A method for preparing a covalent complex of casein peptides and a covalent complex

Abstract

This invention discloses a method for preparing a casein peptide covalent complex and the covalent complex itself, belonging to the field of food manufacturing. The method includes the following steps: pre-preparing complex raw materials, including casein, protease, transglutaminase, chitosan oligosaccharide, and water; uniformly stirring casein and water to obtain a casein solution; enzymatically hydrolyzing the casein solution using the protease to obtain casein hydrolysate; cross-linking the casein hydrolysate and chitosan oligosaccharide using transglutaminase to obtain a cross-linked product; and drying the obtained cross-linked product to obtain the casein covalent complex. The beneficial effects of this invention are: by combining restricted enzymatic hydrolysis with transglutaminase-mediated enzymatic glycosylation, chitosan oligosaccharide and casein peptides achieve covalent bonding, avoiding the use of chemical cross-linking agents, improving system safety and structural stability, and making it less prone to dissociation under pH or ionic changes compared to non-covalent complexes.

Description

Technical Field

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

[0002] Casein is the main protein in dairy products, possessing excellent nutritional value and functional properties, and is widely used in food processing. Casein peptides, obtained through protease hydrolysis, have lower molecular weight, improved solubility, and certain antioxidant activity and physiological functions, making them valuable in the development of functional foods and nutritional preparations. However, casein peptides still suffer from insufficient stability in complex processing environments, limited interfacial activity, and susceptibility to pH and ionic strength, which restricts their 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 certain antibacterial and antioxidant activities, making them promising for applications in the food and biomaterials fields. Currently, the binding of chitosan oligosaccharides with proteins or peptides mostly employs physical complexation methods (such as electrostatic interactions and hydrogen bonding). However, the resulting non-covalent complexes have poor stability and are prone to dissociation under processing, storage, or pH changes, making it difficult to form structurally stable and functionally durable composite systems. While traditional chemical grafting methods can achieve covalent bonding, they require chemical cross-linking agents, posing challenges such as stringent reaction conditions, residue risks, and safety concerns, hindering their widespread application in the food industry.

[0004] No effective solutions have yet been proposed to address the problems in the relevant technologies. Summary of the Invention

[0005] In view of the problems in the related technologies, the present invention proposes a method for preparing a casein peptide covalent complex and the covalent complex, so as to overcome the above-mentioned technical problems existing in the existing related technologies.

[0006] The technical solution of this invention is implemented as follows:

[0007] According to one aspect of the present invention, a method for preparing a casein peptide covalent complex is provided, comprising the following steps:

[0008] S1. Pre-prepared complex raw materials, including casein, protease, transglutaminase, chitosan oligosaccharide and water;

[0009] S2. Mix casein and water evenly to obtain a casein solution;

[0010] S3. Enzymatically hydrolyze the casein solution using protease to obtain casein hydrolysate.

[0011] S4. Cross-link casein hydrolysate and chitosan oligosaccharide using transglutaminase to obtain cross-linked products;

[0012] S5. The obtained cross-linked product is dried to obtain a casein covalent complex.

[0013] Optionally, when casein hydrolysate, chitosan oligosaccharide, and transglutaminase are mixed to obtain cross-linked products, the temperature of both the cross-linking reaction and the mixing reaction is 37°C and the reaction time is 2 h.

[0014] The mass ratio of casein hydrolysate to chitosan oligosaccharide is 1:1 to 4:1.

[0015] Optionally, the casein covalent complex is a chitosan oligosaccharide-casein peptide covalent complex.

[0016] Optionally, the pH value is 7.0 to 8.0 when casein and water are stirred evenly.

[0017] Optionally, the casein solution in step S2 has a mass concentration of 5%.

[0018] Optionally, the protease mentioned in step S3 includes trypsin and papain;

[0019] Optionally, when enzymatically hydrolyzing the casein solution using a protease, the resulting casein hydrolysate includes:

[0020] A trypsin-casein hydrolysate solution was obtained by enzymatic hydrolysis of trypsin and casein solution.

[0021] Papain was used to enzymatically hydrolyze casein solution to obtain papain-casein hydrolysate solution;

[0022] The trypsin-casein hydrolysate solution and the papain-casein hydrolysate solution were thoroughly mixed to obtain the casein hydrolysate.

