with smaller and more dispersed oil droplets.

[0067] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A method for preparing a casein hydrolysate-chlorogenic acid non-covalent complex emulsion, characterized in that, The method includes the following steps: S1. Preparation of casein hydrolysate: The casein solution was enzymatically hydrolyzed by adding neutral protease at 50°C and pH=7. After the enzymatic hydrolysis was completed, the solution was heated in a boiling water bath for 10 min to inactivate the enzyme. Then, it was immediately cooled to room temperature with running tap water, centrifuged, the supernatant was collected and freeze-dried to obtain casein hydrolysate. S2. Preparation of casein hydrolysate-chlorogenic acid non-covalent complex: Chlorogenic acid was dissolved in a 0.01 mol / L phosphate buffer solution with pH=7.0 and magnetically stirred to prepare a chlorogenic acid stock solution. The casein hydrolysate obtained in S1 was mixed with the chlorogenic acid stock solution to obtain a casein hydrolysate-chlorogenic acid non-covalent complex. The non-covalent complex was placed in a shaker and incubated in the dark to obtain a casein hydrolysate-chlorogenic acid non-covalent complex solution. S3. Preparation of casein hydrolysate-chlorogenic acid non-covalent complex emulsion: Add 0.2% xanthan gum solution to the casein hydrolysate-chlorogenic acid non-covalent complex solution obtained in S2 and mix. Homogenize with 5% (v / v) medium-chain triglycerides at room temperature and at 12,000 rpm for 2 min. Then, sonicate under ice bath conditions to prepare the casein hydrolysate-chlorogenic acid non-covalent complex emulsion.

2. The preparation method according to claim 1, characterized in that, The casein solution in S1 is 5%, w / w; the enzymatic hydrolysis time is 2 h; the ratio of neutral protease to casein is 1% (w / w); and the centrifugation conditions are: 10000×g centrifugation for 15 min.

3. The preparation method according to claim 1, characterized in that, The α-glucosidase inhibitory activity of the casein hydrolysate prepared in S1 was 76.14% ± 0.018%, IC50 50 =7.19 mg / mL.

4. The preparation method according to claim 1, characterized in that, The mass fraction of chlorogenic acid mother liquor mentioned in S2 is 10 mg / mL.

5. The preparation method according to claim 1, characterized in that, The final concentration of chlorogenic acid in the casein hydrolysate-chlorogenic acid non-covalent complex described in S2 is 100 μmol / g.

6. The preparation method according to claim 1, characterized in that, The conditions for dark incubation described in S2 are: dark incubation for 2 h at 25°C and 150 r / min on a shaker.

7. The preparation method according to claim 1, characterized in that, The volume ratio of the casein hydrolysate-chlorogenic acid non-covalent complex solution to the xanthan gum solution in S3 is 2:

1.

8. The preparation method according to claim 1, characterized in that, The conditions for ultrasonic treatment described in S3 are: treatment time of 3 min, pulse mode of 2 s on and 2 s off, and power output of 300 watts.

9. A casein hydrolysate-chlorogenic acid non-covalent complex emulsion, characterized in that, The casein hydrolysate-chlorogenic acid non-covalent complex emulsion is prepared by the method described in any one of claims 1 to 8.

10. The application of the casein hydrolysate-chlorogenic acid non-covalent complex emulsion according to claim 9 in the preparation of food with anti-diabetic function and emulsifying properties, characterized in that, The food in question is not intended for therapeutic purposes.

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