[0023] Optionally, when using trypsin and casein solution for enzymatic hydrolysis, the hydrolysis temperature is 37°C and the hydrolysis time is 2 h.

[0024] When using papain to enzymatically hydrolyze casein solution, the hydrolysis temperature is 55℃ and the hydrolysis time is 2 hours.

[0025] Optionally, it also includes heat treatment of casein hydrolysates to inactivate enzymes, including:

[0026] After enzymatic hydrolysis using trypsin and casein solution, enzyme inactivation was performed by heating at 85℃ for 5 min.

[0027] After the casein solution was enzymatically hydrolyzed using papain, the enzyme was inactivated by heating at 100℃ for 5 minutes.

[0028] According to another aspect of the present invention, a chitosan oligosaccharide-casein peptide covalent complex is provided, which is prepared by a method for preparing casein peptide covalent complexes.

[0029] The beneficial effects of this invention are as follows: By combining restricted enzymatic hydrolysis with transglutaminase-mediated enzymatic glycosylation, chitosan oligosaccharides and casein peptides are covalently bound, avoiding the use of chemical cross-linking agents, improving the safety and structural stability of the system, and making it less prone to dissociation under pH or ionic changes compared to non-covalent complexes; moderate enzymatic hydrolysis exposes lysine and hydrophobic group sites, and glycosylation further regulates the secondary and tertiary structures of proteins, enhancing molecular flexibility and interfacial adsorption capacity, which is conducive to the formation of stable interfacial films; the reaction conditions are controllable, and performance can be regulated by adjusting the enzymatic hydrolysis time and glycosylation ratio, making the operation simple and suitable for industrial applications in functional foods and emulsion systems.

[0030] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0031] 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.

[0032] Figure 1 The graph shows the changes in sugar content corresponding to different substrate concentrations, transglutaminase additions, and chitosan oligosaccharide to casein hydrolysate ratios. ad is the hydrolysate hydrolyzed by trypsin, and eh is the hydrolysate hydrolyzed by papain.

[0033] Figure 2 It is an SDS-PAGE electrophoresis pattern of casein and casein hydrolysate, cross-linked products of hydrolysate, and glycosylated products of hydrolysate and chitosan oligosaccharide;

[0034] Figure 3 These are the ultraviolet absorption spectra of different samples;

[0035] Figure 4 These are the intrinsic fluorescence spectra of different samples;

[0036] Figure 5 This is a graph showing the changes in surface hydrophobicity of different samples;

[0037] Figure 6 These are Fourier transform infrared spectra of different samples;

[0038] Figure 7 These are circular dichroism chromatograms of different samples;

[0039] Figure 8 This is a graph showing the content of secondary structures in different samples;

[0040] Figure 9 These are particle size distributions of different samples;

[0041] Figure 10 These are potential diagrams of different samples.

[0042] Figure 11 These are scanning electron microscope images of different samples.

[0043] Figure 12 This is a graph showing the ABTS free radical scavenging rate of different samples;

[0044] Figure 13 This is a graph showing the DPpH free radical scavenging rate of different samples;

[0045] Figure 14 This is a flowchart of a method for preparing a casein peptide covalent complex according to an embodiment of the present invention. Detailed Implementation

[0046] 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.

[0047] Example 1

[0048] Please see Figure 14 This embodiment provides a method for preparing a chitosan oligosaccharide-casein peptide covalent complex, specifically including the following steps:

[0049] S1. Pre-prepared complex raw materials, including casein, proteases (trypsin and papain), transglutaminase, chitosan oligosaccharide and water.

[0050] S2. Mix casein and water evenly to obtain a casein solution.

[0051] Specifically, 5g of casein was weighed and dissolved in water. The pH of the system was adjusted to 7.4 with 4mol / L sodium hydroxide solution. Under these conditions, the mixture was stirred continuously for 24 hours and then brought to a final volume of 100ml to obtain a homogeneous casein solution (mass concentration of 5%).

[0052] S3. Enzymatic hydrolysis of casein solution using proteases to obtain casein hydrolysate. Specifically, this includes:

[0053] Casein solution was enzymatically hydrolyzed using trypsin (hydrolysis temperature 37℃, hydrolysis time 2h, enzyme-to-toxin ratio 1%). After hydrolysis, the enzyme was inactivated at 85℃ for 5min to obtain a trypsin-casein hydrolysate solution. Casein solution was also enzymatically hydrolyzed using papain (hydrolysis temperature 55℃, hydrolysis time 2h, enzyme-to-toxin ratio 1%). After hydrolysis, the enzyme was inactivated at 100℃ for 5min to obtain a papain-casein hydrolysate solution. The two hydrolysate solutions were then thoroughly mixed to obtain casein hydrolysate.

[0054] S4. Cross-linking reaction of casein hydrolysate and chitosan oligosaccharide was carried out using transglutaminase to obtain the cross-linked product. The specific conditions were as follows: substrate concentration was 40 mg / ml, transglutaminase dosage was 10 U / g, mass ratio of casein hydrolysate to chitosan oligosaccharide was 3:1, reaction temperature was 37℃, reaction time was 3 h, and dialysis was performed for 48 h after the reaction was completed.

[0055] S5. The obtained cross-linked product is subjected to vacuum freeze-drying to obtain a chitosan oligosaccharide-casein peptide covalent complex.

[0056] Example 2

[0057] Based on Example 1, the reaction time in step S4 was changed to 2 hours, while the other conditions remained unchanged.

[0058] Example 3

[0059] Based on Example 1, the mass ratio of casein hydrolysate to chitosan oligosaccharide in step S4 was changed to 1:1, while the other conditions remained unchanged.

[0060] Example 4

[0061] Based on Example 1, the mass ratio of casein hydrolysate to chitosan oligosaccharide in step S4 was changed to 4:1, while the other conditions remained unchanged.

[0062] Compare with Example 1

[0063] This comparative example provides a method for preparing a casein peptide covalent complex, which differs from Example 1 in that chitosan oligosaccharide is not added. The method specifically includes the following steps:

[0064] S1. Pre-prepared complex raw materials, including casein, protease, transglutaminase and water;

[0065] S2. Mix casein and water evenly to obtain a casein solution;

[0066] S3. Enzymatically hydrolyze the casein solution using protease to obtain casein hydrolysate;

[0067] S4. Cross-linking reaction of casein hydrolysate with transglutaminase to obtain cross-linked products;

[0068] S5. The obtained cross-linked product is dried to obtain a casein covalent complex.

[0069] Experimental Example 1: Determination of Sugar Content

[0070] The glucosamine uptake levels in the products obtained in Examples 1-8 and Control Example 1 were determined using the DNS method. The results are as follows: Figure 1 As shown.

[0071] Figure 1 The effects of different reaction conditions on the sugar content of two casein hydrolysates (trypsin hydrolysate and papain hydrolysate) with chitosan oligosaccharides are shown. Figure ad shows the results for trypsin hydrolysate, and Figure eh shows the results for papain hydrolysate.

[0072] (1) Effect of substrate concentration on saccharide loading (Figures a and e): As the substrate concentration increased from 30 mg / ml to 60 mg / ml, the saccharide loading of both hydrolysates showed a trend of first increasing and then decreasing. The saccharide loading reached its maximum value when the substrate concentration was 40 mg / ml.

[0073] (2) Effect of TGase addition on sugar content (Figures b and f): As the amount of TGase added increases from 5 U / g to 20 U / g, the sugar content gradually increases, but when the amount of TGase added exceeds 10 U / g, the increase in sugar content tends to slow down.

[0074] (3) Effect of the ratio of chitosan oligosaccharide to casein hydrolysate on the amount of sugar attached (Figures c and g): When the ratio of chitosan oligosaccharide to hydrolysate changes from 1:1 to 1:3, the amount of sugar attached increases significantly, reaching the highest at 1:3 (i.e., hydrolysate: chitosan oligosaccharide = 3:1); when it continues to increase to 1:4, the amount of sugar attached decreases slightly or tends to stabilize.

[0075] (4) Effect of reaction time on sugar content (Figures d and h): As the reaction time is extended from 1 h to 5 h, the sugar content increases rapidly in the first 3 h, and the increase slows down and tends to reach equilibrium after 3 h.

[0076] (5) Comparison of the two hydrolysates: Under the same reaction conditions, the sugar content of trypsin hydrolysate was slightly higher than that of papain hydrolysate.

[0077] Experimental Example 2: SDS-PAGE Analysis

[0078] The products obtained in Example 1 and Control Example 1 were analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 2 As shown in the figure. Electrophoresis results showed that the molecular weight of casein decreased significantly after enzymatic hydrolysis; after treatment with transglutaminase, effective cross-linking reactions occurred between the hydrolysate peptides, forming polymers with larger molecular weights; in the presence of chitosan oligosaccharides, transglutaminase successfully mediated the covalent binding of chitosan oligosaccharides with casein hydrolysates, and the glycosylated product bands exhibited diffuse and upward shift characteristics.

[0079] Experimental Example 3: Ultraviolet Spectroscopy Analysis

[0080] The products obtained in Example 1 and Control Example 1 were subjected to ultraviolet spectral analysis, and the results are as follows: Figure 3 As shown, enzymatic hydrolysis expands the casein structure, exposes aromatic amino acids, and enhances UV absorption; cross-linking and glycosylation further alter the protein conformation and the microenvironment of chromophores, resulting in decreased UV absorption intensity and a red shift.

[0081] Experimental Example 4: Intrinsic Fluorescence Spectroscopy Analysis

[0082] The products obtained in Example 1 and Control Example 1 were subjected to intrinsic fluorescence spectroscopy analysis, and the results are as follows: Figure 4 As shown, enzymatic hydrolysis significantly enhanced fluorescence intensity and caused a red shift; cross-linking and glycosylation significantly decreased fluorescence intensity and caused a blue shift, with the glycosylated product exhibiting the lowest fluorescence intensity.

[0083] Experimental Example 5: Surface Hydrophobicity Analysis

[0084] The surface hydrophobicity of the products obtained in Example 1 and Comparative Example 1 was analyzed, and the results are as follows: Figure 5 As shown, enzymatic hydrolysis significantly increased surface hydrophobicity; cross-linking and glycosylation significantly decreased surface hydrophobicity, with the glycosylated product exhibiting the lowest surface hydrophobicity.

[0085] Experiment Example 6: Fourier Transform Infrared Spectroscopy Analysis

[0086] Fourier transform infrared spectroscopy analysis was performed on the products obtained in Example 1 and Control Example 1, and the results are as follows: Figure 6 As shown. Glycosylated products are at 1050-1150 cm⁻¹ -1 The presence of characteristic absorption peaks for glycosidic bonds in the region confirms the successful covalent grafting of chitosan oligosaccharides.

[0087] Experimental Example 7: Circular Dichroism Chromatography

[0088] The products obtained in Example 1 and Comparative Example 1 were analyzed by circular dichroism chromatography, and the results are as follows: Figure 7-8 As shown, enzymatic hydrolysis disrupted the secondary structure of casein, decreasing the content of α-helices and β-sheets while increasing the content of random coils; cross-linking induced the polypeptide chains to reform an ordered secondary structure; glycosylation further promoted the formation and stabilization of the ordered structure, with the α-helix content reaching its highest level.

[0089] Experimental Example 8: Particle Size and Zeta Potential Analysis

[0090] The particle size and zeta potential of the products obtained in Example 1 and Control Example 1 were analyzed, and the results are as follows: Figure 9-10 As shown, enzymatic hydrolysis significantly reduced particle size and enhanced electronegativity; cross-linking significantly increased particle size and weakened electronegativity; glycosylation further increased particle size while simultaneously increasing the absolute potential value, thus restoring and improving colloidal stability.

[0091] Experiment Example 9: Scanning Electron Microscopy Analysis

[0092] Scanning electron microscopy analysis was performed on the products obtained in Example 1 and Control Example 1, and the results are as follows: Figure 11 As shown, casein raw materials exhibit a dense, blocky aggregate structure; after enzymatic hydrolysis, the structure becomes loose and porous; cross-linking treatment restores the structure to a dense and smooth state; and glycosylation treatment forms a regular three-dimensional porous network structure.

[0093] Experimental Example 10: Determination of Antioxidant Activity

[0094] The products obtained in Example 1 and Control Example 1 were subjected to ABTS and DPpH free radical scavenging activities assays, and the results are as follows: Figure 12-13 As shown, enzymatic hydrolysis significantly improved antioxidant activity; cross-linking treatment led to a decrease in antioxidant activity; glycosylation treatment significantly enhanced antioxidant activity, and the glycosylated product exhibited the strongest free radical scavenging ability among all samples.

[0095] Test results show that the chitosan oligosaccharide-casein peptide covalent complexes prepared in Examples 1-4 of this invention have higher sugar content, better structural stability, better solubility and stronger antioxidant activity compared with the control example (crosslinked product without chitosan oligosaccharide). They also exhibit more stable interfacial properties under different pH and ionic strength conditions.

[0096] In summary, by utilizing the above-mentioned technical solution of the present invention, chitosan oligosaccharide is used as a necessary raw material, and transglutaminase is used to cross-link casein hydrolysate and chitosan oligosaccharide. Under mild, safe, and efficient conditions, covalent bonding between chitosan oligosaccharide and casein peptides is achieved, solving the problems of insufficient stability, short-lasting functional activity, and difficulty in ensuring food safety in the prior art. The resulting complex has broad application prospects in functional foods, emulsion systems, and nutrient delivery.

[0097] 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 casein peptide covalent complex, characterized in that, Includes the following steps: S1. Pre-prepared complex raw materials, including casein, protease, transglutaminase, chitosan oligosaccharide and water; S2. Mix casein and water evenly to obtain a casein solution; S3. Enzymatically hydrolyze the casein solution using protease to obtain casein hydrolysate. S4. Cross-linking reaction of casein hydrolysate and chitosan oligosaccharide with transglutaminase to obtain cross-linked products; S5. The obtained cross-linked product is dried to obtain a casein covalent complex.

2. The method for preparing casein peptide covalent complex according to claim 1, characterized in that, When casein hydrolysate, chitosan oligosaccharide and transglutaminase are mixed and reacted to obtain cross-linked products, the temperature of both the cross-linking reaction and the mixing reaction is 37°C and the reaction time is 2 h. The mass ratio of casein hydrolysate to chitosan oligosaccharide is 1:1 to 4:

1.

3. The method for preparing casein peptide covalent complex according to claim 2, characterized in that, The casein covalent complex is a chitosan oligosaccharide-casein peptide covalent complex.

4. The method for preparing the casein peptide covalent complex according to claim 1, characterized in that, When casein and water are stirred evenly, the pH value is 7.0 to 8.

0.

5. The method for preparing the casein peptide covalent complex according to claim 1, characterized in that, The casein solution in step S2 has a mass concentration of 5%.

6. The method for preparing the casein peptide covalent complex according to claim 1, characterized in that, The proteases mentioned in step S3 include trypsin and papain.

7. The method for preparing the casein peptide covalent complex according to claim 6, characterized in that, When a casein solution is enzymatically hydrolyzed using a protease, casein hydrolysate is obtained, including: A trypsin-casein hydrolysate solution was obtained by enzymatic hydrolysis of trypsin and casein solution. Papain was used to enzymatically hydrolyze casein solution to obtain papain-casein hydrolysate solution; The trypsin-casein hydrolysate solution and the papain-casein hydrolysate solution were thoroughly mixed to obtain the casein hydrolysate.

8. The method for preparing the casein peptide covalent complex according to claim 7, characterized in that, When using trypsin and casein solution for enzymatic hydrolysis, the hydrolysis temperature is 37℃ and the hydrolysis time is 2 h. When using papain to enzymatically hydrolyze casein solution, the hydrolysis temperature is 55℃ and the hydrolysis time is 2 h.

9. The method for preparing the casein peptide covalent complex according to claim 8, characterized in that, It also includes heat treatment of casein hydrolysates to inactivate enzymes, including: After enzymatic hydrolysis using trypsin and casein solution, enzyme inactivation was performed by heating at 85℃ for 5 min. After the casein solution was enzymatically hydrolyzed using papain, the enzyme was inactivated by heating at 100℃ for 5 minutes.

10. A chitosan oligosaccharide-casein peptide covalent complex, characterized in that... It is prepared by any one of the methods of claims 1-9.

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